Note: Descriptions are shown in the official language in which they were submitted.
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LONG ACTING ERYTHROPOIETINS THAT MAINTAIN
TISSUE PROTECTIVE ACTIVITY OF ENDOGENOUS ERYTHROPOIETIN
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/409,020, filed on
September 9, 2002, which is incorporated in its entirety by reference herein.
FIELD OF THE INVENTION
The present invention relates to long acting erythropoietins that
advantageously maintain
tissue protective capabilities after modification. In particular, the present
invention relates to long
acting erythropoietins that are chemically modified in a way that increases
the serum half life but also
maintains the tissue protective function of the native protein ira vivo. The
present invention also
relates to the treatment of anemia and anemia related diseases with the long
acting erythropoietins of
the present invention. Finally, the present invention is directed to assays
useful in the determination
of whether an erythropoietin exhibits tissue protective capabilities.
BACKGROUND OF THE INVENTION
Naturally occurring or endogenous erythropoietin (EPO) is a glycoprotein
hormone produced
mainly in the liver. Endogeneous EPO includes 165 amino acids and has a
molecular weight (in
humans) of about 30,000 to about 34,000 daltons. The glycosyl residues in EPO,
which consist of
three N-linked and one O-linked oligosaccharide chains, are responsible for
about 40 percent of the
protein's total weight. The N-linked oligosaccharide chains are bonded to
amide nitrogens of
asparagine at positions 24, 38 and 83, while the O-linked oligosaccharide
chain is bonded to the
oxygen at the serine residue located at position 126. The EPO protein may
occur in three forms: cx, (3,
and asialo. The a and ,Q forms have the same potency, biological activity, and
molecular weight, but
differ slightly in the carbohydrate components, while the asialo form is an a
or ~i form with the
terminal sialic acid (carbohydrate) removed.
Until recently, the principle function of endogeneous EPO is to act in concert
with other
growth factors to stimulate the proliferation and maturation of responsive
bone marrow erythroid
precursor cells and maintain an individual's hematocrit (percent of whole
blood that contains red
blood cells). The process of producing the red blood cells is called
erythropoiesis, which is a
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precisely controlled physiological mechanism that optimizes the number of red
blood cells for proper
tissue oxygenation without impeding circulation. For example, when oxygen
transport by red blood
cells is reduced, EPO will increase red blood cell production by stimulating
the conversion of
precursor cells in the bone marrow into mature red blood cells, which are then
released into the
circulation. When the number of red blood cells in circulation is over that
needed for normal tissue
circulation, EPO in circulation is decreased. Thus, when the body is in a
healthy state, EPO is
present in very low concentrations in plasma, which is sufficient to stimulate
replacement of red
blood cells lost normally though aging. Plasma EPO levels normally range from
0.01 Units/ml to
0.03 Units/ml.
Given that the kidney produces the majority of the EPO for an individual, the
loss of kidney
function, such as in chronic renal failure (CRF), results in impaired
production of EPO and often
leads to anemia. Similarly, anemia may result from other chronic conditions,
such as cancer, or
treatments associated with these illnesses, such as chemotherapy. Thus, the
administration of
recombinant EPO (discussed in more detail below), which has substantially the
same biological
effects as endogeneous EPO, has been proven useful in restoring hematocrit
levels in individuals with
decreased red blood cells.
In addition to recombinant EPO's role in maintaining hematocrit levels for
chronic conditions,
recombinant EPO has been used to boost red blood cell levels prior to elective
or scheduled surgeries,
thereby reducing or eliminating the need to transfuse blood. For example,
recombinant EPO may be
administered to address concerns about the patient receiving a virus or
pathogen from the blood
supply, or to address religious restrictions regarding blood transfusions.
Furthermore, several lines of recent evidence suggest that EPO, as a member of
the cytokine
superfamily, has other important therapeutic attributes, which are mediated
through interaction with
the EPO receptor (EPO-R). For example, EPO and its receptor may play an
important role in
attenuating tissue injury because the interaction between EPO and the receptor
provides
compensatory responses that serve to improve hypoxic cellular
microenvironment, as well as
modulate programmed cell death caused by metabolic stress. In fact, patients
with chronic renal
failure and/or cancer have generally experienced an improved sense of well-
being and increased
mental acuity following treatment with EPO, an effect previously attributed to
the patient's increased
hematocrit. Recently, however, these improvements have been attributed to
EPO's tissue protective
and enhancing effects, as discussed in International Publication No.
WO/02053580 and U.S. Patent
Publication Nos. 2002/0086816 and 2003/0072737.
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Recent studies also have suggested that systemically administered EPO may
cross the intact
blood brain barrier because the capillaries forming the blood brain barrier
also express the EPO
receptor. As such, an anatomical basis for receptor-mediated transcytosis is
provided from the
peripheral circulation into the brain.
Recombinant EPO (epoetin alfa), which has been commercially available under
tradenames
PROCRIT~ (from Ortho Biotech Inc., Raritan, NJ), and EPOGEN~ (from Amgen,
Inc., Thousand
Oaks, CA), has been used to treat anemia resulting from end stage renal
disease, to treat HIV-infected
patients when used in concert with AZT (zidovudine) therapy, and to
counterbalance the effects of
chemotherapy. While the therapeutic effects of recombinant EPO are numerous,
to date the principal
application of recombinant EPO has been to address chronic anemia. In this
regard, recombinant
EPO is typically administered in an initial dose of between 50-150 units/kg
three times per week for
about six to eight weeks either by an intravenous or subcutaneous inj ection
in order to restore the
suggested hematocrit range within the patient. After the patient achieves a
desired hematocrit level,
such as an amount falling within from about 30 percent to about 36 percent,
that level may be
sustained by maintenance EPO therapy in the absence of iron deficiency and
concurrent illnesses.
While dosage requirements may vary according to the patient's individual
needs, typically
maintenance dosages may be administered about three times a week (less if
larger doses are
provided).
The dosage amount and frequency of the administration of recombinant EPO is
determined in
part upon the half life of the molecule, which may be limited when the
molecule is in vivo. For
example, intravenously administered EPOGEN~ is reportedly eliminated at a rate
consistent with
first order kinetics with a circulating half life ranging from approximately 4
to 13 hours in adult and
pediatric patients with CRF. Thus, in order to be therapeutically effective,
the dosage amount and
frequency of dosing must be tailored to account for the relatively short half
life of the recombinant
EPO.
Additionally, because recombinant EPO is administered either by an intravenous
or
subcutaneous injection, a nurse or physician often is required to administer
recombinant EPO to a
patient. This presents an additional inconvenience to a patient, and is yet
another reason why it may
be desirable to extend the half life of the molecule. As such, efforts to
increase the half life of
recombinant EPO have gained research attention in the past decade based on the
premise that an
extended half life would decrease dosage requirements while still providing
the same or improved
therapeutic benefits.
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In fact, recent experiments on human EPO demonstrated that there is a direct
relationship
between the sialic acid-containing carbohydrate content of EPO, its
circulating half life, and in vivo
bioactivity. As discussed in PCT Publication No. W095/05465, sialic acid
residues cap the ends of
the sugar chains and prevent the detection of galactose by the liver. The N-
linked oligosaccharide
chains typically have up to 4 sialic acids per chain, and the O-linked
oligosaccharide chains have up
to 2 sialic acids per chain. Thus, an unmodifed EPO polypeptide may
accommodate up to a total of
14 sialic acids.
Over time, these sialic acid residues may be cleaved from the protein, thereby
exposing the
galactose chains to detection by the liver. Once the liver detects the
galactose chains, the protein is
filtered from the blood. As such, a stepwise increase in sialic acid content
per EPO molecule is
believed to better shield the galactose chains to provide a corresponding
stepwise increase in
biological activity (measured by the ability of equimolar concentrations of
isolated erythropoietin
isoforms to raise the hematocrit of normal mice). Since unmodified EPO
contains only 14 sialic acid
sites, this approach may have limited ability to extend the half life of EPO.
This led to the hypothesis
that an EPO analog engineered to contain additional oligosaccharide chains
would have enhanced
biological activity. By providing these additional glycosylation sites,
additional oligosaccharide
chains having terminal ends may then be modified with sialic acid residues.
See PCT Publication
Nos. W091105867, W094/09257, and WO01/81405.
For example, a modified EPO analog may have at least one additional N-linked
carbohydrate
chain and/or at least one additional O-linlced carbohydrate chain.
Specifically, WO01/81405
discloses the addition of N-linked carbohydrate chains to the molecule at
amino acids at 30, 51, 57,
69, 88, 89, 136 and/or 138. The modified EPO molecules may have anywhere from
1 to 4 additional
glycosylation sites, which permit the addition of 2 to 16 sialic acid residues
to the molecule.
However, while efforts to increase the serum half life of EPO have proven
successful and are
useful in maintaining hematocrit levels, no attention has been paid to the
effect that these additional
glycosylation sites may have on other functions of EPO.
Thus, it would be beneficial to provide a modified EPO with an extended serum
half life
(long acting) that maintains the functionality of endogenous EPO. In
particular, there is a need in the
art for a long acing EPO compound with the erythropoietic functionality and
tissue protective
functionality for use in pharmaceutical compositions to treat individuals with
anemia and/or related
diseases. In addition, a need exists for assays to determine whether a
particular EPO is antagonistic
to the tissue protective capabilities of endogenous EPO.
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BRIEF SUMMARY OF THE INVENTION
The present invention is directed to a method for regulating the hematocrit
level in humans
including the steps of providing an erythropoietin product having a longer
serum half life than
recombinant human erythropoietin (rhuEPO) and including tissue protective
functionality and
administering a therapeutically effective amount of the erythropoietin
product. In one embodiment,
the step of providing an erythropoietin product further includes the step of
modifying recombinant
erythropoietin with at least one chemical modification to at least one of the
N-linked oligosaccharide
chains or the O-linked oligosaccharide chain, wherein the chemical
modification includes oxidation,
sulfation, phosphorylation, PEGylation, or a combination thereof.
In adddition, the step of administering a therapeutically effective amount of
the erythropoietin
product may include administering the erythropoietin product at a lower molar
amount than rhuEPO
to obtain a comparable target hematocrit.
In one embodiment, the serum half life is at least about 20 percent longer
than the serum half
life of rhuEPO. In another embodiment, the serum half life is at least about
40 percent longer than
the serum half life of rhuEPO.
The present invention is also directed to a man-made erthyropoietin product
including at least
one erythropoietin derivative, wherein at least one N-linked oligosaccharide
chain or at least one O-
linked oligosaccharide chain has at least one chemical modification as a
result of oxidation, sulfation,
phosphorylation, PEGylation, or mixtures thereof, and wherein the
erythropoietin product has a
longer serum half life than rhuEPO. The erythropoietin product preferably has
tissue protective
functionality.
In one embodiment, the at least one chemical modification includes oxidation
of at least one
N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain
to provide at least one
additional acid residue. For example, the at least one chemical modification
may include sulfation of
at least one N-linked oligosaccharide chain or at least one O-linked
oligosaccharide chain to provide
an increased negative charge on the EPO product. In another embodiment, the at
least one chemical
modification includes phosphorylation of at least one N-linked oligosaccharide
chain or at least one
O-linked oligosaccharide chain to provide a.n increased negative charge on the
EPO product. In still
another embodiment, the at least one chemical modification includes addition
of at least one
polyethylene glycol chain to at least one N-linked oligosaccharide chain or at
least one O-linked
oligosaccharide chain.
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The present invention also relates to a method for preparing an erthyropoietin
product having
an extended serum half life and tissue protective activity including the steps
of providing at least one
erythropoietin or erythropoietin derivative; and modifying at least one N-
linked oligosaccharide chain
or at least one O-linked oligosaccharide chain on the at least one endogenous
or recombinant
erythropoietin by oxidation, sulfation, phosphorylation, PEGylation, or a
combination thereof.
The step of modifying may further include the step of replacing at least one
vicinal hydroxyl
on at least one N-linked oligosaccharide chain or at least one O-linked
oligosaccharide chain with at
least one acid residue. In one embodiment, the step of replacing at least one
vicinal hydroxyl on at
least one N-linked oligosaccharide chain or at least one O-linked
oligosaccharide chain with at least
one acid residue further includes replacing a plurality of vicinal hydroxyls
on the least one N-linked
oligosaccharide chain or at least one O-linked oligosaccharide chain with a
plurality of acid residues.
In another embodiment, the step of modifying further includes the steps of
providing an
organic solvent; dissolving the erythropoietin or erythropoietin derivative in
the organic solvent to
form a solution; providing at least one condensing agent; providing at least
one sulfate donor; and
mixing the at least one condensing agent and the at least one sulfate donor
into the solution. In yet
another embodiment, the step of modifying further includes the steps of
providing an organic
solvent; dissolving the erythropoietin or erythropoietin derivative in the
organic solvent to form a
solution; providing at least one condensing agent; providing phosphoric acid;
and mixing the at least
one condensing agent and the at least one phosphoric acid into the solution.
In still another embodiment, the step of modifying further includes the steps
of providing an
organic solvent; dissolving the erythropoietin or erythropoietin derivative in
the organic solvent to
form a first solution; providing at least one oxidizing agent; adding the at
least one oxidizing agent to
the first solution to form a second solution; providing at least one
polyethylene glycol chain; and
mixing the at least one polyethylene glycol chain into the second solution.
The step of providing at
least one polyethylene glycol chain may include providing at least one
polyethylene glycol chain with
at least one primary amino moiety at an end of the chain.
The present invention also relates to a method for treating anemia in patients
at risk for tissue
damage including the steps of providing an erythropoietin product with at
least one chemical
modification to at least one of the N-linked oligosaccharide chains or the O-
linked oligosaccharide
chain, wherein the chemical modification includes oxidation, sulfation,
phosphorylation, PEGylation,
or a combination thereof; administering a therapeutically effective amount of
the erythropoietin
product, wherein the erythropoietin product is administered at a lower molar
amount than rhuEPO to
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obtain a comparable target hematocrit, wherein the erythropoietin product has
tissue protective
functionality.
In this aspect of the invention, the erythropoietin product preferably has a
longer serum half
life than rhuEPO. In one embodiment, the serum half life is at least about 20
percent longer than the
serum half life of rhuEPO. In another embodiment, the serum half life is at
least about 40 percent
longer than the serum half life of rhuEPO.
The present invention further relates to a pharmaceutical composition
including: a
therapeutically effective amount of at least one erythropoietin derivative,
wherein at least one N-
linked oligosaccharide chain or at least one O-linked oligosaccharide chain
has at least one chemical
modification as a result of oxidation, sulfation, phosphorylation, PEGylation,
or mixtures thereof,
wherein the at least one erythropoietin derivative has a longer serum half
life than recombinant
erythropoietin and has tissue protective functionality. In one embodiment, the
pharmaceutical
composition further includes at least one pharmaceutically acceptable carrier.
The at least one
pharmaceutically acceptable carrier may include at least one diluent,
adjuvant, excipient, vehicle, or
mixtures thereof.
In another embodiment, the pharmaceutical composition further includes at
least one wetting
agent, emulsifying agent, pH buffering agent, or a combination thereof. In yet
another embodiment,
the pharmaceutical composition further includes at least one tissue protective
cytokine.
BRIEF DESCRIPTION OF THE FIGURES
Further features and advantages of the invention can be ascertained from the
following
detailed description that is provided in connection with the drawings
described below:
FIG. lA is a comparison of the effectiveness of various forms of EPO in
protecting against
cell death triggered by exposure to trimethyl tin; and
FIG. 1B is a comparison of the effectiveness of various forms of EPO in
protecting against
cell death triggered by exposure to trimethyl tin.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to the use of EPO molecules having an
extended serum half
life (long acting) that are chemically modified with carbohydrate chains so
that the functionality of
endogenous EPO is maintained. As discussed in the background, efforts to
extend the half life of
EPO have generally been focused on adding extra carbohydrate chains to the EPO
molecule to
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protect the galactose chains from exposure. However, the added carbohydrate
chains are believed to
effect the functionality of the EPO analog such that, for example, the
functionality is compromised to
achieve the longer half life. While there are known EPO analogs with longer
half lives than
recombinant EPO that have erythropoietic activity, these analogs do not retain
other recently
discovered therapeutic benefits of EPO, e.g., tissue protective activity.
For example, a 17 amino acid fragment of EPO corresponding to amino acids 30-
47, also
referred to as the O'Brien peptide, has been shown to have tissue protective
activity in vitro, but has
no erythropoietic activity in vitro. Campana, W.M., Misasi, R. & O'Brien,
J.S., Int. J. Mol. Med., l,
235-41 (1998). Therefore, it is believed that a modified EPO molecule having
additional
glycosylation sites within the O'Brien peptide may not have tissue protective
activity in other ifZ vitro
assays. In addition, because the three-dimensional orientation of the EPO
molecule is important to
the functionality, adding glycosylation sites to the molecule may interfere
with the overall
functionality.
Thus, the present invention relates to a long acting EPO with at least one of
erythropoietic
activity, tissue protective activity, transcytosis capability, or a
combination thereof. Preferably, the
long acting EPO of the present invention has erythropoietic activity and at
least one of tissue
protective activity or transcytosis capability.
In one embodiment, the long acting EPO of the present invention has a serum
half life that is
at least about 20 percent longer than the serum half life of recombinant EPO.
In another
embodiment, the serum half life of the long acting EPO of the present
invention is at least about 30
percent longer than the half life of recombinant EPO. In still another
embodiment, the long acting
EPO of the present invention has a serum half life that is at least about 40
percent longer than the
serum half life of recombinant EPO.
Briefly, the long acting EPOs of the present invention include EPO molecules
with
carbohydrate chains that are altered with at least one modification as
compared to a native
(endogenous) EPO, preferably as compared to native human EPO. In one
embodiment, the long
acting EPOS of the present invention undergo a plurality of modifications to
the carbohydrate chains.
In one embodiment, the vicinyl hydroxyls on the carbohydrate chain of a native
EPO are
oxidized into acid residues to produce the long acting EPO molecules of the
present invention. In
another embodiment, the sialic acid residues on the EPO are replaced with less
labile acid residues.
In yet another embodiment, sulfation and/or phosphorylation of the
carbohydrate chain of an EPO
results in a long acting EPO according to the present invention. In still
another embodiment, the long
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acting EPO of the present invention results from the addition of polyethylene
glycol to the
carbohydrate chain of EPO. Any combination of the foregoing modifications is
also contemplated by
the present invention. And, as mentioned above, the present invention also
embraces compositions,
including pharmaceutical compositions, which include one or more of the
aforementioned long acting
EPO molecules.
The long acting EPO molecules of the present invention are contemplated for
inclusion in
pharmaceutical compositions for treating anemia and related diseases,
especially those with
complications resulting from illnesses such as, but limited to, acute renal
failure, sepsis, HIV,
chemotherapy, and the like.
The present invention is also directed to methods for treating anemia and
related diseases, as
well as kits used for the treatment procedure. As used herein, the term
"treatment" refers to both
therapeutic treatment and prophylactic or preventative measures, wherein the
object is to prevent or
slow down (lessen) the targeted pathologic condition or disorder. Those in
need of treatment include
those already with the disorder as well as those prone to have the disorder or
those in whom the
disorder is to be prevented. The present invention contemplates the use of the
long acting EPOS for
chronic administration, acute treatment, and/or intermittent administration.
For the purposes of this
disclosure, "chronic administration" refers to administration of the agents)
in a continuous mode as
opposed to an acute mode, so as to maintain the initial therapeutic effect
(activity) for an extended
period of time and "intermittent administration" is treatment that is not
consecutively done without
interruption, but rather is cyclic in nature.
The long acting EPOS of the present invention, and the uses thereof, are
applicable for any
mammal. As used herein, the term "mammal" refers to any animal classified as a
mammal, including
humans, domestic and farm animals, and zoo, sports, or pet animals, such as
dogs, cats, cattle, horses,
sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human. The
administration of the long
acting EPOS of the present invention include, but is not limited to, oral,
intravenous, intranasal,
topical, intraluminal, inhalation or parenteral administration, the latter
including intravenous,
intraarterial, subcutaneous, intramuscular, intraperitoneal, submucosal,
intradermal, and combinations
thereof.
The present invention further relates to the use of long acting EPO molecules
of the present
invention as a Garner for other molecules into areas of the body that have EPO
receptors. For
example, because certain molecules have poor penetration across the blood
brain barrier, linking
these molecules to the long acting EPOs of the present invention provides a
safe and effective
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transport system of these molecules into the brain. And, as discussed in more
detail later, because
other areas of the body express EPO receptors, such as the retina, the heart,
and the lungs, the long
acting EPO molecules of the present invention may act as a transport system
for molecules having
poor penetration through such areas.
Furthermore, the present invention is directed to assays for determining
whether a particular
EPO maintains the functionality of endogenous EPO. For example, an assay of
the present invention
may determine whether a modified EPO is tissue protective, i.e., an agonist
with regard to
endogenous EPO. As used herein, the term "agonist" is used in the broadest
sense and includes
molecules that mimic the biological activity of a native EPO. In a similar
manner, the term
"antagonist" is used in the broadest sense, and includes any molecule that
partially or fully blocks,
inhibits, or neutralizes the biological activity of native EPO. In one
embodiment, testing of a
particular EPO occurs in an in vitro assay, such as a P19 cell and/or rat
motoneuron assay. In another
embodiment, the assay of the present invention involves the evaluation of a
particular EPO in vivo
using various assays such as the rat focal ischemia, rat retinal ischemia,
spinal chord trauma, and
bicuculline seizure models.
Functionality
As discussed in the background section, various attempts to increase the half
life of EPO have
been successful in that the EPO analogs have a longer half life and maintain
erythropoietic activity.
And, as discussed, the EPO analogs with longer half lives have mainly included
EPO molecules with
additional carbohydrate chains added to the amino acid sequence. However, it
is anticipated that
these additional carbohydrate chains may interfere with other therapeutic
benefits of EPO, such as the
tissue protective functionality and the transcytosis capability of the
molecule. Without being bound
to any particular theory, the placement of the added carbohydrate chains in
the O'Brien peptide may
affect functionality based on importance of this peptide with regard to tissue
protective functionality.
In addition, the added carbohydrate chains, whether within the O'Brien peptide
or outside of the
O'Brien peptide, are believed to have an affect on the three-dimensional
orientation of the molecule.
For example, in the three-dimensional configuration of the molecule, the
additional carbohydrate
chains may block an area of the molecule that is essential to the
functionality. Furthermore, it is
believed that the method of glycosylation (or adding the carbohydrate chains)
may also have an affect
on the functionality of the glycoprotein.
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Tissue Protective Capability
To evaluate the possibility that extra carbohydrate chains may affect the
functionality of the
glycoprotein, the present inventors studied forms of EPO analogs that have
five N-linked
carbohydrate chains (compared to the 3 N-linked carbohydrate chains of
recombinant EPO). In
particular, the inventors used an EPO analog, wherein the analog has extra
glycosylation site at the 32
amino acid, which results in about a 3-fold longer half life than recombinant
EPO (epoetin alfa).
Although this EPO analog appeared within the cerebral spinal fluid after
systemic injection
(Figures lA), it was surprisingly not tissue protective when evaluated in a
subsequent P19 in vitro
assay (Figure 1B). This lack of tissue protective activity was unexpected. In
addition, the lack of
tissue protective activity may produce complications when used for treatment
in anemia patients if
those patients have other conditions requiring the tissue protective
capability. For example, if the
non-tissue protective EPO analog competes with tissue protective endogenous
EPO for the receptor
that triggers the tissue protective response, the extent of injury resulting
from a trauma may actually
be exacerbated due to the use of such EPO analogs. In fact, if a patient on
such an EPO analog
suffered from a stroke, the infarct volume resulting from the stroke may
actually be greater than in an
individual not treated with an EPO analog.
While not wishing to be bound to any particular theory, this fording suggests
that at least one
additional version of an EPO receptor functionally exists in neuronal tissues
for which signaling
differs from that of erythrocyte precursors, and there is a risk that certain
EPO analogs may
antagonize endogenous EPO's ability to bind to this version of the receptor.
The distinctly different
biological activities between endogenous EPO and these EPO analogs suggest
that receptor signaling
occurs via functionally different EPO receptors responding to different
domains of the EPO
molecule. In fact, while the EPO receptor gene protein sequence has been
reported to be identical to
that expressed by the erythroid precursors, the binding affinity of the
neuronal-type receptor for EPO
in vitro is much lower than the EPO receptor of the proerythrocyte. See, e.g.,
Masuda, S., et al., J
Biol Chefn, 268, 11208-16 (1993). Presumably, these differences arise from
accessory proteins and
may indicate that different signaling pathways are employed than those
activated in the erythrocyte
maturation program. Interestingly, this difference in affinity is not modified
by complete
deglycosylation of EPO, a result that is not unexpected if the neurally-active
binding occurs in the
normally non-glycosylated AB loop region of EPO. Id. In addition, EPO produced
by astrocytes
(which is presumably the same product produced by other cells such as neurons)
is also a smaller
version than that produced by the kidney. Masuda, S., J. Bio Claefn, 269,
19488-93 (1994). The
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difference appears to be the result of a different degree of glycosylation.
Id. Whether the desialiated
natural ligand possesses differences in affinity with these known receptor
proteins has not yet been
determined, but is of obvious relevance.
In addition to the presence of EPO receptor modifying accessory proteins, the
EPO receptor is
a complex gene for which a number of edited versions, including a truncated,
soluble receptor, exist.
Yamaji, R., et al., Eur- JBiochefn, 239, 494-500; Yamaji, R., et al., Bichina
Biophys Acta, 1403, 169-
78 (1998); Baryon, C., et al., Gene, 147, 263-8 (1994); Chin, K., et al.,
Brain Res Mol BYain Res, 81,
29-42 (2000); Fujita, M., et al., Lukernia, 11 Suppl 3, 444-5 (1997);
Westenfelder, C., Biddle, D. L. &
Baranowski, R.L., Kid. Intef~nat., 55, 808-820 (1999). Whether any of these
versions subserve the
neural effects of EPO also remains to be determined.
Furthermore, as discussed in the background, the O'Brien peptide has been
shown to have
tissue protective activity in vitro, but no erythropoietic activity in vitro.
In fact, an assay performed
on an EPO analog that contains an added carbohydrate chain within the O'Brien
peptide
demonstrated that the EPO analog lacked tissue protective capabilities. This
result suggests that
certain modifications to the O'Brien peptide, such as the addition of
carbohydrate chains, interferes
with the functionality of the protein. An EPO analog with modifications to the
O'Brien peptide likely
acts as an antagonist towards the endogenous EPO located within the body
because it partially or
fully blocks the endogenous EPO's ability to bind to the EPO receptor. As
such, the risk of
increasing the extent of injury resulting from trauma is likely with the use
of such an EPO analog.
Thus, it is believed that EPO analogs with modifiations to the O'Brien peptide
would also lack tissue
protective capabilities in other in vitro assays such as the rat motoneuron
assay and in in vivo assays
such as the rat focal ischemic, bicuculline seizure, rat retinal ischemia, and
spinal cord trauma assays.
Receptor Mediated Transcytosis
Using the same EPO analog as above, i.e., an EPO analog with five N-linked
carbohydrate
chains (compared to the 3 N-linked carbohydrate chains of recombinant EPO),
the inventors studied
the analog's ability to traverse the blood brain barner. The EPO analog
appeared within the cerebral
spinal fluid after systemic injection (Figures lA and 1B). Without being bound
to any particular
theory, it is believed that the EPO analog is able to cross the intact blood
brain barrier because the
capillaries forming the blood brain barrier also express the EPO receptor and
provide an anatomical
basis for receptor-mediated transcytosis from the peripheral circulation into
the brain. As such, other
systemically administered EPO analogs are also believed to be able to traverse
the blood brain
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barrier, as well as other barriers with capillaries expressing the EPO
receptor.
In sum, because the EPO analogs of the prior art have been shown to maintain
erythropoietic
activity at the sacrifice of ~.t least some of the functionality of endogenous
EPO, there exists a need in
the art for a long acting EPO that maintains all of the known functionality of
endogenous EPO.
Advantageously, the present invention is directed to a long acting EPO of the
present invention that
not only increases the serum half life as compared to recombinant EPO, but
also maintains the
functionality of endogenous EPO, i.e., the tissue protective functionality and
the transcytosis
capability. Various methods of modifying EPO to provide such a beneficial
protein are provided in
the next section.
Modification of Native EPO
The long acting EPOs of the present invention may be formed in a variety of
ways. In
general, the long acting EPOS may be generated by chemically modifying the
carbohydrate (sugar)
chains attached to the EPO. As used herein, the term "carbohydrate chains"
refer to the N-linked and
O-linlced oligosaccharide chains found in endogenous EPO, the additional N-
linked and O-linked
oligosaccharide chains found in EPO analogs, and any other carbohydrate
chains, specifically sugar
chains, attached to EPO.
In one embodiment, endogenous or recombinant EPO is used for modification so
as to prevent
any interference with the tissue protective capabilities of endogenous EPO. In
addition, EPO analogs
are contemplated for modification according to the present invention providing
the additional
glycosylation sites are not located near the O'Brien peptide, i.e., the 30-47
amino acid sequence. As
used herein, the term "EPO analogs" refers to modified EPO molecules that have
at least one
additional N-linked carbohydrate chain and/or at least one additional O-linked
carbohydrate chain. In
one embodiment, an EPO analog used for modification does not include any
additional glycosylation
sites within about 5 amino acids of the O'Brien peptide. In another
embodiment, the EPO analog
does not include any additional glycosylation sites within about 3 amino acids
of the O'Brien peptide.
In still another embodiment, the EPO analog does not include any additional
glycosylation sites
within the O'Brien peptide.
An EPO analog may also be used for modification according to the present
invention
provided that the analog is reviewed in three-dimensional space and it is
confirmed that none of the
additional carbohydrate chains do not block the O'Brien peptide or cause a
loss of tissue protective
functionality. In another aspect, an EPO analog is contemplated for use in
modification according to
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the present invention providing the method of glycosylation does not inhibit
the tissue protective
functionality of the peptide. In still another aspect, an EPO analog may be
used for modification
according to the present invention providing that there is one carbohydrate
chain (or less) in the
O'Brien peptide. For example, endogenous EPO contains a carbohydrate chain at
the 38 amino acid
and an additional carbohydrate chain within the O'Brien peptide has been shown
to inhibit the tissue
protective activity of the protein. Thus, an EPO analog having one
carbohydrate chain or no
carbohydrate chains in the O'Brien peptide is contemplated for use in
modification. In one
embodiment, the carbohydrate chain attached to the 38 amino acid may be
relocated to somewhere
else on the protein.
Nonlimiting examples of modifications according to the present invention
include (1)
'providing additional acid residues on the carbohydrate chains through
oxidation of vicinal hydroxyls;
(2) replacing the sialic acid residues with less labile residues; (3)
increasing the negative charge on
erythropoietin by sulfation and/or phosphorylation; and/or (4) terminate the
carbohydrate chains with
more complex molecules. Thus, the modifications to the carbohydrate chains of
EPO may include
oxidation, sulfation, phosphorylation, and/or PEGylation, among other
procedures, which will be
described in greater detail below and further illustrated in prophetic Example
1.
Oxidizing the Su~-a~ Chains and Replacing- tlae Sialic Acid Residues
A chemically modified long acting EPO of the present invention may include an
EPO in
which the carbohydrates (sugars) are oxidized to provide additional acid
residues. In another
embodiment, the sialic acid residues are replaced with less labile acid
residues. Modifications of this
type result in an increased half life of the molecule, as compared to
endogenous EPO, because the
galactose chains for which the liver screens for and removes the associated
protein from circulation
are protected from detection. A more substantial chemical modification to the
carbohydrate chains
on the EPO leads to a greater increase in the serum half life of the long
acting EPOs of the present
invention. For example, when more vicinal hydroxyls are replaced by acids, a
larger increase in the
serum half life results.
Although one of ordinary skill in the art would recognize several suitable
methods for
converting the galactose units of erythropoietin, one suitable method involves
(1) modifying the
sugar molecules with vicinal hydroxyls with periodate to form aldehydes; and
(2) oxidizing the
aldehydes to generate acids. Reagents suitable for oxidizing the sugar chain
to form aldehydes are
known to those skilled in the art and include, but are not limited to,
periodates, such as sodium
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periodate, and sugar oxidases, such as galactose oxidase. In addition, skilled
artisans would be aware
of suitable reagents for transforming the aldehydes, such as Quantitative
Benedict Solution
(commercially available from Fisher). In one embodiment, the sugar molecules
are oxidized with
sodium periodate and further treated with Quantitative Benedict Solution
(Fisher) to convert the
aldehydes into acids.
In another embodiment, an EPO isomer, one having about 0-13 sialic acid
residues, or an
EPO analog, having at least one carbohydrate chain that lacks a sialic acid
residue, is subjected to
oxidation using galactose oxidase. An asialo form of EPO may be used according
to this aspect of
the invention, i. e., an cx or ,Q form of EPO with the terminal carbohydrate
(sialic acid) removed.
Preferably, asialoerythropoietin is used. Once oxidized, the EPO is subjected
to another oxidative
agent, such as Quantitative Benedict Solution, to transform the aldehydes into
acids.
In yet another embodiment, a ruthenium tetroxide system may be used to
generate the acids
on the sugar chain. Given that these modifications involve the galactose chain
even if the acids
involved in these transformations are stripped from the EPO molecule, the
molecule should be able to
evade removal by the liver since a galactose chain, the component that the
liver screens for, will not
be exposed.
Increasin,~ the Negative ChaY,~e
In another aspect of the present invention, a long acting EPO of the present
invention is
formed by adding sulfates and/or phosphates to the EPO molecule, which will
increase the negative
charge of the molecule and thereby increase the half life of the molecule. In
other words, the
negative charge of the EPO molecule may be increased by sulfation, which
involves the transfer of a
sulfuryl group from a sulfate donor, including protein, glycolipids,
glycosaminoglycans and steroids.
And, the negative charge may also be increased by introducing a phosphoric
group into a
carbohydrate.
One suitable method for sulfation of insulin is discussed in S. Pongor et al.,
Preparation of
High-Potency, Non-aggregating Insulins Using a Novel Sulfation Procedure,
Diabetes, Vol. 32, No.
12, December 1983. For example, insulin sulfation was carried out in an
organic solvent, such as
dimethylformamide (DMF), in the presence of condensing agents, such as N,N'-
dicyclohexyl
carbodiimide (DCC), and a sulfate donor. The degree of sulfation can be
controlled over an eightfold
range by varying the amount of condensing agent. Although conventionally
prepared sulfated insulin
resulted in major insulin bioactivity loss, the bioactivity of the sulfated
insulin made with the Ponger
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process varied between 78 percent and 87 percent of unmodified insulin.
By using a similar procedure for EPO, one of ordinary skill in the art may
control the amount
of sulfation and therefore the serum half life of the chemically modified EPO.
For instance, the
negative charge of EPO may be increased by adding sulfates to the protein by
dissolving EPO or an
EPO analog in at least one water soluble carbodiimide, preferably DCC, at a
temperature of about
4°C. While DCC is preferred as the sulfate donor, those of ordinary
skill in the art would readily
recognize other suitable sulfate donors for use with the present invention.
Similar procedures can be used to control the phosphorylation of the EPO using
phosphoric
acid (H3P04) as the phosphate donor. Again, while phosphoric acid is
preferred, skilled artisans
would be able to readily select other phosphate donors to effect
phosphorylation of EPO.
Terrninatin~- the Cay-bohydr~ate Claains with PEGS
The carbohydrate chains of EPO may also be modified by the addition of at
least one
polyethylene glycol (PEG), a compound with a long and safe clinical history,
which has the following
general formula:
H H H H
HO C-C-~ C -C- ~H
H H n H H
The PEG may also be a methoxy PEG (mPEG) having the following general formula:
H H H H
GHsO G-C-O C -C- OH
,H H n H H
In one embodiment, the PEG is an a.~nino PEG, preferably a methoxy PEG with
primary
amino groups at the termini (mPEG-NHZ). Polyethylene glycol chains with
primary amino groups at
the termini are very useful functionalized polymers. The amino end groups on
mPEG-NHZ are more
reactive toward acylating agents than the hydroxyl groups that are present on
conventional PEGS and
they also readily undergo reductive amination reactions. In another
embodiment, the PEG is an
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electrophilically activated PEG, such as mPEG-succinimidyl propionate (mPEG-
SPA) or mPEG-
succinimidyl butanoate (mPEG-SBA), both of which are commercially available
from Nektar
Therapeutics of Birmingham, Alabama. In yet another embodiment, the PEG is a
methoxy PEG-
hydrazide.
W one embodiment, the addition of the at least one PEG is achieved via
oxidation with
periodate (as disclosed above), followed by the use of cyanoborohydride and an
amino PEG. For
example, EPO in solution may be first oxidized with a periodate, e.g., sodium
periodate, for a
predetermined period of time at room temperature, which produces aldehydes in
the carbohydrate
chains. A suitable periodate is sodium meta-periodate, which is commercially
available from Sigma.
The periodate may then be removed by buffer exchange, at which time the
oxidized sialic acid groups
on N-linked oligosaccharide groups of EPO may be subj ected to at least one
amino PEG in the
presence of cyanoborohydride. Suitable PEGs for use include, but are not
limited to, methoxy-PEG-
hydrazides, which are commercially available from Nektar Therapeutics.
In another embodiment, the addition of the at least one PEG is performed by
the attachment of
PEG groups to terminal galactose residues after oxidation with galactose
oxidase. For example, an
asialo form of EPO (having exposed terminal galactose residues) in buffer is
first subjected to
galactose oxidase (commercially available from Sigma) to generate aldehydes in
the carbohydrate
chains. The buffer may then be removed by buffer exchange, at which time the
oxidized galactose
residues may be subjected to at least one amino PEG in the presence of
cyanoborohydride.
The methods provided above are not intended to be limiting as these or other
methods may be
used to prepare the compounds of the invention. For example, a skilled artisan
would recognize the
applicability of these chemical modifications to creating long acting versions
of other EPO
derivatives such as the tissue protective cytokines disclosed in International
Publication No.
W0102053580 and U.S. Patent Publication Nos. 2002/0086816 and 2003/0072737,
which axe
incorporated by reference herein in their entirety.
PYOduction o~tlae EPO Molecules
A variety of host-expression vector systems may be utilized to produce the
long acting EPO
and EPO-related molecules of the invention. Such host-expression systems
represent vehicles by
which the long acting EPOS of interest may be produced and subsequently
purified, but also represent
cells that may, when transformed or transfected with the appropriate
nucleotide coding sequences,
exhibit the modified erythropoietin gene product in situ. These include but
are not limited to,
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bacteria, insect, plant, mammalian, including human host systems, such as, but
not limited to, insect
cell systems infected with recombinant virus expression vectors (e.g.,
baculovirus) containing the
long acting EPO product coding sequences; plant cell systems infected with
recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti plasmid)
containing
erythropoietin-related molecule coding sequences; or mammalian cell systems,
including human cell
systems, e.g., HT1080, COS, CHO, BHK, 293, 3T3, harboring recombinant
expression constructs
containing promoters derived from the genome of mammalian cells, e.g.,
metallothionein promoter,
or from mammalian viruses, e.g., the adenovirus late promoter; the vaccinia
virus 7.SK promoter.
In addition, a host cell strain may be chosen that modulates the expression of
the inserted
sequences, or modifies and processes the gene product in the specific fashion
desired. Such
modifications and processing of protein products may be important for the
function of the protein.
As known to those of ordinary skill in the art, different host cells have
specific mechanisms for the
post-translational processing and modification of proteins and gene products.
Appropriate cell lines
or host systems can be chosen to ensure the correct modification and
processing of the foreign protein
expressed. To this end, eukaryotic host cells that possess the cellular
machinery for proper
processing of the primary transcript, glycosylation, and phosphorylation of
the gene product may be
used. Such mammalian host cells, including human host cells, include but are
not limited to HT1080,
CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.
For long-term, high-yield production of recombinant proteins, stable
expression is preferred.
For example, cell lines that stably express the recombinant tissue protective
cytokine-related
molecule gene product may be engineered. Rather than using expression vectors
that contain viral
origins of replication, host cells can be transformed with DNA controlled by
appropriate expression
control elements, e.g., promoter, enhancer, sequences, transcription
terminators, polyadenylation
sites, and the like, and a selectable marker. Following the introduction of
the foreign DNA,
engineered cells may be allowed to grow for 1-2 days in an enriched media, and
then are switched to
a selective media. The selectable marker in the recombinant plasmid confers
resistance to the
selection and allows cells to stably integrate the plasmid into their
chromosomes and grow to form
foci that in turn can be cloned and expanded into cell lines. This method may
advantageously be
used to engineer cell lines that express the EPO mutein-related molecule gene
product. Such
engineered cell lines may be particularly useful in screening and evaluation
of compounds that affect
the endogenous activity of the EPO-related molecule gene product.
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Alternatively, the expression characteristic of an endogenous EPO mutein gene
within a cell
line or microorganism may be modified by inserting a heterologous DNA
regulatory element into the
genome of a stable cell line or cloned microorganism such that the inserted
regulatory element is
operatively linked with the endogenous erythropoietin mutein gene. For
example, an endogenous
EPO mutein gene that is normally "transcriptionally silent", i.e., an EPO gene
that is normally not
expressed, or is expressed only at very low levels in a cell line, may be
activated by inserting a
regulatory element that is capable of promoting the expression of an expressed
gene product in that
cell line or microorganism. Alternatively, a transcriptionally silent,
endogenous EPO gene may be
activated by insertion of a promiscuous regulatory element that works across
cell types.
A heterologous regulatory element may be inserted into a stable cell line or
cloned
microorganism, such it is operatively linked with an endogenous erythropoietin
gene, using
techniques, such as targeted homologous recombination, which are well known to
those of skill in the
art, and also described French Patent No. 2646438, U.S. Patent Nos. 4,215,051
and 5,578,461, and
International Publication Nos. W093/09222 and W091/06667, the entire
disclosures of which are
incorporated by reference herein.
Pharmaceutical Compositions
The present invention also relates to pharmaceutical compositions including
the long acting
EPO molecules of the present invention. Because the long acting EPOs of the
present invention
advantageously have erythropoietic activity, as well as tissue protective
capability and transcytosis
capability, they are contemplated for treatment of anemia and related diseases
in individuals also at
risk for various tissue injuries, such as stroke and myocardial infarction. In
addition, the long acting
EPOS of the present invention are contemplated for treatment of anemia and
related diseases in
individuals also experiencing deteroriation of mental faculties, such as
Alzheimer's, Parkinson's and
the like. Furthermore, the long acting EPOS of the present invention are
contemplated for the
treatment of anemia in individuals subject to conditions resulting from the
normal aging process, e.g.,
balance problems leading to falling, easy bruising, and the like. Moreover,
the present invention
relates to the use of the long acting EPOS of the present invention as
carriers for other molecules that
have poor penetration across barriers with capillaries having EPO receptors.
For example, any of the long acting EPOs discussed above may be included in
pharmaceutical
compositions of the invention. In addition, various EPO analogs may be
included in pharmaceutical
compositions of the invention in a blend with at least one tissue protective
cytokine, which will be
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discussed in greater detail below.
The pharmaceutical compositions of the invention contain a therapeutically
effective amount
of the modified EPO, preferably in purified form. The formulation should suit
the mode of
administration. In other words, the pharmaceutical compositions of the
invention include an amount
of the modified EPO of the invention such that the targeted condition is
treatable provided the proper
dose and strategy is employed. And, as discussed in more detail below, the
pharmaceutical
composition should be delivered in a non-toxic dosage amount.
The pharmaceutical compositions of the invention may include a therapeutically
effective
amount of the long acting EPO compound and a suitable amount of a
pharmaceutically acceptable
carrier so as to provide the form for proper administration to the patient. In
a specific embodiment,
the term "pharmaceutically acceptable" means approved by a regulatory agency
of the Federal or a
state government or listed in the U.S. Pharmacopeia or other generally
recognized foreign
pharmacopeia for use in animals, and more particularly in humans. The term
"carner" refers to a
diluent, adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such
pharmaceutical carriers can be sterile liquids, such as saline solutions in
water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as peanut oil,
soybean oil, mineral oil,
sesame oil and the like. A saline solution is a preferred Garner when the
pharmaceutical composition
is administered intravenously. Saline solutions and aqueous dextrose and
glycerol solutions can also
be employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim milk,
glycerol, propylene, glycol,
water, ethanol and the like.
The pharmaceutical compositions of the invention may also contain minor
amounts of wetting
or emulsifying agents, or pH buffering agents. These compositions can take the
form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders, sustained-release
formulations and the like.
The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically
acceptable salts include those formed with free amino groups such as those
derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those
formed with free carboxyl
groups such as those derived from sodium, potassium, ammonium, calcium, fernc
hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
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Corraposition Iracludin,~ Lori Actira E~PO
As briefly mentioned above, any of the long acting EPOs of the present
invention are
contemplated for use in pharmaceutical compositions. In one embodiment, a long
acting EPO
produced from oxidation of vicinal hydroxyls is included in the pharmaceutical
composition of the
invention. In another embodiment, the pharmaceutical composition of the
invention includes at least
one long acting EPO that is a result of replacing the sialic acid residues
with less labile residues. In
yet another embodiment, the long acting EPO included in the pharmaceutical
composition is a result
of increasing the negative charge on EPO by sulfation and/or phosphorylation.
In still another
embodiment, a long acting EPO produced by terminating the carbohydrate chains
with more complex
molecules, e.g., PEG chains, is included in the pharmaceutical compositions of
the invention.
In addition, the present invention contemplates the use of a mixture of long
acting EPOs
produced by any of the methods of the present invention in the pharmaceutical
compositions of the
invention. For example, the pharmaceutical composition of the invention may
include at least one
long acting EPO that is a result of replacing the sialic acid residues with
less labile residues and at
least one long acting EPO that is the result of increasing the negative charge
on EPO by sulfation
and/or phosphorylation.
Transport System
As discussed earlier, the long acting EPOS of the present invention
advantageously are able to
traverse barriers with capillaries having EPO receptors. Thus, another aspect
of the present invention
is a transport system using the long acting EPOS of the present invention as
carriers for molecules
with poor barrier penetration into a targeted area of the body having EPO
receptors. Such transport
systems advantageously provide a novel and safe method of delivery across the
intact barriers.
In one embodiment, the transport system includes the long acting EPOS of the
present
invention and at least one molecule with poor brain penetration to provide a
novel and safe method of
delivery across the intact blood brain barner. In other words, the long acting
EPOS of the present
invention may allow molecules with poor brain penetration to act as molecular
"troj an horses" so as
to enhance brain uptake of either small or large molecule diagnostics or
therapeutic molecules.
In fact, an important problem in the treatment of human brain tumors is posed
by the need to
deliver therapeutic agents to specific regions of the brain, distributing them
within and targeting them
to brain tumors. The molecules that might otherwise be effective in diagnosis
and therapy either do
not cross the blood-brain barrier (BBB) in the brain adjacent to the tumor or
do not cross the blood-
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tumor barrier (BTB) in adequate amounts. Thus, there is a need for novel
delivery strategies that are
unique to the brain and that bypass the vasculature. For example, antibodies
that could be used as
either diagnostic or therapeutic molecules do not cross the BTB in sufficient
quantities to be effective
because of their size. As such, the long acting EPOS of the present invention
may be used as a carrier
for such molecules to allow traversal of the BBB or BTB. One example of a
molecule that may be
used with the long acting EPOS of the present invention is an anti-sense
oligonucleotide, which is
typically used either to inhibit oncogenic signals or to image gene expression
of the brain ifz vivo. In
addition, the long acting EPOS of the present invention may be included in
various gene therapies
(viral or nonviral formulations), which are often too large to cross the BTB
without aid.
Furthermore, the long acting EPOS of the present invention may be used as
carrier-mediated
transporter for various chemotherapeutic agents. Because drug-active efflux
transporters, which are
expressed at the BBB and the BTB, actively efflux chemotherapeutic agents from
the brain back to
the blood, the distribution of these agents in the brain may be inhibited or
prevented. It is partly for
these reasons that most of the classical chemotherapeutic molecules that have
been used to treat
1 S cancer outside the central nervous system (CNS) are ineffective in the
treatment of brain tumors.
Thus, the use of a long acting EPO of the present invention as a carrier for
such chemotherapeutic
agents may be useful not only in carrying the agents into the brain, but also
keeping the agents within
the brain for therapy. In another embodiment, the long acting EPO may be
joined with a drug that
inhibits the active efflux transporter to further ensure the uptake of
chemotherapeutic agents that are
normally effluxed from brain to blood.
In addition, the present invention also contemplates the use of modified EPO
as carriers for
molecules with poor penetration in other areas of the body having EPO
receptors. Non-limiting
examples of such cells include retinal, muscle, heart, lung, liver, kidney,
small intestine, adrenal
cortex, adrenal medulla, capillary endothelial, testes, ovary, pancreas, bone,
skin, and endometrial
cells. In particular, responsive cells include, without limitation, neuronal
cells; retinal cells:
photoreceptor (rods and cones), ganglion, bipolar, horizontal, amacrine, and
Mueller cells; muscle
cells; heart cells: myocardium, pace maker, sinoatrial node, sinoatrial node,
sinus node, and junction
tissue cells (atrioventricular node and bundle of his); lung cells; liver
cells: hepatocytes, stellate, and
I~upffer cells; kidney cells: mesangial, renal epithelial, and tubular
interstitial cells; small intestine
cells: goblet, intestinal gland (crypts) and enteral endocrine cells; adrenal
cortex cells: glomerulosa,
fasciculate, and reticularis cells; adrenal medulla cells: chromaffm cells;
capillary cells: pericyte
cells; testes cells: Leydig, Sertoli, and sperm cells and their precursors;
ovary cells: Graffian follicle
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and primordial follicle cells; pancreas cells: islets of Langerhans, a-cells,
,~-cells, 'y cells, and F-cells;
bone cells: osteoprogenitors, osteoclasts, and osteoblasts; skin cells;
endometrial cells: endometrial
stroma and endometrial cells; as well as the stem and endothelial cells
present in the above listed
organs.
Cof3a~osition Blend of EPO Analog and Tissue Protective C ty okine
As briefly mentioned above, a pharmaceutical composition according to the
present invention
may include an EPO analog having at least one additional N-linked carbohydrate
chain and/or at least
one additional O-linked carbohydrate chain (that exhibits an extended serum
half life but lacks tissue
protective activity) in a blend with at least one tissue protective cytokine.
For example, an EPO
analog having at least two additional N-linked carbohydrate chains, wherein
one of the additional
carbohydrate chains is located in the O'Brien peptide, in combination with a
tissue protective
cytokine, may form a composition of the invention. In another embodiment, the
pharmaceutical
composition of the invention may include at least one tissue protective
cytokine and at least one EPO
analog that contains additional carbohydrate chains that are known, from
reviewing the analog in
three-dimensional space, to block the O'Brien peptide. In yet another
embodiment, a pharmaceutical
composition of the invention includes at least one tissue protective cytokine
and at least one EPO
analog having no tissue protective functionality as a result of the method of
adding the extra
carbohydrate chains to the protein.
An EPO analog with a relocated glycoylation site is contemplated for use in
the
pharmaceutical compositions of the present invention. Without being bound to
any particular theory,
it is believed that if the naturally occurring glycosylation site at amino
acid 38 was relocated
elsewhere on an EPO analog, outside of the 30-47 amino acid segment, the
tissue protective
capabilities of the EPO analog would be enhanced as compared to an EPO analog
with the
glycosylation site at amino acid 38. Thus, the pharmaceutical composition of
the invention may
include an EPO analog with a relocated glycosylation site, from the 38 amino
acid, to elsewhere on
the molecule. The relocated glycosylation site may occur at amino acids 51,
57, 69, 88, 89, 136 or
138, as suggested in PCT Publication No. WO 01/81405. In one embodiment, the
O'Brien peptide
contains 1 or less carbohydrate chains. In an alternative embodiment, the
O'Brien peptide includes 2
or more carbohydrate chains.
Suitable tissue protective cytokines for use with this aspect of the present
invention are
preferably those cytokines that lack an effect on the bone marrow but maintain
the tissue protective
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effect of endogenous, however any cytokine that exhibits tissue protective
capability is contemplated
for use with the present invention. For example, suitable tissue protective
cytokines include
chemically modified EPOS generated by guanidination, amidination,
caxbamylation (carbamoylation),
trinitrophenylation, acylation (acetylation or succinylation), nitration, or
mixtures thereof. In
addition, EPO molecules with a modification of at least one axginine, lysine,
tyrosine, tryptophan, or
cysteine residue or carboxyl groups are also contemplated for use as tissue
protective cytokines
according to this aspect of the present invention.
Moreover, additional tissue protective cytokines for use with the present
invention may be
obtained by limited proteolysis, removal of amino groups, and/or mutational
substitution of arginine,
lysine, tyrosine, tryptophan, or cysteine residues by molecular biological
techniques as disclosed in
Satake et al, 1990, Biochim. Biophys. Acta 1038:125-9, which is incorporated
by reference herein in
its entirety. For example, suitable tissue protective cytokines include at
least one or more mutated
EPOS having a site mutation at C7S, R10I, V11S, L12A, E13A, R14A, R14B, R14E,
R14Q, Y15A,
Y15F, YlSI, K20A, K20E, E21A, C29S, C29Y, C33S, C33Y, P42N, T44I, K45A, K45D,
V46A,
N47A, F48A, F48I, Y49A, Y49S, W51F, W51N, Q59N, E62T, L67S, L70A, D96R, S100R,
S100E,
S100A, S100T, G101A, G101I, L102A, R103A, S104A, S104I, L105A, T106A, T106I,
T107A,
T107L, L108K, L108A, S126A, F142I, R143A, S146A, N147K, N147A, F148Y, L149A,
R150A,
G151A, K152A, L153A, L155A, C160S, I6A, C7A, B13A, N24K, A30N, H32T, N38K,
N83K,
P42A, D43A, K52A, K97A, K116A, T132A, I133A, T134A, K140A, P148A, R150B,
G151A,
K152W, K154A, G158A, C161A, and/or R162A. Examples of the above-referenced
modifications
are described in co-pending U.S. Patent Publication Nos. 2003/0104988,
2002/0086816 and
2003/0072737, which are incorporated by reference herein in their entirety. In
the mutein
nomeclature used herein, the changed amino acid is depicted with the native
amino acid's one letter
code first, followed by its position in the EPO molecule, followed by the
replacement amino acid one
letter code. For example, S 100E refers to a human EPO molecule in which, at
amino acid 100, the
serine has been changed to a glutamic acid.
In another embodiment, the tissue protective cytokine may include one or more
of the above
site mutations, providing that the site mutations do not include I6A, C7A,
K20A, P42A, D43A,
K45D, K45A, F48A, Y49A, K52A, K49A, S100B, R103A, K116A, T132A, I133A, K140A,
N147K,
N147A, R150A, R150E, G151A, K152A, K154A, G158A, C161A, or R162A.
In still another embodiment, the tissue protective cytokines may include
combinations of site
mutations, such as K45D/S100E, A30N/H32T, K45D/R150E, R103E/L108S, K140A/K52A,
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K140A/K52A/K45A, K97A/K152A, K97A/K152A/K45A, K97A/K152A/K45A/K52A,
K97A/K152A/K45A/K52A/K140A, K97A/K152A/K45A/K52A/K140A/K154A,
N24K/N38K/N83K, and N24K/Y15A. In yet another embodiment, the tissue
protective cytokines do
not include any of the above combinations. In another embodiment, the tissue
protective cytokines
may include any of the above-referenced site mutations providing that the site
mutations do not
include any of the following combinations of substitutions: N24K/N38K/N83K
and/or A30N/H32T.
Certain modifications or combinations of modifications may affect the
flexibility of the
mutein's ability to bind with its receptor, such as an EPO receptor or
secondary receptor. Examples
of such modifications or combinations of modifications include, but are not
limited to, K152W,
R14A/YlSA, I6A, C7A, D43A, P42A, F48A, Y49A, T132A, I133A, T134A, N147A,
P148A,
R150A, G151A, G158A, C161A, and R162A. Corresponding mutations are known to
those of
ordinary skill in the art to be detrimental in human growth hormone. Thus, in
one embodiment, the
tissue protective cytokine does not include one or more of the modifications
or combinations of
modifications that may affect the flexibility of the mutein's ability to bind
with its receptor. Further
discussion of such tissue protective cytokines is included in co-pending U.S.
Patent Application No.
attorney docket no. 10165-022-999, filed July l, 2003, entitled "Recombinant
Tissue Protective
Cytokines and Encoding Nucleic Acids Thereof for Protection, Restoration, and
Enhancement of
Responsive Cells, Tissues, and Organs," the entire disclosure of which is
incorporated by reference
herein
Finally, any of the superfamily cytokines that exhibit tissue protective
capabilities may be
used as well so long as they do not interfere with the long acting EPO's
erythropoietic effects or
serum half life. Examples include, but are not limited to, interleukin-3 (IL-
3), interleukin-5 (IL-5),
granulocyte-macrophage colony-stimulating factor (GMCSF), pigment-epithelium
derived factor
(PEDF), and vascular endothelial growth factor (VEGF).
In another aspect of the present invention, a pharmaceutical composition
according to the
present invention may include an EPO analog having at least one additional N-
linked carbohydrate
chain and/or at least one additional O-linked carbohydrate chain (that
exhibits an extended serum
half life but lacks tissue protective activity) in a blend with at least one
small molecule that exhibits
tissue protective functionality. Suitable small molecules include, but are not
limited to, steroids (e.g.,
lazaroids and glucocorticoids), antioxidants (e.g., coenzyme Qlo, alpha lipoic
acid, and NADH),
anticatabolic enzymes (e.g., glutathione peroxidase superoxide dimutase,
catalase, synthetic catalytic
scavengers, as well as mimetics), indole derivatives (e.g., indoleamines,
carbazoles, and carbolines),
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nitric acid neutralizing agents, adenosine / adenosine agonists,
phytochemicals (flavanoids), herbal
extracts (ginko biloba and turmenic), vitamins (vitamins A, E, and C), oxidase
electron acceptor
inhibitors (e.g., xanthene oxidase electron inhibitors), minerals (e.g.,
copper, zinc, and magnesium),
NSAmS (e.g., aspirin, naproxen, and ibuprofen), and combinations thereof. In
addition, a
pharmaceutical composition of the invention may include an EPO analog, a
tissue proetctive
cytokine, and a small molecule with tissue protective activity.
The tissue protective cytokines and/or small molecules are preferably present
in the
pharmaceutical compositions of the invention in an amount sufficient to
maintain or exceed the same
activity in neural or other responsive cellular systems as elicited by
endogenous EPO. In one
embodiment, the tissue protective cytokine andlor small molecule is present in
an amount sufficient
to enhance the tissue protection of the individual by protecting, maintaining,
or enhancing the
viability and function of erythropoietic responsive cells within the
individual. For example, the
pharmaceutical composition of this aspect of the present invention preferably
includes an effective,
non-toxic amount of the tissue protective cytokine, e.g., about 1 ng or
greater. In one embodiment,
the tissue protective cytokine is present in the pharmaceutial composition in
an amount of about 5 mg
or less. In another embodiment, the tissue protective cytokine is present in
the pharmaceutical
composition in an amount of about 500 ng to 5 mg. hl still another embodiment,
the pharmaceutical
composition includes about 1 ~,g to 5 mg of the tissue protective cytokine,
preferably about 500 ~,g to
Smg. In an alternate embodiment, a larger amount of the tissue protective
cytokine is present in the
pharmaceutical composition of the invention, e.g., about 1 mg to 5 mg. As
known to those of
ordinary skill in the art, the amount of pharmaceutical composition
administered to a patient depends
on a number of factors including, but not limited to, the condition of the
patient and the dosing
frequency. This will be discussed in greater detail below with regard to
dosing.
Treatment and Administration Methods
The aforementioned long acting EPOs and pharmaceutical compositions including
the long
acting EPOs are intended for the therapeutic or prophylactic treatment of
anemia, human diseases that
either involve anemia or anemic conditions, or diseases or methods of
treatment that result in anemia.
hz general, the long acting EPOs of the present invention permit less frequent
dosing or the use of
smaller doses of erythropoietin to treat the above diseases without
jeopardizing the patients ability to
recover from other tissue injuries.
The present invention contemplates the use of the long acting EPOS for
systematic or chronic
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administration, acute treatment, and/or intermittent administration. In one
embodiment, the
pharmaceutical compositions of the invention are administered chronically to
protect or enhance the
target cells, tissue or organ. In another embodiment, the pharmaceutical
compositions of the
invention may be administered acutely, i.e., for a single treatment during
injury. In yet another
embodiment, the pharmaceutical compositions of the invention are administered
in a cyclic nature.
The administration of the composition may be parenteral, e.g., via intravenous
injection,
intraperitoneal injection, intra-arterial, intramuscular, intradermal, or
subcutaneous administration;
via inhalation; transmucosal, e.g., oral, nasal, rectal, intravaginal,
sublingual, submucosal, and
transdermal; or combinations thereof. Preferably, the administration of the
pharmaceutical
composition of the invention is parenteral. Such adminstration may be
performed in a dose amount
of about 0.01 pg to about 5 mg, preferably about 1 pg to about 5 mg. In one
embodiment, the dose
amount is about 500 pg to about 5 mg. In another embodiment, the dose amount
is about 1 ng to
about 5 mg. In yet another embodiment, the dose amount is about 500 ng to
about 5 mg. In still
another embodiment, the dose amount is about 1 ~,g to about 5 mg. For example,
the dose amount
may be about 500 ~,g to about 5 mg. In another embodiment, the dose amount may
be about 1 mg to
about 5 mg.
Pharmaceutical compositions of the invention adapted for parenteral
administration include
aqueous and non-aqueous sterile injectable solutions or suspensions, which may
contain antioxidants,
buffers, bacteriostats and solutes that render the compositions substantially
isotonic with the blood of
an intended recipient. In this aspect of the invention, the pharmaceutical
compositions may also
include water, alcohols, polyols, glycerine, vegetable oils, and mixtures
thereof. Pharmaceutical
compositions adapted for parenteral administration may be presented in unit-
dose or multi-dose
containers, for example sealed ampules and vials, and may be stored in a
lyophilized (freeze-dried)
condition requiring only the addition of a sterile liquid carrier, e.g.,
sterile saline solution for
injections, immediately prior to use. Extemporaneous injection solutions and
suspensions may be
prepared from sterile powders, granules and tablets. -In one embodiment, an
autoinjector comprising
an injectable solution of a long acting EPO of the invention may be provided
for emergency use by
ambulances, emergency rooms, and battlefield situations.
In one embodiment, the pharmaceutical composition of the invention is
formulated in
accordance with routine procedures as a pharmaceutical composition adapted for
intravenous
administration to human beings. For example, the pharmaceutical composition
may be in the form of
a solution in sterile isotonic aqueous buffer. Where necessary, the
pharmaceutical composition may
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also include a solubilizing agent and/or a local anesthetic such as lidocaine
to ease pain at the site of
the injection. The ingredients may be supplied either separately or mixed
together in unit dosage
form, for example, as a dry lyophilized powder or water-free concentrate in a
hermetically-sealed
container such as an ampule or sachette indicating the quantity of active
agent. When the
pharmaceutical compositions of the invention are to be administered by
infusion, an infusion bottle
with sterile pharmaceutical grade water or saline may be used for dispensing
the composition. And,
when the pharmaceutical composition are to be administered by injection, an
ampule of sterile saline
may be provided to mix the ingredients may be mixed prior to administration.
Pharmaceutical compositions adapted for oral administration may be provided as
capsules or
tablets; powders or granules; solutions, syrups or suspensions (in aqueous or
non-aqueous liquids);
edible foams or whips; emulsions; or combinations thereof. The oral
formulation may include about
10 percent to about 95 percent by weight active ingredient. In one embodiment,
the active ingredient
is included in the oral formulation in an amount of about 20 percent to about
80 percent by weight.
In still another embodiment, the oral formulation includes about 25 percent to
about 75 percent by
weight of the active ingredient.
Tablets or hard gelatine capsules may include lactose, starch or derivatives
thereof,
magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic
acid or salts thereof.
Soft gelatine capsules may include vegetable oils, waxes, fats, semi-solid,
liquid polyols, or mixtures
thereof. Solutions and syrups may include water, polyols, sugars, or mixtures
thereof.
Moreover, an active agent intended for oral administration may be coated with
or admixed
with a material that delays disintegration and/or absorption of the active
agent in the gastrointestinal
tract. For example, the active agent may admixed or coated with glyceryl
monostearate, glyceryl
distearate, or a combination thereof. Thus, the sustained release of an active
agent may be achieved
over many hours and, if necessary, the active agent can be protected from
being degraded within the
stomach. Pharmaceutical compositions for oral administration may also be
formulated to facilitate
release of an active agent at a particular gastrointestinal location due to
specific pH or enzymatic
conditions.
Pharmaceutical compositions adapted for transdermal administration may be
provided as
discrete patches intended to remain in intimate contact with the epidermis of
the recipient for a
prolonged period of time. In addition, pharmaceutical compositions adapted for
topical
administration may be provided as ointments, creams, suspensions, lotions,
powders, solutions,
pastes, gels, sprays, aerosols, oils, eye drops, lozenges, pastilles, and
mouthwashes and combinations
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thereof. When the topical administration is intended for the skin, mouth, eye,
or other external
tissues, a topical ointment or cream is preferably used. And, when formulated
in an ointment, the
active ingredient, i.e., the long acting EPO, may be employed with either a
paraffinic or a water-
miscible ointment base. Alternatively, the active ingredient may be formulated
in a cream with an
oil-in-water base or a water-in-oil base. When the topical administration is
in the form of eye drops,
the pharmaceutical compositions of the invention preferably include the active
ingredient, which is
dissolved or suspended in a suitable Garner, e.g., in an aqueous solvent.
Pharmaceutical compositions adapted for nasal and pulmonary administration may
include
solid Garners such as powders (preferably having a particle size of about 20
microns to about 500
microns). Powders may be administered by rapid inhalation through the nose
from a container of
powder held close to the nose. In an alternate embodiment, pharmaceutical
compositions intended
for nasal administration according to the present invention may include liquid
carriers, e.g., nasal
sprays or nasal drops. Preferably, the pharmaceutical compositions of the
invention are administered
into the naval cavity directly.
Direct lung inhalation may be accomplished by deep inhalation through a
mouthpiece into the
oropharynx and other specially adapted devices including, but not limited to,
pressurized aerosols,
nebulizers or insufflators, which can be constructed so as to provide
predetermined dosages of the
active ingredient. Pharmaceutical compositions intended for lung inhalation
may include aqueous or
oil solutions of the active ingredient. Preferably, the pharmaceutical
compositions of the invention
are administered via deep inhalation directly into the oropharynx.
Pharmaceutical compositions adapted for rectal administration may be provided
as
suppositories or enemas. In one embodiment, the suppositories of the invention
includes about 0.5
percent to 10 percent by weight of active ingredient. In another embodiment,
the suppository
includes about 1 percent to about 8 percent by weight active ingredient. In
still another embodiment,
the active ingredient is present in the suppository in an amount of about 2
percent to about 6 percent
by weight. In this aspect of the invention, the pharmaceutical compositions of
the invention may
include traditional binders and carrier, such as triglycerides.
Pharmaceutical compositions adapted for vaginal administration may be provided
as
pessaries, tampons, creams, gels, pastes, foams or spray formulations.
The pharmaceutical compositions of the invention may also be administered by
use of a
perfusate, injection into an organ, or locally administered. In such
embodiments, the pharmaceutical
composition preferably has about 0.01 pM to about 30 pM, preferably about 15
pM to about 30 nM,
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of the long acting EPO of the present invention. In one embodiment, the
perfusion solution is the
University of Wisconsin (IJW) solution (with a pH of about 7.4 to about 7.5
and an osmolality of
about 320 mOSm/1), which contains about 1 U/ml to about 25 U/ml EPO; 5 percent
hydroxyethyl
starch (preferably having a molecular weight from about 200,000 to about
300,000 and substantially
free of ethylene glycol, ethylene chlorohydrin, sodium chloride, and acetone),
25 mM KHZP04, 3
mM glutathione; 5 mM adenosine; 10 mM glucose; 10 mM HEPES buffer; 5 mM
magnesium
gluconate; l.SmM CaCl2; 105 mM sodium gluconate; 200,000 units penicillin; 40
units insulin; 16
mg dexamethasone; and 12 mg phenol red. The UW solution is discussed in detail
in U.S. Patent No.
4,798,824, which is incorporated in its entirety by reference herein. In
another embodiment, the UW
solution may contain about 0.01 pg/ml to about 400 ng/ml, preferably about 40
ng/ml to about 300
ng/ml, of recombinant tissue protective cytokine.
It may be desirable to administer the pharmaceutical compositions of the
invention locally to
the area in need of treatment. Such administration may be achieved by local
infusion during surgery;
topical application, e.g., in conjunction with a wound dressing after surgery;
by injection; by means
of a catheter; by means of a suppository; or by means of an implant, said
implant being of a porous,
non-porous, or gelatinous material, including membranes, such as silastic
membranes, or fibers.
In addition, as briefly discussed above with respect to transdermal
administration, a long
acting EPO of the present invention may be delivered in a controlled-release
system. For example,
the polypeptide may be administered using intravenous infusion, an implantable
osmotic pump, a
transdermal patch, liposomes, or other modes of administration. In one
embodiment, a pump may be
used, such as discussed in Saudek et al., 1989, N. Engl. J. Med. 321:574. In
another embodiment,
the compound can be delivered in a vesicle, in particular a liposome, such as
described in
International Publication No. WO 91/04014 and U.S. Patent No. 4,704,355, the
entire disclosures of
which are incorporated by reference herein. W another embodiment, polymeric
materials may be
used to produce a controlled-release system, such as those materials discussed
in Howard et al., 1989,
Neurosurg. 71:105.
Such controlled release systems may be placed in proximity of the therapeutic
target, i. e., the
target cells, tissue or organ, thus requiring only a fraction of the systemic
dose. See, e.g., Goodson,
Medical Applications of Controlled Release, vol. 2, pp. 115-138, 1984. Other
controlled release
systems contemplated for use with the present invention are discussed in the
review by Langer,
Science 249:1527-1533, 1990.
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Dosin
Selection of the preferred effective and non-toxic dose for the administration
methods above
will be determined by a skilled artisan based upon factors known to one of
ordinary skill in the art.
Examples of these factors include the particular form of long acting EPO; the
pharmacokinetic
parameters of the EPO, such as bioavailability, metabolism, half life, etc.
(provided to the skilled
artisan); the condition or disease to be treated; the benefit to be achieved
in a normal individual; the
body mass of the patient; the method of administration; the frequency of
administration, i.e., chronic,
acute, intermittent; concomitant medications; and other factors well known to
affect the efficacy of
administered pharmaceutical agents. Thus the precise dosage should be decided
according to the
judgment of the practitioner and the circumstances of the particular patient.
For example, the Physicians Desk Reference (PDR) shows that, depending on the
patient
population being treated with EPO, different hematocrit levels are targeted to
avoid toxicity.
Physicans Desk Reference, 54th Ed., 519-525 and 2125-2131 (2000). In fact, in
patients with CRF,
the PDR recommends dosing EPO to achieve non-toxic target hematocrits ranging
from 30 percent to
36 percent. hl contrast, for cancer patients on chemotherapy, the PDR teaches
to adjust the dosage at
a different hematocrit level, i.e., if the hematocrit level exceeds 40
percent. The PDR shows that
practitioners monitor the patient's hematocrit during therapy with EPO and, to
avoid toxicity, adjust
the dose and/or withhold treatment if the patient's hematocrit approaches or
exceeds the upper limits
of a target range. Therefore, the skilled practitioner, armed with the
teachings of the present
invention, should be able to administer doses of EPO sufficient to achieve a
therapeutic effect while
avoiding any toxicity complications.
In one embodiment, the long acting EPO of the present invention is
administered chronically
or systemically at a dosage of about 0.1 ~,g/ kg body weight to about 100 ~,g
/kg body weight per
administration. For example, about 1 ~.g/ kg body weight to about 5 ~,g/ kg
body weight is
contemplated for once weekly dosing in the treatment of cancer patients
receiving chemotherapy. In
another embodiment, the dosage of the long acting EPO is about 5 ,ug /kg body
weight to about 50 ~,g
/kg-body weight per administration. W still another embodiment, the long
acting EPO is
administered in an amount of about 10 ~,g /kg body weight to about 30 ~,g /kg
body weight per
administration. In yet another embodiment, the long acting EPO is administered
in an amount of
about 1 ~,g/ lcg body weight or less. For example, about 0.45 ~.g/ kg body
weight to about 0.75 ~.g/ kg
body weight of long acting EPO may be effective when administered once weekly
for treatment of
anemia in CRF patients.
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The effective dose is preferably sufficient to achieve serum levels of the
long acting EPO of
greater than about 10,000 mU/ml (80 ng/ml). In one embodiment, the effective
dose achieves a
serum level of the long acting EPO of about 15,000 mU/ml (120 ng/ml) or
greater. In another
embodiment, the effective dose achieves a serum level of the long acting EPO
of about 20,000
mU/ml (160 ng/ml). The serum levels are preferably measured and achieved at
about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10 hours, or combinations thereof post-administration. Dosages may be
repeated as deemed
necessary by one of ordinary skill in the art. For example, administration may
be repeated daily, as
long as clinically necessary, or after an appropriate interval, e.g., every 1
to 12 weeks, preferably,
every 1 to 3 weeks.
Because the long acting EPOS of the present invention have an increased serum
half life, their
effectiveness in the body is also increased. For example, when a mammalian
patient is undergoing
systemic chemotherapy for cancer treatment, including radiation therapy, the
administration of the
long acting EPO pharmaceutical compositions of the invention during therapy
may decrease the
anemic concerns with less frequent and smaller doses than compared to the
frequency and amount of
present recombinant EPO compositions.
And, as discussed above, when the pharmaceutical compositions of the invention
include a
long acting EPO of the present invention or an EPO analog in a blend with a
tissue protective
cytokine, the compositons may be used to treat anemia and related diseases in
patients that are also at
risk for tissue injury. For example, a patient with anemia that is also a high
risk for heart disease may
be treated with the pharmaceutical compositions of the invention instead of
the EPO analogs
currently available so as to prevent the risk of increased damage from
treatment.
Treatriaent Kits
The invention also provides a pharmaceutical pack or kit that include one or
more containers
filled with one or more of the ingredients of the pharmaceutical compositions
of the invention. In
one embodiment, the effective amount of the long acting EPO and a
pharmaceutically acceptable
carrier may be packaged in a single dose vial or other container.
When the pharmaceutical composition of the invention is adapted for parenteral
administration, for example, the composition may be stored in a lyophilized
condition. Thus, the kit
may include the lyophilized composition, a sterile liquid carrier, and a
syringe for injections. In one
embodiment, the kit includes an ampule containing enough lyophilized material
for several
treatments such that the administrator would weigh out a specific amount of
material and add a
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specific amount of carrier for each treatment session. In another embodiment
the kit may contain a
plurality of ampules each containing specific amounts of the lyophilized
material and a plurality of
containers each containing specific amounts of carrier, such that the
administrator need only mix the
contents of one ampule and one carrier container for each treatment session
without measuring or
weighing. In yet another embodiment, the kit contains an autoinjector
including an injectable
solution of a long acting EPO of the invention. In still another embodiment,
the kit contains at least
one ampule with the lyophilized composition, at least one container of carrier
solution, at least one
container with a local anesthetic, and at least one syringe (or the like). The
ampules and containers
are preferably hermetically-sealed.
When the pharmaceutical compositions of the invention are to be administered
by infusion,
the kit preferably includes at least one ampule with the pharmaceutical
composition and at least one
infusion bottle with sterile pharmaceutical grade water or saline.
A kit according to the present invention may also include at least one
mouthpiece or specially
adapted devices for direct lung inhalation such as pressurized aerosols,
nebulizers, or insufflators. In
this aspect of the invention, the kit may include the device for direct lung
inhalation, which contains
the pharmaceutical composition, or the device and at least one ampule of
aqueous or oil solutions of
the long acting EPO of the present invention.
When the long acting EPO pharmaceutical composition of the invention is
adapted for oral,
transdermal, rectal, vaginal, or nasal, the kit preferably includes at least
one ampule containing the
active ingredient and at least one administration aid. Examples of
administration aids include, but are
not limited to, measuring spoons (for oral administration), sterile cleaning
pads (for transdermal
adminstration, and nasal aspirators (for nasal administration). Such kits may
include a single dose of
the long acting EPO (acute treatment) or a plurality of doses (chronic
treatment).
In addition, the kit may be outfitted with one or more types of solutions. For
example, the
long acting EPO pharmaceutical compositions of the invention may be made in an
albumin solution
and a polysorbate solution. If the kit includes the polysorbate solution, the
words "Albumin free"
preferably appear on the container labels as wells as the kit main panels.
Moreover, the kit may also include a notice in the form prescribed by a
governmental agency
regulating the manufacture, use or sale of pharmaceuticals or biological
products, which notice
reflects approval by the agency of manufacture, use or sale for human
administration.
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EPO Assays
The present invention also relates to assays to determine the erythropoietic
and tissue
protective capabilities of the long acting EPOS of the present invention, as
well as the EPO analogs
used in several of the pharmaceutical compositions of the present invention.
For example, the
erythropoietic affect of a long acting EPO may be verified through the use of
a TF-1 assay, which
will be discussed in greater detail in Example 2. The tissue protective
properties of EPO compounds
may be examined using in vity~o assays and in vivo assays, which are discussed
in greater detail
below. In addition, the present invention also contemplates tests for
determining not only whether a
particular EPO compound has tissue protective activity, but also whether the
EPO compound acts as
an antagonist with respect to endogenous EPO.
The assays of the invention are preferably designed to be completed within a
short period of
time using a minimal amount of the EPO compound. Moreover, the assays provided
herein are
intended to be non-limiting, as one of ordinary skill in the art would
recognize other assays useful for
determining the erythropoietic and tissue protective capabilities of EPO
compounds.
Ey. t~poietic Activi~ Assay
The erythropoietic attributes, i.e., the ability to control hematocrit levels,
of a particular EPO
compound may be determined using various assays. In one embodiment, a TF1 cell
line may be used
to determine whether a particular EPO compound has erythropoietic activity.
The cells may be
pelleted, washed, and resuspended at a concentration of 105 cells in 1 ml of
medium, with
recombinant EPO and an EPO compound of interest added at specific
concentrations. The individual
cultures may be maintained for 24 hours, at which time the cell number is
determined using a
formazan reaction product (CellTiter; Promega, Madison, WI).
The potency of the EPO compound of interest may first be assessed in vivo by
observing its
effect on the hemoglobin concentration using female BALB/c mice. Animals are
administered 500
U/kg-bw EPO, the EPO compound of interest, or an equal volume of vehicle
subcutaneously three
times a week for a total of three weeks (a time interval sufficient to observe
an erythropoietic
response). An EPO compound is determined to be erythropoietic if it raises the
serum hemoglobin
concentration of the mice.
Further assessment of potency may be obtained iya vitYO using TF1
erythroleukemia cells. The
EPO compound of interest is erythropoietic if the relative TF1 cell number
increases beyond that of
the control. In addition, those of ordinary skill in the art would recognize
that other assays for
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determining erythropoietic activity are available. For example, European
Pharmocopeia discusses at
least two assays useful in determining the erythropoietic activity of an EPO
compound, which include
exhypoxic mouse assays and reticulocyte assays.
Tissue Protective Capabili Assays Based on EPO Receptor
In one embodiment, the tissue protective capability assays of the present
invention are based
on the tissue protective receptor for EPO. Once the sequence for the tissue
protective receptor is
isolated, a variety of assays may be used to determined a particular EPO
compound's tissue
protective capability. As known to those of ordinary skill in the art, the
type of assay employed
largely depends on the weight of the EPO compound.
For example, the assays may be competitive assays or sandwich assays or steric
inhibition
assays. Competitive assays rely on the ability of a tracer analogue to compete
with the test sample
analyte for a limited number of binding sites on a common binding partner. As
used herein, the term
"analyte" refers to the EPO compound of interest to be tested for tissue
protective activity. The term
"binding partner" refers to any protein that binds to the analyte (typically
the EPO receptors). As
used herein, "tracer" refers to labeled reagants, such as labeled analyte
analogue, immobilized analyte
analogue, labeled binding partner, immobilized binding partner and steric
conjugates. The tracer
used herein may be any detectable functionality that does not interfere with
the binding of analyte
and its binding partner. Nonlimiting examples include moieties that may be
detected directly, such as
fluorochrome, chemiluminescent, and radioactive labels, as well as moieties
that must be reacted or
derivatized to be detected, such as enzymes. Suitable tracers may be the
radioisotopes P3z, C14, yzs,
H3, y3y and mixtures thereof; fluorophores, such as rare earth chelates,
fluorescein, fluorescein
derivatives, rhodamine, rhodamine derivatives, dansyl, umbelliferone
luciferase (firefly luciferase
and bacterial luciferase (U.S. Pat. No. 4,737,456)), luciferin, 2,3-
dihydrophthalazinediones,
horseradish peroxidase (HRP), alkaline phosphatase, ,Q-galactosidase,
glucoamylase, lysozyme,
saccharide oxidases (glucose oxidase, galactose oxidase, and glucose-6-
phosphate dehydrogenase),
heterocyclic oxidases (uricase and xanthine oxidase) coupled with an enzyme
that employs hydrogen
peroxide to oxidize a dye precursor such as HRP, lactoperoxidase,
microperoxidase, and mixtures
thereof; biotin/avidin; spin labels; bacteriophage labels; stable free
radicals; and combinations
thereof. In one embodiment, the tracer is at least one of horseradish
peroxidase or alkaline
phosphatase.
A skilled artisan is aware of methods of covalently binding the tracers to
proteins or
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polypeptides. For example, coupling agents such as dialdehydes, carbodiimides,
dimaleimides, bis-
imidates, bis-diazotized benzidine, and the like may be used to tag with the
above-described
fluorescent, chemiluminescent, and enzyme labels, some of which are described
in U.S. Patent Nos.
3,940,475 and 3,645,090, the entire disclosures of which are incorporated by
reference herein.
Immobilization of reagents, i.e., separating the binding partner from any
analyte that remains
free in solution, is required for a sandwich assay, and may be accomplished by
either insolubilizing
the binding partner or analyte analogue before the assay procedure, as by
adsorption to a water-
insoluble matrix or surface (U.S. Patent No. 3,720,760), by covalent coupling,
such as glutaraldehyde
cross-linking, or by insolubilizing the partner or analogue afterward, e.g.,
by immunoprecipitation.
Thus, the binding partner may be insolubilized before or after the competition
and the tracer
and analyte bound to the binding partner are separated from the unbound tracer
and analyte. This
separation may be accomplished by decanting (where the binding partner was
preinsolubilized) or by
centrifuging (where the binding partner was precipitated after the competitive
reaction). The amount
of test sample analyte is inversely proportional to the amount of bound tracer
as measured by the
amount of marker substance. Dose-response curves with known amounts of analyte
may be prepared
and compared with the test results to quantitatively determine the amount of
analyte present in the
test sample. When used with enzymes as tracers, the assays are typically
referred to as ELISA
systems.
In sequential sandwich assays, for example, an immobilized binding partmer is
used to adsorb
test sample analyte, the test sample is removed as by washing, the bound
analyte is used to adsorb
labeled binding partner, and' bound material is then separated from residual
tracer. The amount of
bound tracer is directly proportional to test sample analyte. In
"simultaneous" sandwich assays, the
test sample is not separated before adding the labeled binding partner.
Competitive and sandwich methods employ a phase-separation step as an integral
part of the
method, whereas steric inhibition assays are conducted in a single reaction
mixture. Another species
of competitive assay, called a "homogeneous" assay, however, does not require
a phase separation.
Tii such an assay, a conjugate of an enzyme with the analyte is prepared and
used such that when anti-
analyte binds to the analyte the presence of the anti-analyte modifies the
enzyme activity. The tissue
protective receptor is conjugated with a bifunctional organic bridge to an
enzyme such as peroxidase.
Conjugates are selected for use with the EPO so that binding of the EPO
inhibits or potentiates the
enzyme activity of the label. This type of assay is typically referred to as
EMIT.
Steric conjugates are used in steric hindrance methods for homogeneous assay.
These
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conjugates are synthesized by covalently linking a low-molecular-weight hapten
to a small analyte
such that antibody to hapten substantially is unable to bind the conjugate at
the same time as anti-
analyte. The analyte present in the test sample will bind anti-analyte,
thereby allowing anti-hapten to
bind the conjugate, resulting in a change in the character of the conjugate
hapten, e.g., a change in
fluorescence when the hapten is a fluorophore.
More information regarding tissue protective capability assays is discussed in
co-pending U.S.
Patent Application No. 10/188,905, filed July 3, 2002 and in Application
Serial No. 60/456,891, filed
April 25, 2003, both of which are incorporated by reference herein in their
entireties.
Fuf-actiofZal Assays
In the absence of the identification of the sequence for the tissue protective
receptor, the tissue
protective capabilities of an EPO may be determined using functional assays,
both in vivo and ih
vitro. Preferably, one of ordinary skill in the art would perform a single ira
vitro or in vivo assay to
determine the tissue protective capabilities of an EPO compound, but in
certain instances it may be
necessary to perform both to assure that the compound exhibits the same tissue
protective capabilities
in vitro and ira vivo.
In practice, one of ordinary skill in the art would be able to determine
whether an EPO analog
having at least one additional N-linked carbohydrate chain and/or at least one
additional O-linked
carbohydrate chain, using a combination of assays disclosed by the present
invention. First, in vitYo
tests such as the P19 cell and rat motoneuron assays could be used to
determine whether the EPO
compound of interest exhibited tissue protective capabilities. Then, in vivo
studies such as the rat
focal ischemia, bicuculline seizure, or spinal cord trauma models could be
used to verify the results
of the ifa vitro testing.
lu vitro models contemplated by the present invention include, but are not
limited, to those
used to determine the lack of tissue protective capabilities of the
hyperglycosylated-erythropoietin
above: the P19 cell assay, rat motoneuronal cell assay, and the cDNA
microarray, which are
discussed in greater detail below and further illustrated in Example 2. The
examples are intended to
be non-limiting as one of ordinary skill in the art would recognize that there
are other suitable ira vitro
assays for determining the tissue protective capabilities of EPO compounds. In
general, the EPO
compound would be considered tissue protective if, in comparison to a control,
it maintained or
enhanced the viability of the cell. The erythropoietin would be considered
antagonistic if, in
comparison to the control, it detrimentally affected the viability of the
cells within the assay.
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A. In Vitro Assay Based on P19 Cell Line
In one embodiment, the in vitro tissue protective capability assay is based on
a P19 cell line.
For example, P 19 cells may be maintained undifferentiated in DMEM
supplemented with 2 mM L-
glutamine, 100 U/ml penicillin G, 100 ~.g/ml streptomycin sulfate (Gibco) and
10 percent fetal
bovine serum (Hyclone Laboratories), which contains 1.2 g/1 NaHC03 10 mM hepes
buffer. Serum-
free medium may contain the same components as above, with the exception of 5
~g/ml of insulin,
100 ~.g/ml of transferring, 20 nM progesterone, 100 ~M putrescine and 30 nM
Na2Se03 (Sigma) in
place of the fetal bovine serum.
Cells that react with 50 percent confluency are treated overnight with
recombinant EPO and/or
an EPO compound of interest, dissociated with trypsin, washed in serum-free
medium and plated in
25 cm2 tissue culture flasks at a final density of 104 cells/cm2 in serum-free
medium alone, or with the
pretreatment additions. Cell viability may be determined by trypan blue
exclusion.
As known to skilled artisans, the addition of recombinant EPO can prevent cell
loss after serum
withdrawal in undifferentiated neuronal-like P19 cells. For example,
recombinant EPO rescues up to
50 percent of the neuronal-like cells from death if used in a concentration of
0.1 U/ml to 100 U/ml.
Thus, to be considered tissue protective, the EPO compound of interest must
rescue more P19 cells
from death than the control, preferably it must rescue about 25 percent to
about 50 percent of the
cells, most preferably about 40 percent to about 50 percent of the cells.
B. In Vitro Rat Motoneuron Assay
In another embodiment, a rat motoneuron assay is used to determine the tissue
protective
capability of an EPO compound of interest in vitro. For example, primary
motoneurons may be
obtained using spinal cords from 15-day Sprauge Dawley rat embryos and
purified by
immunopanning. The cells are preferably seeded at low density (20000
cells/cmz) onto glass
coverslips in 24 mm well plates precoated with poly-DL-orinthine and laminin
and containing
complete culture medium (Neurobasal, B27 (2 percent), 0.5 mM L-glutamine, 2
percent horse serum,
25 ~.M 2-mercaptoethanol, 25 ~,M glutamate, 1 percent penicillin and
streptomycin, lng/ml BDNF).
After a period of time, preferably about 6 days, EPO (l0U/ml) and the EPO
compound of interest (10
U/ml) or vehicle may be added to the cultures (preferably about 5 days before
determination of
surviving neuronal density). The medium may then be discarded and the cells
may be fixed with 4
percent paraformaldehyde in PBS for 40 minutes, permeabilized with 0.2 percent
Triton X-100,
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blocked with 10 percent fetal calf serum in PBS, incubated with antibodies
against non-
phosphorylated neurofilaments (SMI-32; 1:9000) overnight, and visualized using
the avidin-biotin
method with diaminobenzidine. The viability of motoneurons may be assessed
morphologically by
counting SMI-32 positive cells.
Mixed primary cultures of motoneurons characteristically undergo apoptosis
during
maintenance culture conditions. Addition of recombinant EPO (10 U/ml) to the
culture medium 5
days before assessment of cell number has been shown to significantly increase
the number of
primary motoneurons observed at 5 days. Thus, to be considered tissue
protective, the EPO
compound of interest preferably salvages at least the same number of
motoneurons than the control.
In one embodiment, the EPO compound of interest is considered tissue
protective if a greater number
of motoneurons are saved from death during maintenance culture conditions as
compared to the
control.
C. Ih Vitf~o Assay Based on cDNA Microarray
Another in vitro assay to determine tissue protective capability of an EPO
compound of interest
is a cDNA microarray. This assay may be used to determined if recombinant EPO
and the EPO
compound of interest modify gene expression differently in P19 cells. mRNA
isolated from
undifferentiated P19 cells can show a different pattern of gene modulation
estimated from a mouse
1200 cDNA microarray, depending upon the exposure to the EPOS. For example,
the expression of
1,200 genes in P19 cells may be measured by the use of nylon membrane arrays
from Clontech (Atlas
mouse 1.2). Cells (107/sample) may be treated overnight with saline,
recombinant EPO, an EPO
compound of interest (1mU/ml), or mixtures thereof. The cells are then lysed
for RNA extraction or
subjected to serum deprivation for 3 hours (always in the presence of the same
cytokine added during
pretreatment). After standard total RNA extraction by column chromatography,
with on-column
DNase treatment, polyA + RNA may be purified. Probes may then be constructed
in the presence of
[P3z]-ATP. The labeled probes, having preferably 20 million counts or higher,
rnay be hybridized to
the cDNA nylon membranes at 6~° C. The membranes are washed and exposed
to x-ray film. The
intensity of radioactive signals may be measured with a Phosphor Imager and
analyzed with the Atlas
Image 2.0 computer program (Clontech).
In vivo assays contemplated by the present invention include, but are not
limited to, the tissue
protective assays used to evaluate EPO compounds such as the focal ischemia
model and intra-
hippocampal biculline model. In addition, an in vivo model for evaluating
tissue protection includes
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spinal cord injury assays. Furthermore, the various assays disclosed within
International Publication
No. WO/02053580 and U.S. Patent Publication Nos. 2002/0086816 and 2003/0072737
are
contemplated for use with the present invention.
D. In Vivo Assay Based on Focal Ischemia Model
In one embodiment, the in vivo assay used to determine tissue protective
capabilities of a
particular EPO compound is based on a focal ischemia model. For example, male
Sprague-Dawley
rats (~ 250 gm) may be used with a three vessel focal ischemi model. Briefly,
the rats may be
anesthetized with pentobarbital (60 mg/kg-bw) and maintained at a core
temperature of 37° C using a
water blanket. The right carotid may be occluded by two sutures and
transected. A burr hole
adj acent and rostral to the right orbit allows visualization of the middle
cerebral artery, which may be
cauterized distal to the rhinal artery. To produce a penumbra surrounding this
fixed MCA lesion, the
contra-lateral carotid artery may be occluded for 1 hour using traction
provided by fine forceps.
Saline, recombinant EPO (5000U/kg-bw) or the EPO compound of interest (5000
U/lcg-bw) may be
administered at the onset of the reversible carotid occlusion.
After 24 hours, the brains are removed and serial 1-mm thick sections are cut
through the entire
brain using a brain matrix device (Harvard Apparatus). Each section may then
be subsequently
incubated in a solution of 2 percent triphenyltetrazolium chloride (w/v) in
154 mM NaCI for 30
minutes at 37° C. The volume of injury may be determined using a
computerized image analysis
system (MCID, Imaging Research, St. Catharines, Ontario, Canada). In this
assay, the EPO
compound of interest is considered neuroportective if it ameliorates the
infarct volume due to the rat
MCA focal ischemia to the same or greater extent as the recombinant EPO.
E. In Vivo Assay Based on Intra-hippocampal Biculline Seizure Model
In another embodiment, the tissue protective capability of an EPO compound is
determined in
vivo with intra-hippocampal biculline experiments. For example, male Sprague-
Dawley rats (250-
280 g) are housed at a constant temperature (23° C) and relative
humidity (60 percent) with free
access to food and water and a fixed 12 hour light/dark cycle. The rats are
surgically implanted with
cannula and electrodes under stereotaxic guidance as described in Vezzani, A.,
et al., J. Neu~osci, 19,
5054-65 (1999). Briefly, rats may be anesthetized using Equithesin (1 percent
Phenobarbital / 4
percent chloral hydrate; 3m1/kg i.p.). Two screw electrodes are placed
bilaterally over the parietal
cortex, along with a ground lead positioned over the nasal sinus. Bipolar
nichrome wire insulated
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electrodes (60 ~,m) may then be implanted bilaterally into the denate gyrus of
the dorsal hippocampus
(septal pole) and a cannula (22-gauge) may be unilaterally positioned on top
of the dura for the
intrahippocampal or intracerebroventricular infusion of drugs. The coordinates
from bregma for
implantation of the electrodes should be: (mm) antero-posterior-3.5; lateral
2.4 and 3 below dura with
the nose bar set at -2.5. Paxinos, G. & Watson, C., The Rat Brain in.
Stereotaxic Coordinates,
Academic Press, New York (1986). The electrodes may be connected to a multipin
socket (March
Electronics, NY) and, together with the inj ection cannula, secured to the
skull by acrylic dental
cement.
The experiments are preferably carned out three to seven days after surgery
when the animals
have fully recovered. Animals are then administered recombinant EPO or the EPO
compound of
interest (both 5000 U/kg-bw) or vehicle intraperitoneally 24 hours and again
at 30 minutes before the
induction of bicuculline seizures. The procedures for recording the EEG and
intracerebral inj ection
of drugs have been previously described Vezzani, A., et al., J. Phar~macol Exp
Ther, 239, 256-63
(1986). Briefly, the animals are allowed to acclimatize in a Plexiglass cage
(25x25x60 cm) for a
minimum of 10 minutes before initiating the EEG recording (4-channel EEG
polygraph, model BP8,
Battaglia Rangoni, Bologna, Italy). After about 15 minutes to about 30
minutes, EEG recordings are
made continuously for 120 minutes after 0.8 nmol/0.5 ~l bicuculline methiodide
infusion. All the
injections were made to unanesthetised rats using a needle (28-gauge)
protruding 3 mm below the
cannula.
Seizures may be measured by EEG analysis, which has previously been shown to
provide a
sensitive measure of the anticonvulsant activity of drugs. Vezzani, A., et
al., J. Pharrnacol Exp Ther,
239, 256-63 (1986). For the purposes of this assay, seizures consist of the
simultaneous occurrence
of at least two of the following alterations in all four leads of recordings:
high frequency and/or
multispike complexes and/or high voltage synchronized spike or wave activity.
Synchronous spiking
may be observed intermixed with seizures. The parameters chosen to quantify
seizures are preferably
the latency to the first seizure (seizure onset), the total time spent in
epileptic activity (determined by
adding together the duration of ictal episodes; seizure duration), and the
spiking activity during the
EEG recording period (seizure activity).
The intra-hippocampal bicuculine seizure model using EEG activity as a read-
out has been
shown to be a sensitive and specific predictor of anti-seizure potency of
drugs. Vezzani, A., et al., J.
Phar~macol Exp Ther-, 239, 256-63 (1986). Thus, to be considered tissue
protective the EPO
compound of interest should reduce the frequency and severity of the seizures
to the same or greater
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extent as the recombinant EPO.
F. In Vivo Assay Based on Acute Reversible Glaucoma Rat Model
In yet another in vivo assay according to the present invention, an acute
reversible glaucoma
rat model may be used to determine the tissue protective capability of
particular EPO compounds of
interest. For example, because retinal cells are very sensitive to ischemia,
many of these cells will
die after 30 minutes of ischemic stress. As such, to test whether peripherally-
administered EPO
compounds of interest exhibit tissue protective activities sufficient to
protect cells sensitive to
ischemia, an acute, reversible glaucoma rat model may be used as described by
Rosenbaum et al.,
Tlis. Res. 37: 3443-51, 1997. In particular, saline may be injected into the
anterior chamber of the eye
of adult male rats to a pressure above systemic arterial pressure and
maintained for 60 minutes.
Animals are then administered saline or 5000 U EPO /kg body weight
intraperitoneally 24 hours
before the induction of ischemia, and continued as a daily dose for three
additional days.
Electroretinography may be performed on dark-adapted rats one week after
treatment to
determine whether the EPO compound of interest possesses tissue protective
activity. If the EPO is
tissue protective, there should be good preservation of activity on the
electroretinogram, in contrast to
animals treated with saline alone.
G. Myocardial Infarction Assays
Myocardial infarction assays are also contemplated for use with the present
invention to
determine whether an EPO compound exhibits tissue protective activity in
general or within the heart
specifically. For example, adult male rats may be given EPO (5000 U/kg body
weight) 24 hours
before being anesthetized and prepared for coronary artery occlusion. An
additional dose of EPO may
be given at the start of the procedure, at which time the left main coronary
artery is occluded for 30
minutes and then released. The same dose of EPO is given daily for one week
after treatment, at
which time the animals are studied for cardiac function. Animals receiving a
sham injection (saline)
will demonstrate a large increase in the left end diastolic pressure,
indicative of a dilated, stiff heart
secondary to myocardial infarction, whereas animals receiving the EPO compound
of interest should
exhibit no decrement in cardiac function, compared to sham operated controls
(difference significant
at the p < 0.01 level) if the EPO is tissue protective.
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H. Spinal Cord Iniury Assays
Spinal cord injury assays may also be used with the present invention to
evaluate the tissue
protective abilities of particular EPO compounds of interest. In particular,
rat spinal cord
compression is contemplated for use with the present invention. Wistar rats
(female) weighing about
180 g to about 300 g are preferably used in this study. The animals are
preferably fasted for 12 hours
before surgery, and humanely restrained and anesthesized with an
intraperitoneal injection of
thiopental sodium (40 mglkg-bw). After infiltration of the skin (bupivacaine
0.25 percent), a
complete single level (T-3) laminectomy is performed through a 2 cm incision
with the aid of a
dissecting microscope. Traumatic spinal cord injury is induced by the
extradural application of a
temporary aneurysm clip exerting a 0.6 newton (65 grams) closing force on the
spinal cord for 1
minute. After removal of the clip, the skin incision is closed and the animals
allowed to recover fully
from anethesia and returned to their cages. The rats are monitored
continuously with bladder
palpation at least twice daily until spontaneous voiding resumed. '
Animals in a control group receive normal saline (via intravenous injection)
immediately after
the incision is closed. The remaining animals receive the EPO compound of
interest in an amount of
16 micrograms/kg-bw iv. The motor neurological function of the rats is then
evaluated using a
locomotor rating scale. In this scale, animals are assigned a score ranging
from 0 (no observable
hindlimb movements) to 21 (normal gait). The rats are tested for functional
deficits at 1 hour, 12
hours, 24 hours, 48 hours, 72 hours, and 1 week after injury by the same
examiner who is blind to the
treatment each animal receives. If the EPO compound of interest is tissue
protective, the rats that are
given the EPO should exhibit a quicker and beter overall recovery from the
injury than the rats that
are given the saline injection.
I. Rabbit Spinal Cord Ischemia Testing
In another embodiment, rabbit spinal cord ischemia testing allows testing for
tissue protective
capability. For example, New Zealand White rabbits (36, 8-12 months old, male)
weighing 1.5 kg to
2.5 kg are used in this study. The animals are fasted for 12 hours and
humanely restrained.
Anesthesia induction is via 3 percent halothane in 100 percent oxygen and
maintained with 0.5
percent to 1.5 percent halothane in a mixture of 50 percent oxygen and 50
percent air. An
intravenous catheter (22 gauge) is placed in the left ear vein. Ringers
lactate is infused at a rate of 4
ml/kg body weight (bw) per hour during the surgical procedure. Preoperatively,
cefazoline 10
mg/kg-bw is administered intravenously for prophylaxis of infection. The
animals are placed in the
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right lateral decubitus position, the skin prepared with povidone iodine,
infiltrated with bupivacaine
(0.25 percent) and a flank skin incision is made parallel to the spine at the
12th costal level. After
incision of the skin and subcutaneous thoracolumbar fascia, the longissimus
lumborum and
iliocostalis lumborum muscles are retracted. The abdominal aorta is exposed
via a left retroperitoneal
approach and mobilized just inferior to the left renal artery. A piece of PE-
60 tubing is looped
around the aorta immediately distal to the left renal artery and both ends
passed through a larger
rubber tube. By pulling on the PE tubing, the aorta is non-traumatically
occluded for 20 minutes.
Heparin (400 IU' is administrated as an intravenous bolus before aortic
occlusion. After 20
minutes of occlusion, the tube and catheter are removed, the incision is
closed and the animals are
monitored until full recovery, at which time, they are serially assessed for
neurological function. A
control group of animals receives normal saline intravenously immediately
after release of aortic
occlusion. Another group of animals receives 6.5 ~,g/kg-bw of the EPO compound
of interest
intravenously immediately after reperfusion (n = 6 for each group).
Motor function is assessed according to the criteria of Drummond and Moore by
an
investigator blind to the treatment at 1 hour, 24 hours, and 48 hours after
reperfusion. A score of 0 to
4 is assigned to each animal as follows: 0 = paraplegic with no evident lower
extremity motor
function; 1 = poor lower extremity motor function, weak antigravity movement
only; 2 = moderate
lower extremity function with good antigravity strength but inability to draw
legs under body; 3 =
excellent motor function with the ability to draw legs under body and hop, but
not normally; 4 =
normal motor function. The urinary bladder is evacuated manually in paraplegic
animals twice a day.
If the EPO compound of interest is tissue protective, the animals that are
given the EPO
should exhibit a quicker and better overall recovery from the injury than the
animals receiving the
saline injection.
As briefly discussed earlier, several types of tissues possess EPO receptors
and, therefore,
may be responsive to the tissue protective affects of EPO. Thus, depending
upon the proposed
clinical application for the EPO compound of interest, a slcilled artisan
would recognize that similar
in vitro assays involving these additional responsive cells may be performed,
or in vivo assays
involving the associated organs may also be performed. For example, in vitro
assays based on serum
deprivation can be performed using myocardial, retinal, and Leydig cells and a
protocol similar to
that outlined above for the P19 assay.
Ifz vivo assays can be directed to individual organs as well. For example, to
evaluate an
EPO's affect upon retinal cells, one of ordinary skill in the art may perform
the retinal ischemia assay
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described above. In addition, to evaluate an EPO analog's effect upon
myocardial cells, a skilled
artisan could readily modify the myocardial infarction model discussed above.
Those of ordinary
skill in the art will be sufficiently skilled to select the appropriate assay
or model to evaluate whether
a particular EPO possesses tissue protective activities with regard to an
erythropoietic responsive cell,
tissue or organ.
EXAMPLES
The following prophetic examples are merely illustrative of the preferred
embodiments of the
present invention, and are not to be construed as limiting the invention, the
scope of which is defined
by the appended claims.
Example 1: Chemically Modified EPO
A. Oxidation of Sugar Chains
The sugar units of EPO may be converted into acids by the following procedure.
EPO and an
amount of sodium periodate sufficient to provide the amount of oxidation
desired (the greater the
amount of sodium periodate the greater the extent of the oxidation) may be
placed within a 100 mM
sodium acetate buffer. This solution may then be incubated on ice for about 20
minutes and dialyzed
thoroughly using distilled water. The product may then be removed from the
dialysis tubing and
collected into a fresh tube (Product I).
A Quantitative Benedict Solution (18 g copper sulfate, 100g sodium carbonate
(anhydrous),
200 g potassium citrate, 125 g potassium thiocyanante, 25 g potassium
ferrocyanide) may be
dissolved into distilled water to a final volume of 1 liter. Several drops of
methylene blue may then
be added to the Quantitative Benedict Solution.
Product I may then be added to the Quantitative Benedict Solution until the
color of the
solution becomes clear indicating the solution is fully oxidized. The solution
may then be desalted
and concentrated using an Ultrafree Centrifugal Filter Unit. The sample
(Product II) may then be
further dialyzed thoroughly using distilled water.
B. Oxidation of Asialo Form EPO with Galactose Oxidase
50 to 500 ~,g asialo form of EPO , 10 ~.1 lU/~.1 galactose oxidase, and 100 ~1
10 mM sodium
phosphate buffer may be mixed in a 15 ml conical centrifuge tube (110 ~,l
total volume). This
mixture may then be incubated for 2 hours at 37°C, at which time the
solution may be dialyzed
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thoroughly using distilled water. The product may be removed from the dialysis
tubing and collected
into a fresh tube (Product III).
A Quantitative Benedict Solution (as described above) may be dissolved into
distilled water to
a final volume of 1 liter. Several drops of methylene blue may then be added
to the Quantitative
Benedict Solution.
Product III may be added to the Quantitative Benedict Solution until the color
of the solution
becomes clear indicating the solution is fully oxidized. The solution may then
be desalted and
concentrated using an Ultrafree Centrifugal Filter Unit. The sample (Product
IV) may then be further
dialyzed thoroughly using distilled water.
C. Sulfation of EPO
EPO may be dissolved in N,N-dimethylformamide (DMF-SA) at 4° C. N,N'-
dicyclohexyl
carbodiimide (DCC) dissolved in DMF may then be added and the solution shaken
for 4 hours at 4°
C. Cracked ice may be added and the pH may be adjusted to 7.5 with 10 N NaOH.
The volume of
the solution may be adjusted and the sample may be centrifuged for 1000 x g
for 15 minutes in a type
HN-S2 centrifuge (DAMONIEC, Needham Hts., Massachusettes). The supernatant may
then be
extensively dialyzed. More information regarding sulfation is discussed in S.
Pongor et al.,
Preparation of High-Potency, Non-aggregating Insulins Using a Novel Sulfation
Procedure, Diabetes,
Vol. 32, No. 12, December 193, the entirety of which is incorporated herein by
reference.
D. Attachment of PEG chains to EPO
EPO may be modified through the attachment of PEG chains to oxidized
carbohydrates, such
as those obtained above in A (Product I). The degree of modification may be
controlled by varying
the periodate concentration during oxidation.
PEG-EPO conjugates may be prepared by first oxidizing EPO (2-4 mg/ml in 50 mM
sodium
acetate) for 30 minutes at room temperature with 1 mM to 100 mM sodium meta-
periodate (Sigma).
The phosphate buffer may then be removed by buffer exchange in 100 mM sodium
acetate, ph 5.4.
Methoxy-PEG-hydrazide of various molecular weights (Nektar Therapeutics) may
then be
added at a 5 fold to 100 fold molar excess (polymer: protein). The
intermediate hydrazine linkage
may then be further reduced by the addition of 15 mM sodium cyanoborohydride
(Sigma) and
allowed to react overnight at 4°C. The resultant conjugates may then be
fractionated / purified by
techniques known in the art.
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E. Attachment of PEG chains to Asialo EPO
An asialo form of EPO may be modified through the attachment of PEG chains to
newly
created terminal galactose residues after oxidiation with galactose oxidase,
such as those obtained
above in B (Product III).
Recombinant human EPO (rhuEPO) may be desialized using Sialidase A (Prozyme,
Inc.)
according to the manufacturer protcol. The chemical modification is preferably
confirmed by
running the reaction product on a SDS polyacrylamide gel. Staining the
resultant bands should show
that the modified EPO has an apparent molecular weight of about 31 kDa, while
the unmodified EPO
has a molecular weight of about 34 kDa. The sialic acid residues remaining on
the EPO are
preferably less than 0.1 mole/mole of EPO.
After the asialo form of EPO is obtained, the newly exposed galactose residues
on EPO (2-4
mg/ml in 10 mM sodium phosphate buffer) may be oxidized with 100 units of
galactose oxidase in
PBS (Sigma) per ml of EPO solution. The reaction mixture may then be incubated
at 37°C for 2
hours.
The phosphate buffer may then be removed by buffer exchange in 100 mM sodium
acetate,
ph 5.4. Methoxy-PEG-hydrazide of various molecular weights (Nektar
Therapeutics) may then be
added at a 5 fold to 100 fold molar excess (polymer: protein). The
intermediate hydrazine linkage is
then preferably further reduced by the addition of 15 mM sodium
cyanoborohydride (Sigma) and
allowed to react overnight at 4°C. The resultant conjugates may then be
fractionated l purified by
techniques known in the art.
F. Attachment of PEG chains to Asialo EPO
An asialo form of EPO may be modified through the attachment of PEG chains to
newly
created terminal galactose residues after oxidiation with galactose oxidase,
such as those obtained
above in B (Product III).
RhuEPO (1 mg) may be desialized using Neuraminidase (Seikagaku Corporation of
Japan, 1
U of lyophilized powder is dissolved in 100 ~,L of 75 mM NaP04 (pH 6.5)) at a
ratio of 1mg EPO to
0.05 units of Neuraminidase (5 ~,L). Five units (5 ~,L) of galactose oxidase
(450 ~,L dissolved in 75
mM NaP04 (pH 6.5) (Sigma)) may then be added to the mixture.
The phosphate buffer may then be removed by buffer exchange in 100 mM sodium
acetate,
ph 5.4. PEG-NH2 (750 molecular weight, Nektar Therapeutics) and 15 mM sodium
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cyanoborohydride (Sigma) may then be added to the mixture and allowed to react
overnight at 4°C.
The PEG-NH2 is preferably added at a 250 fold molar excess (polymer: protein)
(80 mg of PEG-
NHZ). The resultant conjugates may then be fractionated / purified by
techniques known in the art.
Example 2: Functional Assays
A. Erythropoietic Assay
The erythropoietic attributes, i.e., the ability to control hematocrit levels,
of a particular EPO
compound were determined using the following assay.
TF1 is a human erythroleukemic cell line with complete dependence on growth
factors,
including EPO. Kitamura, at al., Blood 73, 375-80. TFl cells were obtained
from ATCC and
maintained in RPMI 1640 with the following: 2mM L-glutamine, 10 mM Hepes, 1 mM
sodium
pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 5 ng/ml GM-CSF, and 10
percent fetal bovine
serum until experimentation. TFl cells obtained during active growth were
pelleted, washed three
times with medium alone, and resuspended at a concentration of 105 cells in 1
ml of medium, with or
without GM-CSF, with EPO or an EPO analog having at least one additional N-
linked carbohydrate
chain and/or at least one additional O-linked carbohydrate chain added at
specific concentrations.
The individual cultures were maintained for 24 hours and the cell number was
determined using a
formazan reaction product (CellTiter; Promega, Madison, WI) according to the
manufacturer's
protocol.
The potency of the EPO compound was first assessed in vivo by observing its
effect on the
hemoglobin concentration using female BALB/c mice. Animals were administered
500 U/kg-bw
EPO, the EPO compound of interest, or an equal volume of vehicle
subcutaneously three times a
week for a total of three weeks (a time interval sufficient to observe an
erythropoietic response). An
EPO compound is determined to be erythropoietic if it raises the serum
hemoglobin concentration of
the mice. Further assessment of potency was obtained in vitYO using TFl
erythroleulcemia cells. The
studies confirmed that an EPO is erythropoietic if the relative TF1 cell
number increases beyond that
of the control.
Those of ordinary skill in the art would recognize that other assays, such as
the exhypoxic
mouse assay and the reticulocyte assay (European Pharmocopeia), are also
suitable for use with the
present invention to determine erythropoietic activity.
B. Tissue Protective Assay
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The tissue protective attributes of an EPO analog having at least one
additional N-linked
carbohydrate chain and/or at least one additional O-linked carbohydrate chain
were determined using
the following assay.
Neuronal cultures were established from the hippocampus of 18-day rat fetuses.
Brains were
removed and freed from meninges and the hippocampus was isolated. Cells were
then dispersed by
incubation for 5 minutes at 37° C in a 2.5 percent trypsin solution
followed by titration. The cell
suspension was diluted in serum-free Neurobasal media containing 1 percent B-
27 supplement
(Gibco, Rockville, MD, USA) and plated onto polyornithine-coated coverslips at
a density of 80,000
cells per coverslip. Cells were then pre-treated with EPO overnight and then
exposed with or without
1) EPO, 2) an EPO analog having at least one additional N-linked carbohydrate
chain and/or at least
one additional O-linked carbohydrate chain, or 3) an asialo form of EPO to 5
~.M TMT for 24 hours.
Cultures were used between 10 and 14 days in vitro.
The viability of the cells was measured by the 3-(4,5-dimethyl-thiazol-2-yl)-
2,5-
diphenyltetrazolium bromide (MTT) assay. Denziot, F., and Lang, R. 1986. Rapid
Colormetric
Assay for Cell Growth and Survival. Modifications to the tetrazolium dye
procedure giving
improved reliability. Jlynmunol Metlaods 89: 271-277. Briefly, MTT tetrazolium
salt was dissolved
in serum-free medium to a final concentration of 0.75 mg/ml and added to the
cells at the end of the
treatment for 3 hours at 37° C. The medium was then removed and the
formazan was extracted with
1N HCl:isopropanol (1:24) . Absorbance at 560 nm was read on a microplate
reader.
As demonstrated in FIG. lA, the EPO analog having at least one additional N-
linked
carbohydrate chain and/or at least one additional O-linked carbohydrate chain
did not exhibit a tissue
protective function.
The invention described and claimed herein is not to be limited in scope by
the specific
embodiments herein disclosed, since these embodiments are intended as
illustrations of several
aspects of the invention. Any equivalent embodiments are intended to be within
the scope of this
invention. Indeed, various modifications of the invention in addition to those
shown and described
herein will become apparent to those skilled in the art from the foregoing
description. Such
modifications are also intended to fall within the scope of the appended
claims. All references cited
herein are incorporated by reference herein in their entireties for all
purposes.
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