Indian Patents
Title of Invention

A PEPTIDE FOR TREATING A PATIENT HAVING A CONDITION MEDIATED BY NEUROTOXICITY, NEURODEGENERATION OR NEUROLOGICAL DAMAGE
Abstract The invention relates to a peptide for treating a patient having a condition mediated by neurotoxicity, neurodegeneration or neurological damage comprising one or more monomeric peptides of 8 to about 40 amino acids in length that bind to EPO receptor, each monomeric peptide comprising a sequence of amino acids X4X5XaXbX6XcXdX7 (SEQ ID NO: 47), wherein Xa is G or A; Xb is P or A; Xc is T or A; Xd is selected from W, A, and F; X4 is selected from R, H, Y, L, and W, or X4 is nonexistent; X5 is selected from F, M, and I; X6 is independently selected from the 20 genetically coded L-amino acids of the stereoisomeric D-amino acids; and X7 is selected from D, V, E, I, and L.
Full Text NEUROPROTECTIVE PEPTIDES
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of United States Provisional Application Serial
No. 60/207654, filed May 26, 2000.
FIELD OF THE INVENTION
The present invention is directed to methods of treating diseases and conditions
involving the nervous system by administration of compositions having the therapeutic
activity of human erythropoietin. These compositions include therapeutic agents such as
peptides, peptide dimers, polypeptides, and proteins that have the full range of biological
activity of human erythropoietin or only certain biological activities of erythropoietin. The
present invention also provides improved therapeutic regimens wherein the therapeutic agent
is administered at concentrations below those required to stimulate hematopoiesis.
BACKGROUND OF THE INVENTION
Erythropoietin (EPO) is a glycoprotein hormone produced by the kidney in response
to tissue hypoxia that stimulates red blood cell production in the bone marrow. The gene for
erythropoietin has been cloned and expressed in Chinese hamster ovary (CHO) cells as
described in United States Patent No. 4,703,008. Recombinant human erythropoietin (r-
HuEPO or Epoetin alfa) has an amino acid sequence identical to that of human urinary
erythropoietin, and the two are indistinguishable in chemical, physical and immunological
tests. Recombinant human erythropoietin acts by increasing the number of cells capable of
differentiating into mature erythrocytes, triggering their differentiation and augmenting
hemoglobin synthesis in developing erythroblasts (Krantz SB. Blood (1991) 77: 419-434,
Beckman BS, Mason-Garcia M. The Faseb Journal (1991) 5: 2958-2964).
Epoetin alfa has been well tolerated in studies conducted to date. Hypertensive
encephalopathy and seizures have occasionally been described in dialysis patients treated with
Epoetin alfa, particularly during the early phase of therapy when hematocrit is rising. (Eschbach
JW, Egrie JC, Downing MR, Browne JK, Adamson JW. New Engl J Med (1987) 316: 73-78,
Winearls CG, Oliver DO, Pippard MJ, et al. Lancet (1986) 2 (8517): 1175-1177). Such reports
became more rare as experience of use of the compound developed. Occasionally, cancer
patients treated with Epoetin alfa have experienced an increase in blood pressure associated with
a significant increase in hematocrit. The risk, however, appears substantially lower than in
chronic renal failure patients.
No antibody titers against Epoetin alfa could be demonstrated and confirmed in subjects
treated with Epoetin alfa for up to 2 years, indicating the absence of immunological sensitivity to
Epoetin alfa. Skin rashes and urticaria have been observed rarely and when reported have been
mild and transient in nature, but these events suggest allergic hypersensitivity to some
components of the Epoetin alfa formulation.
Epoetin alfa is approved for sale in many countries for the treatment of anemia in chronic
renal failure (dialysis and predialysis), anemia in zidovudine treated HIV positive patients (US),
anemia in cancer patients receiving platinum-based chemotherapy, as a facilitator of
autologous blood pre-donation, and as a peri-surgical adjuvant to reduce the likelihood of
requiring allogeneic blood transfusions in patients undergoing orthopedic surgery.
EPO influences neuronal stem cells, likely during embryonic development, and
possibly during in vitro experiments of differentiation. (Juul et al Pediatr Dev Pathol (1999)
2(2) 148 - 158. Juul et al Pediatr Res (1998) 43(1) 40 - 49.) Further, neonates and infants
that suffer CNS injury via hypoxia, meningitis, and intraventricular hemorrhage, show an
EPO induced neuroprotective effect (Juul et al Ped Res (1999) 46(5) 543 - 547.)
EPO helps prevent apoptosis of neural tissue in cases of injury that create hypoxia.
This may be the result of EPO produced locally by astrocytes (Morishita et al Neuroscience
(1996) 76(1) 105 - 116). Neuroprotection has been demonstrated in gerbil hippocampal and
rat cerebrocortical tissue (Sakanaka et al PNAS (1998) 95(8) 4635 - 4640. Sadamoto et al
Biochem Biophys Res Commun (1998) 253(1) 26 - 32).
EPO induces biological effects of PC12 cells, including changes in Ca2+, changes in
membrane potential, and promotion of neuronal survival. This has been interpreted that EPO
can stimulate neural function and viability (Koshimura et al J. Neurochem (1999) 72(6)
2565-2572. Tabria et al Int J Dev Neurosci (1995) 13(3/4) 241 - 252.).
O'Brien et al propose that a 17 amino acid peptide sequence of EPO can act through
the EPO -R (Erythropoietin receptor) to induce biological activity in NS20Y, SK-N-MC, and
PC 12 cells, which includes sprouting, differentiation and neuroprotection. Curiously this
peptide does not promote proliferation of hematologic cells, thus it appears inactive in cell
lines well understood for their sensitivity to EPO activity. (Campana et al Int J Mol Med
(1998) 1(1) 235 - 241 and United States Patent No.'s 5,700,909, issued on 12/23/1999,
5,571,787, issued on 11/5/1996, 5,714,459, issued on 2/3/1998, and 5,696,080, issued on
12/9/1997, all to O'Brien et al.)
EPO may influence neuronal stem cell commitment to drive differentiation of neurons
as opposed to astrocytes or oligodendrocytes. This is compared to a similar activity of EPO,
where it functions to drive commitment of hematopoietic stem cells to produce red blood
cells (RBCs). There is an apparent relationship between CNS hypoxic injury, resulting in the
production of EPO from astrocytes that commits neuronal stem cells to differentiate into
neurons, while simultaneously acting as a neuroprotective function for existing neurons.
(WIPO publication number WO99/21966, published on 5/6/1999, Weiss et al.)
SUMMARY OF THE INVENTION
The present invention is directed to methods of treating diseases and conditions
involving the nervous system by administration of compositions having the neurological
therapeutic activity of human erythropoietin.
In a first embodiment, the present invention is directed to a method for treating a
patient having a disorder characterized by neurotoxicity, neurodegeneration or neurological
damage, comprising administering to said patient a therapeutically effective amount of a
peptide comprising one or more monomeric peptides of 8 to about 40 amino acids in length
that bind to EPO receptor, each monomeric peptide comprising a sequence of amino acids
X4X5XaXbXc, (SEQ ID NO: 47), wherein
Xa is G or A;
Xb is P or A;
Xc is T or A;
Xd is selected from W, A, and F;
X4 is selected from R, H, Y, L, and W, or X4 is nonexistent;
X5 is selected from F, M, and I;
X6 is independently selected from the 20 genetically coded L-amino acids or the
stereoisomeric D-amino acids; and
X7 is selected from D, V, E, I, and L.
In particular, said sequence is X3X4X5GPX6TWX7X8(SEQ ID NO: 1), wherein
X3 is selected from C, E, A, a-amino-?-bromobutyric acid, and homocysteine (Hoc);
and
X8 is selected from C, K, A, a-amino-?-bromobutyric acid, and homocysteine (Hoc).
In a second embodiment, the present invention is directed to peptides which
behave as cell-surface receptor agonists and antagonists, as well as dimers and multimers
of such peptides which exhibit binding and signal initiation of growth factor-type
receptors. In one embodiment, the present invention provides peptides which behave as
EPO agonists. These peptides may be dimers or multimers of such peptides, preferably 14
to about 20 residues in length, comprising a core amino acid sequence of X3X4
X5GPX6TWX7X8 (SEQ ID NO: 1) wherein each amino acid is indicated by standard one
letter abbreviation; X3 can be C, E, A, a-amino-?-bromobutyric acid, or Hoc, where Hoc
is homocysteine; X4 can be R, H, Y, L, or W, or X4 is nonexistent; X5 can be M, F, or I;
X6 is independently any one of the 20 genetically coded L-amino acids or the
stereoisomeric D-amino acids; X7 can be D, E, I, L, or V; and X8 can be C, K, A, a-
amino-?-bromobutyric acid, or Hoc, where Hoc is homocysteine.
Preferably, the monomeric peptide unit of the dimer or multimer comprises a core
sequence of amino acids YX2X3X4X5GPX6TWX7X8 (SEQ ID NO: 2), wherein each of X2 and
X6 is independently any one of the 20 genetically coded L-amino acids; X3 is C; and X8 is C.
Preferably, the monomeric peptide unit of the dimer comprises a core sequence of
amino acids X1YX2X3X4XjGPX6TWX7X8X9XI0X11 (SEQ ID NO: 3), wherein each of X1, X2,
X6, X9, X10, and X11 is independently selected from the 20 genetically coded L-amino acids.
Particularly, X3 can be C, E, A; X4 can be R, H, or Y, or X4 is nonexistent; X5 can be M, F, or
I; X7 can be D or V; and X8 can be C, K, or A.
In a more preferred embodiment, both X3 and X8 are C and thus, the monomeric
peptide unit of the dimer comprises a core sequence of amino acids X1 YX2 CX4 X5 GPX6
TWX7 CX9 X10 X11 (SEQ ID NO: 4). Particularly, the monomeric peptide unit comprises
a core sequence of amino acids X1 YX2 CX4 X5 GPX6 TWX7 CX9 X10 X11 (SEQ ID NO:
5), where X4 can be R or H; X5 can be F or M; X6 can be I, L, T, M, or V; X7 is D or V;
X9 can be G, K, L, Q, R, S, or T; and X10 can be A, G, P, R, or Y. More particularly, the
monomeric peptide unit of the dimer will comprise a core sequence of amino acids X1
YX2 CX, X5 GPX6 TWX7 CX9 X10 X11 (SEQ ID NO: 6), where X1 can be D, E, L, N, S,
T, or V; X2 can be A, H, K, L, M, S, or T; X, is R or H; X9 can be K, R, S, or T; and X10 is
P.
Preferably, the monomeric peptide unit of the dimer will comprise a core sequence of
amino acids X1 YX2 CX4 X5 GPX6 TWX7 CX9 X10 X11 (SEQ ID NO: 6), where X1 can be D,
E, L, N, S, T, or V; X2 can be A, H, K, L, M, S, or T; X4 is R or H; X, can be K, R, S, or T;
and X,o is P.
Particularly preferred monomeric peptide units of the dimers include:
GGLYLCRFGPVTWDCGYKGG (SEQ ID NO:7);
GGTYSCHFGPLTWVCKPQGG (aka EMP-1) (SEQ ID NO:8);
GGDYHCRMGPLTWVCKPLGG (SEQ ID NO:9);
VGNYMCHFGPITWVCRPGGG (SEQ ID NO: 10);
GGVYACRMGPITWVCSPLGG (SEQ ID NO: 11);
VGNYMAHMGPITWVCRPGG (SEQ ID NO: 12);
GGTYSCHFGPLTWVCKPQ (aka EMP-16) (SEQ ID NO: 13);
GGLYACHMGPMTWVCQPLRG (aka EMP-36) (SEQ ID NO: 14);
TIAQYICYMGPETWECRPSPKA (aka EMP-38) (SEQ ID NO: 15);
YSCHFGPLTWVCK (aka EMP-20 (SEQ ID NO:: 16);
YCHFGPLTWVC (aka EMP-23) (SEQ ID NO: 17);
SCHFGPLTWVCK (aka EMP-24) (SEQ ID NO: 18);
GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO: 19);
GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO:20);
GGTYSCFGPLTWVCKPQGG (aka EMP-27) (SEQ ID NO:21);
TYSCHFGPLTWVCKPQGG (aka EMP-17) (SEQ ID NO:22);
TYSCHFGPLTWVCKPQ (aka EMP-18) (SEQ ID NO:23);
YSCHFGPLTWVCKP (aka EMP-19) (SEQ ID NO::24);
YSCHFGPLTWVC (aka EMP-21) (SEQ ID NO::25);
YSCHFGALTWVCK (aka EMP-22) (SEQ ID NO :26);
GGCRIGPITWVCGG (aka EMP-25) (SEQ ID NO:27);
HFGPLTWV (aka EMP-26) (SEQ ID NO:28);
GGTTSCHFGPLTWVCKPQGG (aka EMP-7) (SEQ ID NO:29);
GGTFSCHFGPLTWVCKPQGG (aka EMP-8) (SEQ ID NO:30);
GGTYSCHFGALTWVCKPQGG (aka EMP-10) (SEQ ID N0:31);
GGTYSCHFGPATWVCKPQGG (aka EMP-11) (SEQ ID NO.32);
GGTYSCHFGPLAWVCKPQGG (aka EMP-12) (SEQ ID NO:33);
GGTYSCHFGPLTAVCKPQGG (aka EMP-13) (SEQ ID NO:34);
GGTYSCHFGPLTFVCKPQGG (aka EMP-14) (SEQ ID NO:35);
GGTYSCHFGPLTWVCKAQGG (aka EMP-15) (SEQ ID NO:36);
GGTXSCHFGPLTWVCKPQGG (aka EMP-28, X = D-Tyr) (SEQ ID NO:37);
GGTXSCHFGPLTWVCKPQGG (aka EMP-29, X =p-NO2-Phe)
(SEQ ID NO:38);
GGTXSCHFGPLTWVCKPQGG (aka EMP-30, X = p-NH2-Phe)
(SEQ ID NO:39);
GGTXSCHFGPLTWVCKPQGG (aka EMP-31, X =p-F-Phe) (SEQ ID NO:40);
GGTXSCHFGPLTWVCKPQGG (aka EMP-32, X =p-I-Phe) (SEQ ID NO:41);
GGTXSCHFGPLTWVCKPQGG (aka EMP-33, X = 3,5-dibromo-Tyr)
(SEQ ID NO:42);
Ac-GGTYSCHFGPLTWVCKPQGG (aka EMP-34) (SEQ ID NO:43);
GGLYACHMGPMTWVCQPLGG (aka EMP-35) (SEQ ID NO:44);
LGRKYSCHFGPLTWVCQPAKKD (aka EMP-37) (SEQ ID NO:45); and
GGTYSEHFGPLTWVKKPQGG (aka EMP-39) (SEQ ID NO:46).
Preferably, monomelic peptide units of the dimers include:
GGTYSCHFGPLTWVCKPQGG (aka EMP-1) (SEQ ID NO:8);
GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO: 19);
GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO:20); and
YCHFGPLTWVC (aka EMP-23) (SEQ ID NO: 17).
In accordance with the present invention the monomelic units of the dimers can be the
same or different.
In a preferred embodiment polyethylene glycol (PEG) may be employed as a linker to
form the dimeric peptides of the present invention through a covalent bond.
In another embodiment, the present invention is directed to pharmaceutical
compositions comprising at least one peptide of the invention and a pharmaceutical carrier to
be used in a method of treating or preventing neurotoxicity.
In a further embodiment, the present invention provides a method for therapeutically
treating a mammal having a disease or condition resulting from a neurotoxic or
neurodegenerative or neuro-damaging event by administration of at least one of the peptides
of the present invention.
In a still further embodiment, a method for therapeutically treating a mammal having
a neurotoxic, neuro-damaging or neurodegenerative condition which may be modulated by
EPO by using at least one of the peptides of the present invention is provided.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 Panel A and Panel B show that EPO receptor is expressed in rat hippocampal
and cortical cultures.
Figure 2 shows that EPO receptor is expressed in neuronal cell lines: PC 12 and SK-
N-MC cells.
Figure 3 shows EPO induced gene expression in PC 12 cells. (Top) Total RNA
isolated from PC-12 cells treated with 1 nm EPO for 24 hr was subject to RT-PCR to
quantify the changes in gene expression of specific BCL family members. Pre-treatment with
EPO resulted in a 6 fold increase in the expression of the anti-apoptotic gene BCLXL and a
greater than 5 fold decrease in the expression of the pro-apoptotic Bak. These results are
consistent with the gene chip results suggesting a possible mechanism for EPO's protective
effects. (Bottom) Agarose gels showing RT-PCR products representing the regulation of
BclXL and Bak. m - markers, 1 - RT-PCR negative control, lane 2 - No Treatment, lane 3 -
50 ng/ml NGF, lane 4 - EPO 1 nm.
Figure 4 shows that rhEPO protects rat cerebral cortical neurons against glutamate
toxicity.
Figure 5 shows that rhEPO protects rat PC12 cells against glutamate-induced cell
death. 7 day cultures of PC-12 cells were treated with erythropoietin for 24 hours before
being exposed to a toxic concentration of glutamate (200 urn). Cultures were allowed to
recover for 24 hours and cell survival was determined using a Trypan Blue exclusion assay. 1
to 10 pm Erythropoietin given 24 hours prior to a 15 minute exposure to glutamate
significantly increased cell survival (p<.001 student t-test. the protective activity of epo> decreased at higher doses.
Figure 6 shows that rhEPO protects rat PC 12 cells against NGF withdrawal-induced
cell death. Cultures of PC-12 cells were grown in the presence of NGF for 7 days and then
treated with EPO for 24 hours before they were switched to media without NGF. Cell
survival was determined by counting the number of viable cells immediately following the
removal of NGF and comparing it to the number of viable cells at 12 hr, 24 hr, 48 hr and 72
hr following growth factor withdrawal. Cell viability was determined based on morphological
characteristics including phase brightness, presence of axons and absence of membrane
Webbing. Treatment with EPO increased the number of viable cells at each time point
following growth factor withdrawal with an optimum concentration of 10 pm.
Figure 7 shows that rhEPO promotes neurite outgrowth in rat cerebral cultures.
Figure 8 shows that rhEPO promotes neurite outgrowth in rat hippocampal cultures.
Figure 9 shows that EMP-1 promotes neurite outgrowth in rat cerebral cortical
cultures.
Figure 10 shows that EMP-1 promotes neurite outgrowth in rat hippocampal cultures.
Figure 11 shows that EMP-6 promotes neurite outgrowth in rat cerebral cortical
cultures.
Figure 12 shows that EMP-6 promotes neurite outgrowth in rat hippocampal cultures.
Figure 13 shows that EMP-9 promotes neurite outgrowth in rat cerebral cortical
cultures.
Figure 14 shows that EMP-9 promotes neurite outgrowth in rat hippocampal cultures.
Figure 15 shows that EMP-23 promotes neurite outgrowth in rat cerebral cortical
cultures.
Figure 16 shows that EMP-23 promotes neurite outgrowth in rat hippocampal
cultures.
Figure 17 shows that EMP-27 promotes neurite outgrowth in rat cerebral cortical
cultures.
Figure 18 shows that EMP-27 promotes neurite outgrowth in rat hippocampal
cultures.
Figure 19 shows that EPO protects against ischemic injury: study I continuous iv
infusion via osmotic mini-pump.
Figure 20 shows plasma determinations for study I.
Figure 21 shows that EPO does not protect against ischemic injury: study II single iv
bolus dose.
Figure 22 shows plasma determinations for study II.
Figure 23 shows that EPO protects against ischemic injury: study III repeat iv bolus
dosing.
Figure 24 shows plasma determinations: study III.
DETAILED DESCRIPTION
"Erythropoietin" (EPO) used herein includes those peptides, peptide dimers,
polypeptides, and proteins that have the full range of biological activity (for example,
hematopoietic and neurological activities) of human erythropoietin or only certain biological
activities (for example, hematopoietic or neurological activities only) of erythropoietin, as
well as erythropoietin analogs, erythropoietin isoforms, erythropoietin mimetics,
erythropoietin fragments, hybrid erythropoietin proteins, fusion proteins, oligomers and
multimers of the above, homologues of the above, glycosylation pattern variants of the
above, and muteins of the above, regardless of the biological activity of same, and further
regardless of the method of synthesis or manufacture thereof including, but not limited to,
recombinant, whether produced from cDNA or genomic DNA, synthetic, transgenic, and
gene activated methods. Specific examples of erythropoietin include, Epoetin alfa (EPREX*,
ERYPO®, PROCRIT®), NEORECORMON, Novel erythropoiesis stimulating protein (NESP
or ARANESP, a hyperglycosylated analog of recombinant human erythropoietin (Epoetin)
described in European patent application EP640619), human erythropoietin analog - human
serum albumin fusion proteins such as those described in the international patent application
WO 99/66054, erythropoietin mutants such as those described in the international patent
application WO 99/38890, erythropoietin omega, which may be produced from an Apa I
restriction fragment of the human erythropoietin gene described in United States Patent No.
5,688,679, altered glycosylated human erythropoietin such as those described in the
international patent application WO 99/11781, PEG conjugated erythropoietin analogs such
as those described in WO 98/05363 or United States Patent No. 5,643,575. Specific
examples of cell lines modified for expression of endogenous human erythropoietin are
described in international patent applications WO 99/05268 and WO 94/12650. The
generally preferred form of EPO is purified, recombinant human EPO (rhEPO), currently
formulated and distributed under the trademarks of EPREX®, ERYPO®, or PROCRIT®.
The abbreviation "EMP" as used herein refers to peptide mimetics of EPO, particularly
certain peptides described in United States Patent No.'s 5,767,078 and 5,773,569.
Following is a list of amino acid abbreviations used in the present specification for
various peptides. The individual amino acid residues are identified according to a single letter
and three letter code that is readily known and used by those of ordinary skill in the art.
AMINO ACID ABBREVIATIONS
3-Letter 1-Letter
alanine ala A
valine val V
leucine leu L
isoleucine ile I
proline pro P
phenylalanine phe F
tryptophan trp W
methionine met M
glycine gly G
serine ser S
threonine thr T
cysteine cys C
tyrosine tyr Y
asparagine asn N
glutamine gln Q
aspartic acid asp D
glutamic acid glu E
lysine lys K
arginine arg R
histidine his H
In a first embodiment, the present invention is directed to methods of treating
neuronal cells with a pharmaceutical composition comprising a therapeutically active peptide
that behaves as cell-surface receptor agonists as well as dimers and multimers of such
peptides that exhibit binding and signal initiation of growth factor-type receptors. In one
embodiment, the present invention provides peptides that behave as EPO agonists.
Particularly, these peptides may be dimers or multimers that have two 'monomelic' peptide
units of 8 to 40 or more amino acids, preferably 14 to about 20 residues in length, comprising
a core amino acid sequence of X3 X4 X5 GPX6 TWX7 X8 (SEQ ID NO: 1) where each amino
acid is indicated by standard one letter abbreviation; X3 can be C, E, A, a-amino-?-
bromobutyric acid, or Hoc, where Hoc is homocysteine; X4 can be R, H, Y, L, or W, or X4 is
nonexistent; X5 can be M, F, or I; X6 is independently any one of the 20 genetically coded L-
amino acids or the stereoisomeric D-amino acids; X7 can be D, E, I, L, or V; and X8 can be C,
K, A, a-amino-?-bromobutyric acid, or Hoc, where Hoc is homocysteine, provided that either
X3 or X8 is C or Hoc. Preferably, the monomeric peptide unit of the dimer or multimer
comprises a core sequence YX2X3X4GPX6TWX7X8 (SEQ ID NO: 2) where each amino
acid is indicated by standard one letter abbreviation; each X1, X2, Xg, X9, X10, and X11 is
independently selected from the 20 genetically coded L-amino acids; X3 can be C, E, A, a-
amino-?-bromobutyric acid, or Hoc, where Hoc is homocysteine; X4 can be R, H, Y, L, or W,
or X4 is nonexistent; X5 can be M, F, or I; X7 can be D, E, I, L, or V; and X8 can be C, K, A,
a-amino-?-bromobutyric acid, or Hoc, where Hoc is homocysteine. More preferably, either
X3 or X8 is C or Hoc.
Preferably, the monomelic peptide unit of the dimer or multimer comprises a core
sequence of amino acids YX2X3X4X5GPX6TWX7X8 (SEQ ID NO: 2), wherein each of X2 and
X6 is independently any one of the 20 genetically coded L-amino acids; X3 is C; and X8 is C.
Preferably, the monomelic peptide unit of the dimer comprises a core sequence of
amino acids X1YX2X3X4X5GPX6TWX7X8X9X10X11 (SEQ ID NO: 3), wherein each of X1, X2,
X6, X9, X10, and X11 is independently selected from the 20 genetically coded L-amino acids.
Particularly, X3 can be C, E, A; X4 can be R, H, or Y, or X4 is nonexistent; X5 can be M, F, or
I; X7 can be D or V; and X8 can be C, K, or A.
In a more preferred embodiment, both X3 and X8 are C and thus, the monomeric
peptide unit of the dimer comprises a core sequence of amino acids X, YX2 CX4 X5 GPX8
TWX7 CX9 X10 X11 (SEQ ID NO: 4). Particularly, the monomeric peptide unit comprises
a core sequence of amino acids X, YX2 CX4 X5 GPX6 TWX7 CX9 X10 X11 (SEQ ID NO:
5), where X4 can be R or H; X5 can be F or M; X6 can be I, L, T, M, or V; X7 is D or V;
X9 can be G, K, L, Q, R, S, or T; and X10 can be A, G, P, R, or Y. More particularly, the
monomeric peptide unit of the dimer will comprise a core sequence of amino acids X,
YX2 CX4 X5 GPX6 TWX7 CX9 X10 X11 (SEQ ID NO: 6), where X1 can be D, E, L, N, S,
T, or V; X2 can be A, H, K, L, M, S, or T; X4 is R or H; X, can be K, R, S, or T; and X10 is
P.
Preferably, the monomeric peptide unit of the dimer will comprise a core sequence of
amino acids X1 YX2 CX4 X5 GPX6 TWX7 CX9 X10 X11 (SEQ ID NO: 6), where X, can be D,
E, L, N, S, T, or V; X2 can be A, H, K, L, M, S, or T; X4 is R or H; X, can be K, R, S, or T;
and X10 is P.
Particularly preferred monomelic peptide units of the dimers include:
GGLYLCRFGPVTWDCGYKGG (SEQ ID NO:7);
GGTYSCHFGPLTWVCKPQGG (aka EMP-1) (SEQ ID NO:8);
GGDYHCRMGPLTWVCKPLGG (SEQ ID NO:9);
VGNYMCHFGPITWVCRPGGG (SEQIDNO:10);
GGVYACRMGPITWVCSPLGG (SEQ ID NO: 11);
VGNYMAHMGPITWVCRPGG (SEQ ID NO: 12);
GGTYSCHFGPLTWVCKPQ (aka EMP-16) (SEQ ID NO: 13);
GGLYACHMGPMTWVCQPLRG (aka EMP-36) (SEQ ID NO: 14);
TIAQYICYMGPETWECRPSPKA (aka EMP-38) (SEQ ID NO: 15);
YSCHFGPLTWVCK (aka EMP-20 (SEQ ID NO: 16);
YCHFGPLTWVC (aka EMP-23) (SEQ ID NO: 17);
SCHFGPLTWVCK (aka EMP-24) (SEQ ID NO: 18);
GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ED NO: 19);
GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ED NO:20);
GGTYSCFGPLTWVCKPQGG (aka EMP-27) (SEQ ED NO:21);
TYSCHFGPLTWVCKPQGG (aka EMP-17) (SEQ ED NO:22);
TYSCHFGPLTWVCKPQ (aka EMP-18) (SEQ ED NO:23);
YSCHFGPLTWVCKP (aka EMP-19) (SEQ ED NO:24);
YSCHFGPLTWVC (aka EMP-21) (SEQ ID NO:25);
YSCHFGALTWVCK (aka EMP-22) (SEQ ED NO:26);
GGCRIGPITWVCGG (aka EMP-25) (SEQ ED NO:27);
HFGPLTWV (aka EMP-26) (SEQ ED NO:28);
GGTTSCHFGPLTWVCKPQGG (aka EMP-7) (SEQ ED NO:29);
GGTFSCHFGPLTWVCKPQGG (aka EMP-8) (SEQ ED NO:30);
GGTYSCHFGALTWVCKPQGG (aka EMP-10) (SEQ ED NO:31);
GGTYSCHFGPATWVCKPQGG (aka EMP-11) (SEQ ED NO:32);
GGTYSCHFGPLAWVCKPQGG (aka EMP-12) (SEQ ED NO:33);
GGTYSCHFGPLTAVCKPQGG (aka EMP-13) (SEQ ED NO:34);
GGTYSCHFGPLTFVCKPQGG (aka EMP-14) (SEQ ED NO:35);
GGTYSCHFGPLTWVCKAQGG (aka EMP-15) (SEQ ED NO:36);
GGTXSCHFGPLTWVCKPQGG (aka EMP-28, X = D-Tyr) (SEQ ID NO:37);
GGTXSCHFGPLTWVCKPQGG (aka EMP-29, X =p-NO2-Phe)
(SEQ ID NO:38);
GGTXSCHFGPLTWVCKPQGG (akaEMP-30,X=p-NH2-Phe)
(SEQ ID NO:39);
GGTXSCHFGPLTWVCKPQGG (aka EMP-31, X =p-F-Phe) (SEQ ID NO:40);
GGTXSCHFGPLTWVCKPQGG (aka EMP-32, X = p-I-Phe) (SEQ ID N0:41);
GGTXSCHFGPLTWVCKPQGG (aka EMP-33, X = 3,5-dibromo-Tyr)
(SEQ ID NO:42);
Ac-GGTYSCHFGPLTWVCKPQGG (aka EMP-34) (SEQ ID NO:43);
GGLYACHMGPMTWVCQPLGG (aka EMP-35) (SEQ ID NO:44);
LGRKYSCHFGPLTWVCQPAKKD (aka EMP-37) (SEQ ID NO:45); and
GGTYSEHFGPLTWVKKPQGG (aka EMP-39) (SEQ ID NO:46).
Most preferably, monomeric peptide units of the dimers include:
GGTYSCHFGPLTWVCKPQGG (aka EMP-1) (SEQ ID NO:8);
GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO: 19);
GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO:20); and
YCHFGPLTWVC (aka EMP-23) (SEQ ID NO: 17).
EPO is administered by any suitable means as appropriate for the particular patient being
treated, as would be apparent to one skilled in the art. The phrase "therapeutically effective" as
used herein will vary from patient-to-patient, and depending on the particular range of
biological activities possessed by the EPO molecule being administered. Typically, for EPO
having hematopoietic activity, a therapeutically effective amount will be from about 1 to 500
I.UAg body weight and more preferably from 50 to 300 I.UAg body weight especially
when erythropoietin is administered via subcutaneously. For EPO molecules not possessing
hematopoietic activity the therapeutically effective dose may be more or less that an EPO
molecule having hematopoietic activity. The preferred methods of administration are
intravenous (iv) and subcutaneous (sc), with subcutaneous being generally preferred.
Hematopoieucally active EPO is administered within the range of about 50 - 1000 U/kg per
dose, one to five times per week. In another embodiment, the EPO composition is administrated
directly to the nervous system. This administration route includes, but is not limited to, the
intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal
and/or peri-spinal routes of administration, which can employ intracranial and intravertebral
needles, and catheters with or without pump devices. Infusion doses can range, for example,
from about 1.0 to 50,000 U/kg/min of EPO composition over a period ranging from several
minutes to several days. Hematopoietically active EPO administration is delayed or withheld if
the patient, male or female, exhibits a hemoglobin level in excess of about 15 g/dL.
The present invention provides in one embodiment a method to treat acute and
chronic neurodegenerative disorders comprising administration of EPO or analogs thereof.
Acute neurodegenerative disorders include, but are not limited to, various types of acute
neurodegenerative disorders associated with neuronal cell death or compromise including
cerebrovascular insufficiency, focal or diffuse brain trauma, diffuse brain damage, and spinal
cord injury. Examples of acute neurodegenerative disorders are: cerebral ischemia or
infarction including embolic occlusion and thrombotic occlusion, reperfusion following acute
ischemia, perinatal hypoxic-ischemic injury, cardiac arrest, as well as intracranial
hemorrhage of any type (such as epidural, subdural, subarachnoid and intracerebral), and
intracranial and intravertebral lesions (such as contusion, penetration, shear, compression and
laceration), and whiplash shaken infant syndrome. Chronic neurodegenerative disorders that
can be treated with one or more methods of the present invention include, but are not limited
to, Alzheimer's disease, Pick's disease, diffuse Lewy body disease, progressive supranuclear
palsy (Steel-Richardson syndrome), multisystem degeneration (Shy-Drager syndrome),
chronic epileptic conditions associated with neurodegeneration, motor neuron diseases
including amyotrophic lateral sclerosis, degenerative ataxias, cortical basal degeneration,
ALS-Parkinson's-Dementia complex of Guam, subacute sclerosing panencephalitis,
Huntington's disease, Parkinson's disease, synucleinopathies (including multiple system
atrophy), primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease /
spinocerebellar ataxia type 3 and olivopontocerebellar degenerations, Grilles De La Tourette's
disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy (Kennedy's
disease), primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease,
Kugelberg-Welander disease, Tay-Sach's disease, Sandhoff disease, familial spastic disease,
Wohlfart-Kugelberg-Welander disease, spastic paraparesis, progressive multifocal
leukoencephalopathy, familial dysautonomia (Riley-Day syndrome), and prion diseases
(including, but not limited to Creutzfeldt-Jakob, Gerstmann-Straussler-Scheinker disease,
Kuru and fatal familial insomnia).
Because of the combination of neuroprotection and neurite outgrowth induced by
rhEPO, other clinical conditions can be treated with one or more methods of the present
invention include treating and / or preventing the neurological (including, but not limited to,
cognitive) and psychiatric (including, but not limited to, psychopathology, depression, or
anxiety), manifestations associated with peripheral diseases including, but not limited to,
EPO deficiency (e.g., renal disease), blood loss of any kind (including, but not limited to,
hemodialysis, peritoneal dialysis, diagnostic sampling, occult gastrointestinal bleeding), renal
failure and end-stage renal disease, renal transplantation, and other conditions associated with
anemia and neurological and neuropsychiatric manifestations, including, but not limited to,
hematological and non-hematological malignancies / cancer, symptoms or complications in
patients receiving chemotherapy (including, but not limited to, cisplatin) and other drugs
(including, but not limited to, zidovudine), other hematological disorders (including, but not
limited to, sickle cell anemia and thalassemia), inflammatory and infectious disorders
(including, but not limited to, human immunodeficiency viral infections), chronic systemic
autoimmune diseases (including, but not limited to, systemic lupus erythematosus), Henoch
Schonlein Purpura, and hemolytic uremic syndrome. Also included in the present invention
are the treatment and / or prevention of neurological and neuropsychiatric manifestations
resulting from chemical, toxic, infectious and radiation injury of the nervous system and as a
result of prematurity, as well as the treatment and / or prevention of neurological and
neuropsychiatric consequences of encephalopathies including, but not limited to, those of
anoxic-ischemia, hepatic, glycemic, uremic, electrolyte and endocrine origin.
Also, because of the combination of neuroprotection and neurite outgrowth induced
by rhEPO, this molecule can also be applicable for the treatment and / or prevention of
plexopathies (including plexus palsies), multifocal neuropathies, sensory neuropathies, motor
neuropathies, sensory-motor neuropathies, infections neuropathies, autonomic neuropathies,
sensory-autonomic neuropathies, demyelinating neuropathies (including, but not limited to,
Guillain-Barre syndrome and chronic inflammatory demyelinating polyradiculoneuropathy),
other inflammatory and immune neuropathies, neuropathies induced by drugs, neuropathies
induced by pharmacological treatments, neuropathies induced by toxins, traumatic
neuropathies (including, but not limited to, compression, crush, laceration and segmentation
neuropathies), metabolic neuropathies, endocrine and paraneoplastic neuropathies, and other
neuropathies such as Charcot-Marie-Tooth disease (type 1a, 1b, 2, 4a, 1-X linked),
Friedreich's ataxia, metachromatic leukodystrophy, Refsum's disease,
adrenomyeloneuropathy, Ataxia-telangiectasia, Dejerine-Sottas neuropathy (types A and B),
Lambert-Eaton syndrome, and disorders of the cranial nerves.
The following examples illustrate the present invention without, however, limiting
the same thereto.
EXAMPLE 1
rhEPO is expressed in primary rat neuronal cultures and in neuronal cell lines
Primary Neuronal Cell Culture
Dissociated hippocampal and cortical cell cultures were established from embryonic
day 18 rat fetuses as previously described (Mattson et al., 1994). Briefly, fetuses were
removed via cesarean section from pregnant moms (Sprague-Dawley) anesthetized with
halothane according to the AVMA Panel on Euthanasia. Pups were decapitated and the
brains were removed and placed in HEPES-buffered Hank's Balanced Salt solution (HBSS;
Gibco). The hippocampi and cortices were dissected out and pooled according to tissue-type.
Tissue was trypsinized for 15 min (1mg/ml trypsin-HBSS; Worthington), rinsed with fresh
HBSS, incubated in trypsin inhibitor (1mg/ml; Sigma) for 5 min, rinsed again with fresh
HBSS and then triturated in 1ml fresh HBSS with a fire-polished glass pipette. Dissociated
cells were seeded at 30,000 cells/well onto poly-D-lysine coated 96-well plates
(Collaborative BioScience). Each well contained 100µl of Eagle's Minimal Essential Media
(MEM; Gibco) supplemented with 26mM NaHCO3 (Sigma), 56mM glucose (Sigma), 15mM
KCl (Sigma), 1mM sodium pyruvate (Sigma), 1.1mM L-glutamine (Sigma), 10% (v/v) heat-
inactivated fetal bovine serum (Hyclone), and 0.001% gentamicin sulfate (Sigma) (pH 7.4).
Cells were allowed to attach for 24h in a humidified 37°C 5% CO2 incubator before
experimental treatment. The culture media was aspirated and exchanged with fresh media
every three days.
Immunocytochemistry
Parallel hippocampal and cortical cultures were treated as described above and
were processed for immunocytochemistry (ICC) as described previously (Smith-
Swintosky et al., 1997). Briefly, cells were plated onto four-chamber poly-D-lysine
coated glass slides (LabTek, Napersville, EL). On the seventh day in culture, the culture
media was removed and the cells were washed once with Dulbecco's phosphate buffered
saline (DPBS; Sigma) and then fixed with 10% phosphate-buffered formalin for 15min at
room temperature. After fixation, the cultures were rinsed with DPBS and placed in
blocking serum for 10min (normal horse serum; 1:50 dilution in DPBS; Vector Labs,
Burlingame, CA). Cultures were rinsed again and then incubated for 30min in an anti-
mouse monoclonal antibody specific to the EPO receptor (EBP-7); 1:75 dilution in
antibody diluent (Zymed, South San Francisco, CA). Cultures were rinsed several times
with DPBS, then exposed to biotinylated secondary antibody for 30min (Vector Labs).
Cultures were rinsed a final time and then incubated for 30min in avidin-biotinylated
horseradish peroxidase complex (mouse IgG ABC kit, Vector Labs). The presence of the
primary antibody was detected using 3'3-diaminobenzidine tetrahydrochloride (DAB,
Biomeda, Foster City, CA) - two exposures for 5min each. Cells were then
counterstained with hematoxylin, dehydrated, cleared, coverslipped and photographed
under an Olympus BX-2 light microscope.
Results
Robust staining for EPO receptor was observed in both neurons and glia within
hippocampal and cortical cultures (Figure 1a and Figure 1b). EPO receptor expression levels
appear to increase with time in culture.
Discussion
These results show that EPO plays a role in early development of the nervous system,
particularly the hippocampus and cerebral cortex.
Immunohistochemistry of neuronal cell lines
The neuronal cell lines PC-12, derived from a pheochromocytoma of the rat adrenal
gland (Greene and Tischler, 1976), and SK-N-MC, obtained from a neuroepithelioma of a
brain of human origin (Spengler et al., 1973), were used. PC-12 cells can be reversibly
induced to the neuronal phenotype in the presence of nerve growth factor (NGF). PC-12 cells
were grown on poly-D-Lysine coated tissue culture dishes in DMEM containing 10% horse
serum and 5% FBS and in the presence of 0.1 µg/ml NGF for 7 days to induce the neuronal
phenotype. SK-N-MC cells were cultured in minimal essential media supplemented with 1.0
mM Sodium pyruvate, 1.5 g/L sodium bicarbonate, 2mM glutamine and 10% FBS for 4 days.
PC-12 and SK-N-MC cells were cultured in a 96 well plate from Greiner, conducive for
microscopy. On the day of the experiment cells were fixed in 10% Formalin containing 10%
sucrose and incubated in blocking buffer (40 mM Tris HCL, Ph 8.0, 27mM NaCl, and
0.2%Tween 20). Receptors for erythropoietin were detected by incubating the cells with a
rabbit polyclonal, anti-erythropoietin receptor antibody (C-20 from Santa Cruz) and a FITC
conjugated secondary antibody. Labeled cells were visualized using a fluorescent microscope
(ATTO).
Results
A polyclonal antibody against the erythropoietin receptor labeled both SK-N-MC and
PC-12 cells as seen in Figure 2 (right panels). All SK-N-MC cells visible at low
magnification appeared to be labeled with the antibody. The majority of PC-12 cells that
were detectable were labeled with the anti-erythropoietin receptor antibody. A few intact PC-
12 cells that retained a characteristic neuronal phenotype in this preparation showed staining
throughout the axonal process as well as in the cell body. Secondary antibody alone did not
label either cell type (Figure 2, left panels).
Therefore, these results demonstrate that these cell lines, SK-N-MC cells from a
human neuroepithelioma and PC-12 cells from a rat pheochromocytoma, express the
erythropoietin receptor. These cell lines are therefore responsive to erythropoietin and can
provide a good system to study the effects of erythropoietin on neurons.
EXAMPLE 2
EPO induced gene expression in PC 12 cells
Cell Culture
PC-12 cells (from a rat Pheochromocytoma) were cultured on poly-D-Lysine coated
tissue culture plastic in DMEM containing 10 % FBS and 5% Horse serum. To induce the
neuronal phenotype in PC-12 cells, serum was removed and the cells were treated with NGF
(50 ng/ml). Cells were grown for 7 days in the presence of the NGF then used for
experiments.
EPO Treatment and RNA isolation
PC-12 cells were cultured as described in Example 1 in a 10 cm poly-D-lysine coated
tissue culture dish. Cells were incubated in the presence of lU/ml of EPO for 24 hr. Total
RNA was then isolated using a Qiagen RNAeasy mini prep kit and used for RT-PCR.
Quantitative RT-PCR
Real time reverse transcription and PCR were performed in a single reaction using a
light cycler and an RNA amplification kit from Roche Molecular Biochemicals. RNA was
quantitated and added in equal amounts to reaction mix that includes the dsDNA specific dye
SYBR green I. Specific PCR reaction products are quantitated by detecting the amount of
fluorescence in the reaction at each PCR cycle. Final analysis was performed using the data
analysis software included with the light cycler instrument.
Summary
Pre-treatment of PC-12 cells with EPO (lU/ml) for 24 hr resulted in significant
changes in the gene expression of the bcl-2 family members bclXL and bak as seen in Figure
3. Cells treated with EPO showed a 6-fold increase in the expression of the anti-apoptotic
gene, bclXL, and a 5-fold decrease in the expression of the pro-apoptotic gene, bak. EPO has
been shown previously to increase the survival of red blood cell progenitor cells by
increasing bclXL expression (Silva et al., 1996). These results show that EPO uses a similar
mechanism to protect neurons from undergoing apoptosis in response to injury. The
regulation of bak shows that EPO can effect the expression of additional genes to elicit this
effect.
EXAMPLE 3
rhEPO neuroprotection and neurite outgrowth effects
on rat hippocampal and cortical cells and PC12 cells
Primary Neuronal Cell Culture
Dissociated hippocampal and cortical cell cultures were established from embryonic
day 18 rat fetuses as previously described (Mattson et al., 1994). Briefly, fetuses were
removed via cesarean section from pregnant moms (Sprague-Dawley) anesthetized with
halothane according to the AVMA Panel on Euthanasia. Pups were decapitated and the
brains were removed and placed in HEPES-buffered Hank's Balanced Salt solution (HBSS;
Gibco). The hippocampi and cortices were dissected out and pooled according to tissue-type.
Tissue was trypsinized for 15 min (1mg/ml trypsin-HBSS; Worthington), rinsed with fresh
HBSS, incubated in trypsin inhibitor (lmg/ml; Sigma) for 5 min, rinsed again with fresh
HBSS and then triturated in 1ml fresh HBSS with a fire-polished glass pipette. Dissociated
cells were seeded at 30,000 cells/well onto poly-D-lysine coated 96-well plates
(Collaborative BioScience). Each well contained 100µl of Eagle's Minimal Essential Media
(MEM; Gibco) supplemented with 26mM NaHCO3 (Sigma), 56mM glucose (Sigma), 15mM
KCl (Sigma), 1mM sodium pyruvate (Sigma), 1.1 mM L-glutamine (Sigma), 10% (v/v) heat-
inactivated fetal bovine serum (Hyclone), and 0.001% gentamicin sulfate (Sigma) (pH 7.4).
Cells were allowed to attach for 24h in a humidified 37°C 5% CO2 incubator before
experimental treatment. The culture media was aspirated and exchanged with fresh media
every three days.
Glutamate Toxicity Assay
Cortical cells were seeded at 200,000 cells/dish onto polyethylenimine-coated 35mm
culture dishes. Each dish contained 1.5ml MEM supplemented as described above. On the
seventh day in culture, four fields per pre-marked dish were visualized with a Nikon Diaphot
inverted microscope (10x magnification) and photographed prior to experimental treatment.
Immediately following, the cultures were treated with vehicle or recombinant human
erythropoietin (rhEPO; lot #41C514; 50µM stock in 0.2M citrate, 0.585g.L NaCl diluted to
appropriate concentrations in Dulbecco's phosphate buffered saline (DPBS; Sigma) + 0.1%
bovine serum albumin (BSA; Sigma)). Twenty-four hours later the cultures were treated
with 100µM glutamate (Sigma). Twenty-four hours post-glutamate, the four fields from each
dish were photographed again. Cell survival was measured by counting viable cells in each
field pre- and post-experimental treatment. Neurons were considered viable if they had
neurites that were uniform in diameter and smooth in appearance, and somata that were
smooth and round to oval in shape. Data were expressed as percent of control (vehicle;
mean ± SD).
PC12 Cell Culture
PC-12 cells (from a rat Pheochromocytoma) were cultured on poly-D-Lysine coated
tissue culture plastic in DMEM containing 10 % FBS and 5% Horse serum. To induce the
neuronal phenotype in PC-12 cells, serum was removed and the cells were treated with NGF
(50 ng/ml). Cells were grown for 7 days in the presence of the NGF then used for
experiments.
Glutamate Toxicity
PC-12 cells were cultured as described above. 24 hr prior to insult, cells were treated
with rhEPO at concentrations ranging from 1 pm to 1 nm. On the day of the experiment, cells
were exposed to 200 uM glutamate for 30 min. Cells were then washed 2 times with fresh
media to remove the glutamate and cultured in fresh media containing NGF but no EPO.
After 24 hr cells were assayed for viability using a trypan blue exclusion assay. Briefly,
media was removed and the cells were incubated in 0.4% Trypan Blue for 5 min. Cells were
then washed gently with PBS, then fixed with 10% formalin. Cell viability was determined
by counting the total number of cells vs. the number of trypan blue positive (dead) cells.
NGF withdrawal
PC-12 cells were cultured as described above in a 96 well poly-d-lysine coated
multi-well plate and treated with rhEPO (1 pm to 10 nm) for 24 hr prior to NGF
withdrawal. On the day of the experiment the cells were washed with buffer 3 times to
remove NGF and then cultured in fresh media without NGF. Immediately following
NGF washout cells were counted (t=0) to determine the number of living cells. Cell
viability was based on morphological characteristics including phase brightness, presence
of axons, and absence of blebbing. Cell counts were performed at 12 hr, 24 hr, 48 hr and
72 hr and the number of viable cells were scored.
Neurite Outgrowth Assay
Twenty-four hours after plating, cultures were treated with vehicle (PBS+0.1% BSA),
100ng of various growth factors (brain derived neurotrophic factor (BDNF; Promega), glial-
derived neurotrophic factor (GDNF; Promega), nerve growth factor (NGF; Boehringer
Mannheim), basic fibroblast growth factor (bFGF; Boehringer Mannheim), insulin-like
growth factor -1 (IGF-1; Boehringer Mannheim), neurotrophin-3 (NT3; Calbiochem),
neurotrophin-4 (NT4; Calbiochem), ciliary neurotrophic factor (CNTF; Calbiochem),
epidermal growth factor (EGF; Calbiochem), vascular endothelial growth factor (VEGF;
Calbiochem)), or rhEPO (prepared same as above; 10fM - 10nM)). Each treatment condition
was run in quadruplicate or octuplicate. On the third day in culture, the media was aspirated
off and replaced with fresh media and test compound. At one week in culture, the cells were
fixed with 10% phosphate-buffered formalin for 15min, then rinsed with DPBS (Sigma) and
placed in blocking serum for 30 min (horse serum; 1:50 dilution in DPBS; Vector Labs).
Cultures were rinsed again with DPBS and then incubated in primary antibody for 2 hr
(microtubule-associated protein-2 (MAP-2) is a selective marker for dendritic processes;
anti-mouse monoclonal (Chemicon); 1:1000 dilution of MAP-2 in antibody diluent
(Zymed)). Negative control wells were incubated in antibody diluent alone. Background
signal was determined by blank wells (cell-free) incubated with or without antibody.
Cultures were rinsed again with DPBS and then placed in fluorescein for 1 hr (FITC; anti-
mouse IgG; rat adsorbed; 1:50 dilution in DPBS; Vector Labs). Cultures were rinsed a final
time with DPBS and then the plates were read on a Cytofluor 4000 fluorescence plate reader.
Neurite outgrowth was expressed as percent change from control (vehicle; mean fluorescence
± SD).
Results
Neuroprotection study with primary neuronal cultures: Pretreatment of cultures with
rhEPO for 24h prior to glutamate administration resulted in a significant increase in neuronal
survival (Figure 4). Cell survival was maximally increased approximately 200% over
parallel cultures treated with glutamate alone. The neuroprotective effect of rhEPO was
concentration-dependent, with the greatest effects observed at pM concentrations in which
cell survival was greater than or equal to vehicle (no glutamate) treated cultures.
Neuroprotection study with PC 12 cells: Pre-treatment with EPO resulted in a
significant decrease in cell death induced by both glutamate toxicity and growth factor
withdrawal in PC-12 cells (Figures 5 and 6). The peak concentration for the neuroprotective
effect in both experiments was 10 pm. The dose response curve for the neuroprotective effect
of EPO was bi-phasic with the ability of EPO to protect cells against cytotoxicity decreasing
at concentrations above 10 pm, with no significant effect at 1 ran. These results suggest that
EPO is able to prevent an apoptotic response in neurons exposed to a variety of cytotoxic
insults
Neurite outgrowth study with primary neuronal cultures: Cultures treated with rhEPO
resulted in a significant increase in neurite outgrowth as measured by MAP2-FITC
immunofluorescence. The neurite outgrowth promoting effect was concentration dependent
with maximal activity observed at pM levels (Figures 7 and 8). The results indicate that
rhEPO treatment induced a larger outgrowth response in the hippocampal cultures (12-44%
over control) than in the cortical cultures (15-29% over control). A comparison between
rhEPO and known growth factors indicates that they exhibit regional differences in their
neurite outgrowth promoting abilities. rhEPO's ability to increase neurite outgrowth in
cortical cultures is greater than or equal to that of known growth factors. This observation is
compelling and important since few factors have such effects on cortical cells. On the other
hand, many growth factors exert strong outgrowth responses in the hippocampus (38-86%
over control). Compared to such growth factors, rhEPO showed moderate yet robust
outgrowth promoting activity; however, its activity was superior to several growth factors
including BDNF, NGF and VEGF.
Discussion
The neuroprotection studies confirmed previous evidence that rhEPO is protective at
pM concentrations against glutamate toxicity and serum withdrawal in vitro.
Surprisingly we discovered that rhEPO promotes neurite outgrowth in primary
mammalian neural cells. The effect was robust for hippocampal and cortical cells. The
effect was potent with efficacy observed at sub-picomolar concentrations, far more potent
than any previous EPO related observation. Moreover, in cerebral cortical neurons, which
respond to few growth factors, rhEPO was superior in inducing neurite outgrowth relative to
the majority of known growth factors.
From a therapeutic perspective, the observation that rhEPO promotes neuroprotection
and neurite outgrowth in cerebral cortical neurons is very important. During
neurodegeneration, neural cells can be in different stages of the process. Some may be
stressed, others experience significant neurite retraction and loss of synaptic input, and
eventually all affected cells will succumb to death. A therapeutic agent that can intervene in
this process at multiple levels can be of great benefit to the recovery of the neural cells and
eventually neural function. The present data support that rhEPO accomplishes this task by
protecting the cells, by enhancing their survival, by promoting re-establishment of synaptic
contacts and connections, and by stabilizing the neuronal and neural circuitry.
It should also be specified that the data are particularly important, considering that
very few growth factors are effective in cerebral cortical neurons, and also that very few
growth factors display the dual activity as neuroprotectants and promoters of neurite
outgrowth in cortical neurons. It is particularly relevant that this dual activity of rhEPO was
observed at sub-picomolar / picomolar concentrations.
EXAMPLE 4
EPO MIMETIC PEPTIDES STIMULATE NEURITE OUTGROWTH
Cell Culture
Dissociated hippocampal and cortical cell cultures were established from embryonic
day 18 rat fetuses as previously described (Mattson et al., 1994). Briefly, fetuses were
removed via cesarean section from pregnant moms (Sprague-Dawley) anesthetized with
halothane according to the AVMA Panel on Euthanasia. Pups were decapitated and the
brains were removed and placed in HEPES-buffered Hank's Balanced Salt solution (HBSS;
Gibco). The hippocampi and cortices were dissected out and pooled according to tissue-type.
Tissue was trypsinized for 15 min (1mg/ml trypsin-HBSS; Worthington), rinsed with fresh
HBSS, incubated in trypsin inhibitor (1mg/ml; Sigma) for 5 min, rinsed again with fresh
HBSS and then triturated in 1ml fresh HBSS with a fire-polished glass pipette. Dissociated
cells were seeded at 30,000 cells/well onto poly-D-lysine coated 96-well plates
(Collaborative BioScience). Each well contained 100µl of Eagle's Minimal Essential Media
(MEM; Gibco) supplemented with 26mM NaHCO3 (Sigma), 56mM glucose (Sigma), 15mM
KCl (Sigma), 1mM sodium pyruvate (Sigma), 1.1mM L-glutamine (Sigma), 10% (v/v) heat-
inactivated fetal bovine serum (Hyclone), and 0.001% gentamicin sulfate (Sigma) (pH 7.4).
Cells were allowed to attach for 24h in a humidified 37°C 5% CO2 incubator before
experimental treatment. The culture media was aspirated and exchanged with fresh media
every 3 days.
Neurite Outgrowth Assay
Twenty-four hours after plating, cultures were treated with vehicle (PBS+0.1% BSA),
100ng of various growth factors (brain derived neurotrophic factor (BDNF; Promega), glial-
derived neurotrophic factor (GDNF; Promega), nerve growth factor (NGF; Boehringer
Mannheim), basic fibroblast growth factor (bFGF; Boehringer Mannheim), insulin-like
growth factor -1 (IGF-1; Boehringer Mannheim), neurotrophin-3 (NT3; Calbiochem),
neurotrophin-4 (NT4; Calbiochem), ciliary neurotrophic factor (CNTF; Calbiochem),
epidermal growth factor (EGF; Calbiochem), vascular endothelial growth factor (VEGF;
Calbiochem)), or Epo mimetic peptides (EMP1, EMP6, EMP9, EMP23 and EMP27; diluted
in DPBS+0.1% BSA; 10fM - 10nM; Table 1). Each treatment condition was run in
quadruplicate. On the third day in culture, the media was aspirated off and replaced with
fresh media and test compound. At one week in culture, the cells were fixed with 10%
phosphate-buffered formalin for 15min, then rinsed with DPBS (Sigma) and placed in
blocking serum for 30 min (horse serum; 1:50 dilution in DPBS; Vector Labs). Cultures
were rinsed again with DPBS and then incubated in primary antibody for 2 hr (microtubule-
associated protein-2 (MAP-2) is a selective marker for dendritic processes; anti-mouse
monoclonal (Chemicon); 1:1000 dilution of MAP-2 in antibody diluent (Zymed)). Negative
control wells were incubated in antibody diluent alone. Background signal was determined
by blank wells (cell-free) incubated with or without antibody. Cultures were rinsed again
with DPBS and then placed in fluorescein for 1 hr (FITC; anti-mouse IgG; rat adsorbed; 1:50
dilution in DPBS; Vector Labs). Cultures were rinsed a final time with DPBS and then the
plates were read on a Cytofluor 4000 fluorescence plate reader. Neurite outgrowth was
expressed as percent change from control (vehicle; mean fluorescence ± SEM).
Results
Neurite outgrowth study: Cultures treated with EMP's resulted in a significant
increase in neurite outgrowth as measured by MAP2-FITC immunofluorescence. The neurite
outgrowth promoting effect was concentration dependent with maximal activity observed at
pM levels (Figures 9-18). The results indicate that EMP's displayed different activity
profiles. At the concentrations tested, EMP-1 and 6 displayed typical bell-shaped dose-
response profiles in hippocampal cultures with peak activity observed between 30-300pM
(83-117% increase over vehicle). EMP-9 and 27 exhibited a flat response profile in
hippocampal cultures with peak activity observed at similar concentrations as EMP-1 and 6,
but the amplitude of the response was greatly attenuated (29-32% increase over vehicle).
EMP-23 had a modest response to EPO in hippocampal cultures with peak activity observed
between 30pM-lnM that led to a 43-46% increase over vehicle response. In the cortical
cultures, EMP-1,9 and 27 exhibited response profiles similar to most known growth factors -
a flat response overall with maximal activity occurring between 30-300pM reaching 32-40%
above the vehicle response. EMP-6 and 23 displayed typical bell-shaped dose-response
profiles with peak activity observed between 30-300pM resulting in a 68-87% increase in
outgrowth over vehicle response levels. Overall, EMP-6 promoted robust neurite outgrowth
activity in both hippocampal and cortical cultures; whereas, EMP-1 showed selective effects
in hippocampal cultures over cortical cultures and EMP-23 effects were greater in cortical
cultures than hippocampal cells. EMP-9 and 27 neurite outgrowth responses were less
impressive overall.
A comparison between the EMP's and known growth factors indicated that they
exhibit regional differences in their neurite outgrowth promoting abilities. The EMP's ability
to increase neurite outgrowth in cortical cultures was greater than or equal to that of known
growth factors (Figure 9-18). This observation is compelling and important since few factors
have such effects on cortical cells. On the other hand, many growth factors exert strong
outgrowth responses in the hippocampus (38-86% over control). Compared to such growth
factors, the EMP's showed moderate yet robust outgrowth promoting activity; however, they
displayed superior activity over BDNF, NGF and VEGF.
Discussion
EMP's promote neurite outgrowth in mammalian cells. The effect was robust for
hippocampal and cortical cells. The neurite outgrowth promoting effect, was superior to that
of various growth factors. The effect was potent with efficacy observed at picomolar
concentrations.
It should also be specified that the data are particularly important, considering that
very few growth factors or mimetics are effective in cerebral cortical neurons in promoting
neurite outgrowth. It is particularly relevant that this activity of the EMPs was observed at
sub-picomolar / picomolar concentrations.
EXAMPLE 5
EPQ protects aginst ischemic injury
Subjects
Male spontaneous hypertensive rats (Charles River) weighing between 250-300g
were weighed and then anesthetized with ketamine (100mg/ml)/xylazine (20mg/ml) cocktail
(1.2ml/kg; i.p.)- The level of anesthetic was assessed by corneal reflex (air puff to eye) and
leg jerk in response to tail or foot pinch. Once the rat was anesthetized, it was placed on a
small animal surgical board and restrained during the surgical procedure. The rat's body
temperature was monitored continuously with a rectal probe and maintained at 37°C with a
homeostatic heating pad.
Experimental model of cerebral ischemia
Rats were rendered ischemic by tandem occlusion of the left common carotid artery
and the left middle cerebral artery for 2h followed by 22h of reperfusion using a modification
of the technique described by Brint and co-workers (J. Cereb Blood Flow Metab 8:474-485,
1988). Specifically, the left CCA was isolated through an incision in the ventral surface of
the neck. For isolation of the ipsilateral MCA, a second incision was made between the
lateral canthus of the left eye and the corresponding external auditor)' canal to bare the
underlying skull. The MCA was exposed through a 5mm burrhole drilled 2-3mm rostral to
the fusion of the zygomatic arch and the squamosal bone under direct visualization with a
Zeiss operating microscope. The dura was opened with a sterile 26g needle and a platinum
alloy wire (0.1mm diameter) was inserted beneath the MCA just superior to the inferior
cortical vein. The MCA was temporarily occluded by elevation and compression of the vessel
across the alloy wire, as described by Aronowski and colleagues (Stroke, 25:2235-2240,
1994). Concurrently, the CCA was occluded with an aneurysm clip. The duration of
occlusion of the CCA and the MCA was 2h. At the end of this period, the wire and the clip
were carefully removed to allow reperfusion of the vessels and the incision area was sutured
shut The rat was placed in an isolation cage to recover before returning to bis home cage. .
Study Design
Study I: EPO and vehicle delivered via osmotic mini-pump
Twenty-hours prior to the onset of ischemia, an osmotic mini-pump (Model 1003D;
Alza) filled with vehicle or EPO was placed between the scapula of the rat. A
catheter attached to the pump was inserted and tethered within the right jugular vein
for continuous infusion of drug at a rate of 1µl/hr. Rats were divided into five groups:
(1) sham-operated vehicle-treated, (2) ischemic vehicle treated, (3) ischemic
l.32U/day EPO treated, (4) ischemic 132U/day EPO treated and (5) ischemic
1321U/day EPO treated. On Day Two, rats were rendered ischemic as described
above. Twenty-two hours later, the rats were evaluated for behavioral performance, a
blood sample was collected for terminal plasma levels of EPO and then the rat was
euthanized, brain removed, sectioned and stained for histological analysis.
Study D: EPO and vehicle delivered as a single intravenous bolus injection
On Day One, the rats were rendered ischemic as described above. Rats were divided
into four groups: (1) ischemic vehicle treated, (2) ischemic 1000U/kg EPO treated, (3)
ischemic 2500U/kg EPO treated and (4) 5000U/kg EPO treated. Fifteen minutes
post-occlusion, the rats received vehicle or EPO as an intravenous bolus injection.
Twenty-two hours later, the rats were evaluated for behavioral performance, a blood
sample was collected for terminal plasma levels of EPO and then the rat was
euthanized, brain removed, sectioned and stained for histological analysis.
Study III: EPO and vehicle delivered as repeat intravenous bolus injections
On Day One, the rats were rendered ischemic as described above. Rats were divided
into two groups: (1) ischemic vehicle treated and (2) ischemic 2500U/kg EPO treated.
Drug was administered as an intravenous bolus at 15min, 2h, 4h and 6h post-
occlusion for a total dose of 10,000U/kg. Twenty-two hours later, the rats were
evaluated for behavioral performance, a blood sample was collected for terminal
plasma levels of EPO and then the rat was euthanized, brain removed, sectioned and
stained for histological analysis.
Outcome measures
Plasma Determinations: Blood samples were collected from each rat via the orbital
sinus at the time of sacrifice. Plasma was separated out, frozen and analyzed by EPO ELISA
for determination of plasma concentration (U/ml).
Infarct Volume: Brains were removed, blocked into lmm slabs and stained with
2,3,5-triphenyl tetrazolium chloride dye (TTC; Sigma) for 15 min at room temperature.
Stained sections were stored in 10% buffered formalin at 4oC. Sections were visualized by a
Nikon SMZ-U microdissecting scope. Images of each brain section were captured with a
CCD camera and processed using Image Pro Phase HI software in order to calculate infarct
volume.
Results
Study I: EPO given at 132 or 1321U/day as a continuous infusion via osmotic mini-
pump significantly reduced infarct volume (Figure 19). Plasma concentrations correlated
with the protective effect (Figure 20).
Study II: EPO given as a single iv bolus 15 min post-occlusion did not protect against
ischemic damage in this model at 1000, 2500 or 5000U/kg (Figure 21). Plasma
concentrations were low in each group (Figure 22).
Study III: EPO given as a repeat iv bolus of 2500U/kg at lSmin, 2h, 4h and 6h post-
occlusion led to a significant decrease in infarct volume (Figure 23). Plasma concentrations
are presently being determined (Figure 24).
Discussion
Data support that continuous or repeat dosing with low to moderate concentrations of
EPO can significantly reduce infarct volume in spontaneous hypertensive rats rendered
ischemic via the transient tandem occlusion of the CCA and MCA. The results also support
that there is a critical relationship between the amount and timing of EPO administration for
the protective effect to occur. Low doses of EPO given over an extended period of time can
be more beneficial than high doses given the same way or as a single bolus infusion. This is
in agreement with the in vitro data indicating that EPO maximal protective effect is observed
at low doses (pM) and actually looses efficacy at higher doses (µM).
REFERENCES
Johnson, D.L., Farrell, F.X., Barbone, F.P., McMahon, F.J., Tullai, J., Hoey K., Wrighton,
N.C., Livnah, O., Middleton, S.A., Loughney, D., Stura, E.A., Dower, W.J., Mulcahy, L.S.,
Wilson, LA. & JoIIiffe L.K., Identification of a 13 Amino Acid Peptide Mimetic of
Erythropoietin and Description of Amino Acids Critical for the Mimetic Activity of EMPl,
Biochemistry 37, 3699-3710 (1998).
Johnson, D.L., Farrell, F.X., Barbone, F.P., McMahon, F.P., Tullai, J., Kroon, D., Freedy, J.,
Zivin , R.A., Mulcahy, L.S., & Jolliffe, L.K. Amino Terminal Dimerization of an
Erythropoietin Mimetic Peptide Results in Increased Erythropoietic Activity, Chemistry &
Biology 4, 939-950 (1997).
Johnson, Dana L.; Zivin, Robert A. Agonist peptide dimers. US Patent No. 5,767,078
Campana, W.M., Misasi, R. and O'Brien, J.S. (1998) Identification of a neurotrophic
sequence of erythropoietin. Inter. J. Mol. Med. 1:235-241.
Patent application WO 95/03821, O'Brien, J.S., Kishimoto, Y. and Altman, D.E. Title:
Prosaposin and cytokine-derived peptides as therapeutic agents.
Sadamoto, Y, Igase, K, Sakanaka, M., Sato, K, Otsuka, H., Sakaki, S., Masuda, S. and
Sasaki, R. (1998). Erythropoietin prevent place navigation disability and cortical infarction
in rats with permanent occlusion of the middle cerebral artery. Biochem Biophys Res
Comm. 253:26-32.
Tabira, T., Konishi, Y. and Gallyas, F. (1995). Neurotrophic effect of hematopoietic
cytokines on cholinergic and other neurons in vitro. Int. J. Dev. Neurosci. 13:241-252.
Morishita, E., Masuda, S., Nagao, M., Yasuda, Y. and Sasaki, R. (1997). Erythropoietin
receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin
prevent in vitro glutamate-induced neuronal death. Neurosci 76:105-116.
Sakanaka, M, Wen T-C, Matsuda, S, Masuda, S., Morishita, E., Nagao, M., and Sasaki, R.
(1998) In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc.
Natl. Acad. Sci. 95:4635-4640.
Marti, H.H., Gassman, M, Wenger, R.H., Kvietikova, I., Morganti-Kossmann, M.C.,
Kossmann, T., Trentz, O., and Bauer, C. (1997) Detection of erythropoietin in human
liquor: Intrisic erythropoietin production in the brain. Kidney Int. 51:416-418.
Konishi, Y., Chui, D-H, Hirose, H., Kunishita, T. and Tabira, T. (1993) Trophic effect of
erythropoietin and other hematopoietic factors on central cholinergic neurons in vitro and in
vivo. Brain Res. 609:29-35.
Bernaudin, M., Marti, H.H., Roussel, S., Divoux, D., Nouvelot, A., MacKenzie, E.T. and
Petit, E. (1999). A potential role for erythropoietin in focal permanent cerebral ischemia in
mice. J. Cereb. Blood Flow Metab. 19:643-51.
Nakamura, T., Ebihara, L, Shimada, N. and Koide, H. (1998) Elevated levels of
erythropoietin in cerbrospinal fluid of depressed patients. Amer. J Med Sc:i 315:199-201.
Masuda, S., Okano, M., Yamagishi, K., Nagao, M., Ueda, M. and Sasaki, R. (1994) A novel
site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes. J.
Biol. Chem. 269:19488-93.
Patent application WO 98-CA991, Weiss, S and Sorokan, S.T. Title: Erythropoietin-
mediated neurogenesis.
Campana, W.M., Misasi, R. and O'Brien, J.S. (1998) Identification of a neurotrophic
sequence of erythropoietin. Inter. J. Mol. Med. 1:235-241.
Patent application: WO 95/03821, O'Brien, J.S., Kishimoto, Y. and Altaian, D.E. Title:
Prosaposin and cytokine-derived peptides as therapeutic agents.
Konishi, Y., Chui, D-H, Hirose, H., Kunishita, T. and Tabira, T. (1993) Trophic effect of
erythropoietin and other hematopoietic factors on central cholinergic neurons in vitro and in
vivo. Brain Res. 609:29-35.
Mattson, M.P., Barger, S.W., Begley, J, and Mark, R.J. (1994) Calcium, free radicals and
excitotoxic neuronal death in primary cell culture. Methods Cell Biol. 46:187-216.
Smith-Swintosky VL, Cheo-Isaacs CT, D'Andrea MR, Santulli RJ, Darrow, AL, Andrade-
Gordon P. (1997) Protease-acrivated receptor 2 (PAR-2) is present in the rat hippocampus
and is associated with neurodegeneration. J. Neurochem., 69:1890-1896.
Silva, M., Grillot, D., Benito, A., Richard, C, Nunez, G., Fernandez-Luna, J.L. (1996).
Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through
Bcl-XL and Bcl-2. Blood 88, pp 1576-1582.
We Claim
1. A peptide for treating a patient having a condition mediated by
neurotoxicity, neurodegeneration or neurological damage comprising one
or more monomeric peptides of 8 to about 40 amino acids in length that
bind to EPO receptor, each monomeric peptide comprising a sequence of
amino acids X4X5XaXbX6XcXdX7 (SEQ ID NO: 47), wherein
Xa is G or A;
Xb is P or A;
Xc is T or A;
Xd is selected from W, A, and F;
X4 is selected from R, H, Y, L, and W, or X4 is nonexistent;
X5 is selected from F, M, and I;
X6 is independently selected from the 20 genetically coded L-amino acids
of the stereoisomers D-amino acids; and
X7 is selected from D, V, E, I, and L.
2. The peptide as claimed in claim 1, wherein said sequence is
X4X5GPX6TWX7 (SEQ ID NO: 48).
3. The peptide as claimed in claim 2, wherein said sequence is
X3X4X5GPX6TWX7X8 (SEQ ID NO: 1), wherein
X3 is selected from C, E, A, a-amino-?-bromobutyric acid, and
homocysteine (Hoc); and,
X8 is selected from C, K, A, a-amino-?-bromobutyric acid, and
homocysteine (Hoc).
4. The peptide as claimed in claim 3, wherein either X3 or X8 is C or
homocysteine (Hoc).
5. The peptide as claimed in claim 4 wherein X3 or X8 is C.
6. The peptide as claimed in claim 3, wherein
X3 is selected fro C, E, and A;
X4 is selected from R, H, and Y, or X4 is nonexistent;
X6 is selected from V, L, I, M, E, and A; and
X7 is D or V; and
X8 is selected from C, K and A.
7. The peptide as claimed in claim 3, wherein said peptide is a dimer of each
of said monomelic peptides comprising a sequence of amino acids
YX2X3X4X5GPX6TWX7X8 (SEQ ID NO: 2), wherein
X2 and X6 are each independently selected from the 20 genetically coded
L-amino acids;
X3 is C; and
X8 is C.
8. The peptide as claimed in claim 7, wherein
X2 is selected from L, S, H, M, A, and I, or X2 is nonexistent; and
X6 is selected from V, L, I, M, E, and A.
9. The peptide as claimed in claim 7, wherein each of said monomeric
peptides comprising a sequence of amino acids
X1YX2X3X4X5GPX6TWX7X8X9X10X11 (SEQ ID NO: 3), wherein each of X1, X2,
X6,X9, X10, and X11 is independently selected from the 20 genetically coded
L-amino acids.
10.The peptide as claimed in claim 9, wherein
X3 is selected from C, E, and A;
X4 is selected from R, H, and Y, or X4 is nonexistent;
X7 is D or V;
X8 is C or K;
X9 is K, G, L, Q, R, S, or T; and
X10 is A, G, P, R, or Y.
11.The peptide as claimed in claim 10, wherein
X1 is D, E, L, N, S, T, or V;
X2 is selected from L, S, H, M, A, and I, or X2 is nonexistent;
Xg is selected from K, Q, R, S, and G; and
X10 is selected from P, Y, and A.
12. The peptide as claimed in claim 3, wherein said peptide is a dimer of each
of said monomeric peptides comprising a sequence of amino acids
XX2X3X4X5GPX6TWX7X8 (SEQ ID NO: 49), wherein X' is selected from D-
Tyr, p-NO2Phe, p-NH2-Phe, p-F-Phe, p-I-phe, and 3, 5-dibromo-Tyr.
13.The peptide as claimed in claim 12, wherein said sequence is
X'CHFGPLTWVC.
14.The peptide as claimed in claim 1, wherein each of said monomeric
peptides comprise a sequence independently selected from
GGLYLCRFGPVTWDCGYKGG (SEQ ID NO: 7);
GGTYSCHFGPLTWVCKPQGG (aka EMP-1) (SEQ ID NO: 8);
GGDYHCRMGPLTWVCKPLGG (SEQ ID NO: 9);
VGNYMCHFGPITWVCRPGGG (SEQ ID NO: 10);
GGVYACRMGPITWVCSPLGG (SEQ ID NO: 11);
VGNYMAHMGPITWVCRPGG (SEQ ID NO: 12);
GGTYSCHFGPLTWVCKPQ (aka EMP-16)(SEQ ID NO: 13);
GGLYACHMGPMTWVCQPLRG (aka EMP-36)(SEQ ID NO: 14);
TIAQYICYMGPETWECRPSPKA (aka EMP-38)(SEQ ID NO: 15);
YSCHFGPLTWVCK (aka EMP-20)(SEQ ID NO: 16);
YCHFGPLTWVC (aka EMP-23)(SEQ ID NO: 17);
SCHFGPLTWVCK (aka EMP-24)(SEQ ID NO: 18);
GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO: 19);
GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO: 20);
GGTYSCFGPLTWVCKPQGG (aka EMP-27)(SEQ ID NO: 21);
TYSCHFGPLTWVCKPQGG (aka EMP-17)(SEQ ID NO: 22);
TYSCHFGPLTWVCKPQ (aka EMP-18)(SEQ ID NO: 23);
YSCHFGPLTWVCKP (aka EMP-19)(SEQ ID NO: 24);
YSCHFGPLTWVC (aka EMP-21)(SEQ ID NO: 25);
YSCHFGALTWVCK (aka EMP-22)(SEQ ID NO: 26);
GGCRIGPrrWVCGG (aka EMP-25)(SEQ ID NO: 27);
HFGPLTWV (aka EMP-26)(SEQ ID NO: 28);
GGTTSCHFGPLTWVCKPQGG (aka EMP-7) (SEQ ID NO: 29);
GGTFSCHFGPLTWVCKPQGG (aka EMP-8) (SEQ ID NO: 30);
GGTYSCHFGALTWVCKPQGG (aka EMP-10)(SEQ ID NO: 31);
GGTYSCHFGPATWVCKPQGG (aka EMP-11)(SEQ ID NO: 32);
GGTYSCHFGPLAWVVCKPQGG (aka EMP-12)(SEQ ID NO: 33);
GGTYSCHFGPLTAVCKPQGG (aka EMP-13)(SEQ ID NO: 34);
GGTYSCHFGPLTFVCKPQGG (aka EMP-14)(SEQ ID NO: 35);
GGTYSCHFGPLTWVCKAQGG (aka EMP-15)(SEQ ID NO: 36);
GGTXSCHFGPLTWVCKPQGG (aka EMP-28, X=D-Tyr)
(SEQ ID NO: 37);
GGTXSCHFGPLTWVCKPQGG (aka EMP-29, X=p-NO2-Phe)
(SEQ ID NO: 38);
GGTXSCHFGPLTWVCKPQGG (aka EMP-30, X=p-NH2-Phe)
(SEQ ID NO: 39);
GGTXSCHFGPLTWVCKPQGG (aka EMP-31, X=p-F-Phe)
(SEQ ID NO: 40);
GGTXSCHFGPLTWVCKPQGG (aka EMP-32, X=p-I-Phe)
(SEQ ID NO: 41);
GGTXSCHFGPLTWVCKPQGG (aka EMP-33, X=3, 5-dibromo-Tyr)
(SEQ ID NO: 42);
Ac-GGTYSCHFGPLTWVCKPQGG (aka EMP-34)(SEQ ID NO: 43);
GGLYACHMGPMTWVCQPLGG (aka EMP-35)(SEQ ID NO: 44);
LGRKYSCHFGPLTWVCQPAKKD (aka EMP-37) (SEQ ID NO: 45); and
GGTYSEHFGPLTWVKKPQGG (aka EMP-39) (SEQ ID NO: 46).
15.The peptide as claimed in claim 14, wherein each of said monomeric
peptides is independently selected from:
GGTYSCHFGPLTWVCKPQGG (aka EMP-1) (SEQ ID NO: 8);
GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO: 19);
GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO: 20); and
YCHFGPLTWVC (aka EMP-23) (SEQ ID NO: 17).
16.The peptide as claimed in claim 1, wherein said peptide is a dimer formed
by a polyethylene glycol linker through a covalent bond.
17.The peptide as claimed in claim 16, wherein each monomeric peptides of
said dimer are covalently bound N-terminus to N-terminus.
18.The peptide as claimed in claim 16, wherein each monomeric peptides of
said dimer are covalently bound N-terminus to C-terminus.
19.The peptide as claimed in claim 1, wherein said monomeric peptides are
dimerized on activated benodiazepins, oxazalones, azalactones,
aminimides or diketopiperazine.
20.The peptide as claimed in claim 19, wherein said monomeric peptides are
covalently bound N-terminus to N-terminus.
21.The peptide as claimed in claim 19, wherein said monomeric peptides are
covalently bound N-terminus to C-terminus.
22.The peptide as claimed in claim 2, comprising at least one peptide dimer.
23. The peptide as claimed in any of the preceding claims, wherein said
peptide dimer, is a cell surface receptor agonist selected from a group
consisting of GH agonist, PDGF agonist, EGF agonist, G-CSF agonist, TPO
(thrombopoietin) agonist, VEGF agonist, FGF agonist, insulin agonist, IL-3
agonist, IL-5 agonist, IL-6 agonist and IL-2 agonist.
24.The peptide as claimed in claim 23, wherein said agonist comprises a
sequence of amino acids YX2X3X4X5GPX6TWX7X8 (SEQ ID NO: 2), wherein
each of X2 and X6 is independently selected from the 20 genetically coded
L-amino acids; X3 is C; X4 is R, H, L or W; X5 is M, F or I; X7 is D, E, I, L or
V; and X8 is C.
25.The peptide as claimed in claim 23, wherein said agonist comprises a
sequence of amino acids X1YX2X3X4X5GPX6TWX7X8X9X10X11 (SEQ ID NO: 3)
wherein each of X1, X2, X6, X9, X10 and Xn is independently selected from
any one of the 20 genetically coded L-amino acids; X3 is C; X4 is R, H, L or
W; X5 is M, F or I; X7 is D, E, I, L or V; and X8 is C.
26.The peptide as claimed in claim 23, wherein said agonist comprises a
sequence of amino acids X1YX2X3X4X5GPX6TWX7X8X9X10X11 (SEQ ID NO: 3)
wherein each of X1, X2, and X11 is independently selected from any one of
the 20 genetically coded L-amino acids; X3 is C; X4 is R or H, X5 is F or M;
X6 is I, L, T, M or V; X7 is D or V; X9 is G, K, L, Q, R, S, or T; and X10 is A,
G, P, R, or Y.
27.The peptide as claimed in claim 23, wherein said agonist comprises a
sequence of amino acids X1YX2X3X4X5GPX6TWX7X8X9X10X11 (SEQ ID NO: 3)
wherein each of X1 is D, E, L, N, S, T or V; X2 is A, H, K, L, M, S or T; X3 is
C; X4 is R or H; X5 is M, F or I; X6 and X11 are independently any one of the
20 genetically coded L-amino acids; X7 is D, E, I, L or V; X8 is C; X9 is K, R,
S, or T; and X10 is P.
28.The peptide as claimed in claim 23, wherein said agonist is selected from
a group consisting of:
GGLYLCRFGPVTWDCGYKGG (SEQ ID NO: 7);
GGTYSCHFGPLTWVCKPQGG (aka EMP-1) (SEQ ID NO: 8);
GGDYHCRMGPLTWVCKPLGG (SEQ ID NO: 9);
VGNYMCHFGPITWVCRPGGG (SEQ ID NO: 10);
GGVYACRMGPITWVCSPLGG (SEQ ID NO: 11);
VGNYMAHMGPITWVCRPGG (SEQ ID NO: 12);
GGTYSCHFGPLTWVCKPQ (aka EMP-16)(SEQ ID NO: 13);
GGLYACHMGPMTWVCQPLRG (aka EMP-36)(SEQ ID NO: 14);
TIAQYICYMGPETWECRPSPKA (aka EMP-38)(SEQ ID NO: 15);
YSCHFGPLTWVCK (aka EMP-20)(SEQ ID NO: 16);
YCHFGPLTWVC (aka EMP-23)(SEQ ID NO: L7);
SCHFGPLTWVCK (aka EMP-24)(SEQ ID NO: 18);
GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO: 19);
GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO: 20);
GGTYSCFGPLTWVCKPQGG (aka EMP-27)(SEQ ID NO: 21);
TYSCHFGPLTWVCKPQGG (aka EMP-17)(SEQ ID NO: 22);
TYSCHFGPLTWVCKPQ (aka EMP-18)(SEQ ID NO: 23);
YSCHFGPLTWVCKP (aka EMP-19)(SEQ ID NO: 24);
YSCHFGPLTWVC (aka EMP-21)(SEQ ID NO: 25);
YSCHFGALTWVCK (aka EMP-22)(SEQ ID NO: 26);
GGCRIGPITWVCGG (aka EMP-25)(SEQ ID NO: 27);
HFGPLTWV (aka EMP-26)(SEQ ID NO: 28);
GGTTSCHFGPLTWVCKPQGG (aka EMP-7) (SEQ ID NO:: 29);
GGTFSCHFGPLTWVCKPQGG (aka EMP-8) (SEQ ID NO: 30);
GGTYSCHFGALTWVCKPQGG (aka EMP-10)(SEQ ID NO: 31);
GGTYSCHFGPATWVCKPQGG (aka EMP-11)(SEQ ID NO: 32);
GGTYSCHFGPLAWVCKPQGG (aka EMP-12)(SEQ ID NO: 33);
GGTYSCHFGPLTAVCKPQGG (aka EMP-13)(SEQ ID NO: 34);
GGTYSCHFGPLTFVCKPQGG (aka EMP-14)(SEQ ID NO: 35);
GGTYSCHFGPLTWVCKAQGG (aka EMP-15)(SEQ ID NO: 36);
GGTXSCHFGPLTWVCKPQGG (aka EMP-28, X=D-Tyr)
(SEQ ID NO: 37);
GGTXSCHFGPLTWVCKPQGG (aka EMP-29, X=p-NO2-Phe)
(SEQ ID NO: 38);
GGTXSCHFGPLTWVCKPQGG (aka EMP-30, X=p-NH2-Phe)
(SEQ ID NO: 39);
GGTXSCHFGPLTWVCKPQGG (aka EMP-31, X=p-F-Phe)
(SEQ ID NO: 40);
GGTXSCHFGPLTWVCKPQGG (aka EMP-32, X=p-I-Phe)
(SEQ ID NO: 41);
GGTXSCHFGPLTWVCKPQGG (aka EMP-33, X=3, 5-dibromo-Tyr)
(SEQ ID NO: 42);
Ac-GGTYSCHFGPLTWVCKPQGG (aka EMP-34)(SEQ ID NO: 43);
GGLYACHMGPMTWVCQPLGG (aka EMP-35)(SEQ ID NO: 44);
LGRKYSCHFGPLTWVCQPAKKD (aka EMP-37) (SEQ ID NO: 45); and
GG7YSEHFGPLTWVKKPQGG (aka EMP-39) (SEQ ID NO: 46).
29.The peptide as claimed in claim 23, wherein said peptide dimers are
formed with a polyethylene glycol linker through a covalent bond.
The invention relates to a peptide for treating a patient having a condition
mediated by neurotoxicity, neurodegeneration or neurological damage
comprising one or more monomeric peptides of 8 to about 40 amino acids in
length that bind to EPO receptor, each monomeric peptide comprising a
sequence of amino acids X4X5XaXbX6XcXdX7 (SEQ ID NO: 47), wherein Xa is G or A;
Xb is P or A; Xc is T or A; Xd is selected from W, A, and F; X4 is selected from R, H,
Y, L, and W, or X4 is nonexistent; X5 is selected from F, M, and I; X6 is
independently selected from the 20 genetically coded L-amino acids of the
stereoisomeric D-amino acids; and X7 is selected from D, V, E, I, and L.

Documents:

in-pct-2002-1487-kol-granted-abstract.pdf

in-pct-2002-1487-kol-granted-claims.pdf

in-pct-2002-1487-kol-granted-correspondence.pdf

in-pct-2002-1487-kol-granted-description (complete).pdf

in-pct-2002-1487-kol-granted-drawings.pdf

in-pct-2002-1487-kol-granted-examination report.pdf

in-pct-2002-1487-kol-granted-form 1.pdf

in-pct-2002-1487-kol-granted-form 18.pdf

in-pct-2002-1487-kol-granted-form 26.pdf

in-pct-2002-1487-kol-granted-form 3.pdf

in-pct-2002-1487-kol-granted-form 5.pdf

in-pct-2002-1487-kol-granted-reply to examination report.pdf

in-pct-2002-1487-kol-granted-specification.pdf

in-pct-2002-1487-kol-granted-translated copy of priority document.pdf


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Patent Number 225483
Indian Patent Application Number IN/PCT/2002/1487/KOL
PG Journal Number 46/2008
Publication Date 14-Nov-2008
Grant Date 12-Nov-2008
Date of Filing 03-Dec-2002
Name of Patentee ORTHO MCNEIL PHARMACEUTICAL INC
Applicant Address US ROUTE 202, RARITAN, NEW JERSEY 08869
Inventors:
# Inventor's Name Inventor's Address
1 SMITH-SWINTOSKY VIRGINIA 3163 LINE LEXINGTON ROAD, HATFIELD, PA 19440
2 RENZI MICHAEL 485 OAK DRIVE, HARLEYSVILLE, PA 19438
3 PLATA-SALAMAN CARLOS 1313 SQUIRE DRIVE, AMBLER, PA 19002
4 JOLLIFFE LINDA 16 DAVENPORT WAY, HILLSBOROUGH, NEW JERSEY 08844
5 FARRELL FRANCIS 4934 JULIE COURT, DOYLESTOWN, PA 18890
6 JOHNSON DANA L 1343 LONELY COTTAGE ROAD, UPPER BLACK EDDY, PA 19872
PCT International Classification Number A61K 38/00
PCT International Application Number PCT/US01/16654
PCT International Filing date 2001-05-23
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/207,654 2000-05-26 U.S.A.