Note: Descriptions are shown in the official language in which they were submitted.
2 1 6 2 ~ 1 ~ PCT~S94/05773
094/28013
PEPTIDES FOR SUPPRESSION OF MYELOID PROGENITOR
1 CELL PROLIFERATION AND TREATMENT OF SEPTIC SHOCK
The proliferation and differentiation of
hematopoietic progenitor cells are regulated by an
interactive network of stimulatory and inhibitory
5 molecules. The biomolecules have the capacity to
directly stimulate the proliferation and/or
differentiation of progenitor cells, or to act
indirectly by enhancing or suppressing the production or
action of other cytokines, Many of the cytokines are
lO multifunctional, and may manifest stimulatory or
suppressive activity depending upon the target cell or
assay system.
A number of well-characterized cytokines have
been identified which have stimulatory effects on
15 myelopoiesis. Erythropoietin (EPO~, granulocyte-colony
stimulating factor (G-CSF), and granulocyte macrophage-
colony stimulating factor (GM-CSFJ are known to be
stimulatory to the production of myèloid cells, and have
clinical utility in the treatment o~ chemotherapy-
20induced myelosuppression.
Molecules which act to suppress myelopoiesisare also known, and have been reviewed by Broxmeyer
(1992) Am. J. Pediatr. Hematol. Oncol. 14:22. Both
purified natural and recombinant molecules have been
25implicated as physiologically active suppressor
molecules. Some of these molecules act directly at the
progenitor cell level to decrease the proliferativ~
capabilities of such cells, while others act indirectly
to decrease production andtor release of stimulatory
30cytokines.
2 1 6 2 6 l 0 PCT~S9~/05773
W094/28Q13
l For example, purified and recombinant forms of
H ferritin suppress growth factor induced clonal
proliferation of myeloid progenitors in vitro, and H
ferritin has suppressive activity when ~mi nistered to
5 mice. Both immature and more mature progenitor cells
are responsive to inhibition by H ferritin. Macrophage
inflammatory protein (MIP)-la has suppressive activity
on more immature progenitor cells, including human and
murine colonies forming units of granulocytes,
10 erythrocytes, macrophages, and megakaryocytes (CFU-GEMM)
and murine CFU-A. The interferons (IFNs), tumor
necrosis factors (TNFs), prostagl~n~ins PGEl and PGE2,
inhibin, and transforming growth factor (TGF)-~ have
also been implicated as suppressor factors for myeloid
15progenitor cells. The iron-binding protein lactoferrin
exerts indirect suppressive activity by decreasing the
production or release of colony stimulating factors or
IL-1 from monocytes and macrophages.
Bone marrow and spleen cells from mice treated
20with lactoferrin, H ferritin or PGE release a molecule
in vitro with suppressive activity for early myeloid
pro~enitors (Gentile et al., 1989, Blood 74: 228a; U.S.
Patent No. ~,149,544 to Gentile et al.). The suppressor
molecule has an apparent molecular weight of 8 kD, which
25is similar to MIP-la, but it is apparently biochemically
and immunologically distinct from MIP-la.
Peptide suppressors of myelogenesis have also
been reported. U.S. Patent No. 4,384,991 describes an
inhibitor purified from granulocytes which inhibits the
30proliferation of normal and leukemic myeloid cells. The
inhibitor is reported to have the amino acid composition
2162610
094/28013 PCT~S94/05773
TaUlASXlSerzThrlGlX3Gly2Ala ( P04 ) - Lu et al. (1989)
Exp. Hematol. 17: 93~ report the suppressive activity
of the synthetic pentapeptide Glu-Glu-Asp-Cys-Lys.
Moore _ al. (1988) J. Immunol. 141:2699 report that a
5 tuftsin analog, (ALA1)-tuftsin, has an inhibitory effect
on a restricted population of progenitor cells
responsive to macrophage colony stimulating factor (M-
CSF or CSF-1) and lipopolysaccharide (LPS).
Cytokines which have stimulatory effects on
lO myelopoiesis have clinical utility in the treatment of
chemotherapy-induced myelosuppression. Negative
regulators are potentially useful in treatment of
hematopoietic disorders by dampening blood cell
production in hyperproliferative states and for
15 selectively placing normal progenitors out of cycle and
thus in a reversibly protected state from the effects of
S-phase specific chemotherapeutic drugs. ~ccordingly,
there is a need in the art for clinically useful
suppressors of myelogenesis.
Gram-negative sepsis is a progressive,
injurious systemic inflammatory response to infection in
which bacterial endotoxin triggers biochemical events
that lead to serious complications such as shock, adult
respiratory distress syndrome, and disseminated
25intravascular coagulation. Despite the ongoing
development of new therapies for sepsis and septic
shock, mortality remains unacceptably high.
Accordingly, there remains a need in the field for
effective therapies for the treatment and prevention of
30sepsis and septic shock.
In accordance with the present invention,
novel peptides have been discovered which are capable of
.
2 1 6 2 6 10 PCT~S94/05773
W094/280~
--4--
1 suppressing the proliferation of myeloid progenitor
cells. The peptides of the present invention are useful
in the treatment of myeloid proliferative disorders,
such as leukemia, and for protecting hematopoietic cells
5 prior to chemotherapy by suppressing myelopoiesis. The
peptides of the present invention are also useful in the
treatment and prevention of septic shock.
The present invention is directed, in one
embodiment, to biologically active peptides comprising
10 at least five amino acids and further comprising the
seguence Ala-Lys-Pro-Arg. In a preferred embodiment,
the peptide comprises the sequence Lys-Ala-Lys-Pro-Arg.
In another embodiment, the peptide comprises the
se~uence Ala-Lys-Pro-Arg-Ala. In a preferred
15 embodiment, the biologically active peptide is Glu-Thr-
Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro. In
still another embodiment, the peptide is Glu-Thr-Val-
Ile-Met-Lys-Ala-Lys-Pro-Arg. In yet another embodiment,
the peptide is Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro. The
20present invention also encompasses pharmaceutical
compositions cont~; ni ng a peptide comprising the
sequence Ala-Lys-Pro-Arg, and the use of the peptides
comprising the sequence Ala-Lys-Pro-Arg in suppressing
the proliferation of myeloid progenitor cells and in the
25prevention and treatment of septic shock. In another
embodiment, the present invention provides a method of
treatment of myelopoietic hyperproliferative disorders.
The present invention further provides a method of
reducing chemotherapy-induced myelosuppression. Yet
30another aspect of the present invention provides a
method of prevention of endotoxin-induced septic shock.
2 1 6 2 6 ~ ~ PCT/USg4/05773
094/28013
--5--
l Figure 1 is a graph providing the results of a
competitive ELISA comparing capacities of soluble
ferritin and 14-mer peptide to interfere with binding of
anti-peptide IgG with affixed peptide antigen.
5 The present invention is directed to
biologically active peptides which suppress the
proliferation of myeloid progenitor cells. The peptides
of the present invention contain from five to twenty or
more amino acids, and comprise the contiguous amino
lO acids Ala-Lys-Pro-Arg (SEQ ID N0:1). The residues Ala-
Lys-Pro-Arg are referred to herein as the tetramer. As
will be evident to the ordinarily skilled artisan, the
tetramer can be at any position in the peptide such that
the peptide maintains myelosuppressive activity.
15 Similarly, the additional residues in the peptide can be
any amino acids so long as myelosuppressive activity is
maintained. The myelosuppressive activity of the
peptides of the present invention can be determined as
discussed hereinbelow. In a preferred embodiment, the
20 peptide comprises the sequence Lys-Ala-Lys-Pro-Arg (SEQ
ID N0:2). In another embodiment the peptide comprises
the sequence Ala-Lys-Pro-Arg-Ala (SEQ ID N0:3). In yet
another ~mho~ t the peptide comprises the sequence
Glu-Thr-Val-Ile-Met-Lys Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro
25(SEQ ID N0:4). In a preferred embodiment, the
biologically active peptide is Glu-Thr-Val-Ile-Met-Lys-
Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro. In another preferred
embodiment the peptide is Glu-Thr-Val-Ile-Met-Lys-Ala-
Lys-Pro-Arg (SEQ ID N0:5). In still another preferred
30embodiment the peptide is Ala-Lys-Pro-Arg-Ala-Asn-Phe-
Pro (SEQ ID N0:6)
2 1 6 2 6 1 0 PCT~S94/05773
WO g4/28013
--6--
1 The peptides of the present invention may be
synthesized by methods known in the art. For example,
the peptides may be derived by chemical or enzymatic
cleavage from proteins or polypeptides containing the
5 subject peptides. Preferably, the peptides are
chemically synthesized by known methods including
solution or solid phase synthetic procedures such as
Merrifield synthesis, in which a protected amino acid is
bound to a resin particle as an ester bond. Solid phase
10 synthesis is commonly preferred for synthesis of longer
peptides, and short peptides can be made efficiently by
solution synthesis. After synthesis, the peptides can
be purified by art-recognized methods such as gel
electrophoresis, silica gel or alumina chromatography,
15 and high pressure liquid chromatography.
The myelosuppressive activity of the peptides
of the present invention can be determined by standard
assays which measure the proliferation of myeloid
progenitor cells. Such assays are known to one of
20 ordinary skill in the art. The peptides of the present
invention suppress the proliferation of a broad range of
progenitor cell subsets, including mature subsets of
granulocyte-macrophage progenitor cells ( CFU-GM)
macrophage progenitors (CFU-M), and granulocyte
25progenitors tCFU-G), and early subsets of CFU-GM,
erythroid (BFU-E) and multipotential (CFU-GEMM)
progenitors. Accordingly, any one of a variety of known
assays which measure the proliferation of such
progenitor cells is appropriate for determining the
30suppressive activity of the present peptides. Further,
the activity of the instant peptides is not species
specific, and thus activity can be measured in assays
-
2 1 6 2 6 ~ 0 PCT~S94/05773
094/28013
--7--
l using mammalian cells including, for example, human or
mouse myeloid progenitor cells.
A typical assay measures granulocyte-
macrophage colony and cluster formation and is described
5 in U.S. Patent No. ~,149,~44. Briefly, a single cell
suspension of bone marrow cells from normal endotoxin
resistant mice is prepared and cultured in soft agar
medium. The concentration of cells per assay is
typically 1 x 105 cells/ml. Proliferation of CFU-GM is
lO stimulated by the addition to each culture of murine GM-
CSF. Each culture further contains a peptide of the
present invention or control medium. Cultures are
incubated in a fully humidified C02 environment, and
total colonies (more than 50 cells) and clusters (4 to
15 50 cells) are scored after 5 to 8 days. The inhibitory
activity of a peptide is measured as the amount that
CSF-stimulated colony and cluster formation is decreased
relative to assays with control medium.
Suitable variations of the above-described
20 assay include measurement of suppression of colony
formation of other subsets of mouse or human progenitor
cells stimulated with appropriate growth factors or
stimulants. Such variations include measurements of
suppression of colony formation by immature subsets of
25mouse CFU-GM stimulated with recombinant murine GM-CSF
alone or the combination of recombinant murine GM-CSF
and recombinant murine Steel Factor (also known as mast
cell growth factor and stem cell factor). Measurement
of suppression of colony formation of mature macrophage
30progenitors ( CFU-M) stimulated with murine CSF-l, or
immature erythroid ( 8FU-E) or multipotential ( CFU-GEMM)
progenitors stimulated with recombinant human EPO can
W094/280~ 2 1 6 2 6 1 0 PCT~S94/05773
1 also be used to determine the activity of the peptides
of the present invention. Other appropriate assays
include, but are not limited to, measurement of
suppression of colony formation by immature subsets of
5 human CFU-GM stimulated with the combination of
recombinant human GM-CSF and recombinant human Steel
Factor, or mature CFU-GM stimulated with recombinant
human GM-CSF, or mature granulocyte progenitor cells
(CFU-G) stimulated with recombinant human G-CSF, or
lO immature BFU-E stimulated with recombinant human EPO in
combination with either recombinant human IL-3 or
recombinant human Steel Factor, or immature CFU-G~MM
stimulated with recombinant human EPO and recombinant
human Steel Factor.
As discussed hereinabove, the inhibitory
activity of a peptide is measured as the amount that
cytokine-stimulated colony and cluster formation is
decreased, and can be expressed as the percent change in
colony formation from control medium. In accordance
20with the present invention, a peptide is considered to
have suppressor activity if it is capable of
significantly inhibiting colony formation (p < 0.05)
relative to control medium in any of the above-described
or similar assays.
The myelosuppressive activity of the peptides
of the present invention can also be assessed by
injecting the peptides into an experimental animal, such
as a mouse, and determining effects on proliferation and
absolute numbers of progenitor cells in femur and
3Ospleen, and on nucleated cellularity in bone marrow,
spleen and blood. For example, the effect of the
peptides of the present invention on proliferation of
094/280~ 2 1 6 2 6 l 0 PCT~S94/05773
_9_
l myelopoietic progenitor cells can be determined as
described by Maze et al. ~1992) J. Immunol. 149:1004.
Briefly, mice are injected intravenously
(i.v.) with either sterile pyrogen-free saline or a
5 peptide of the instant invention diluted in sterile
pyrogen-free saline. Twenty-four hours after the single
dose i.v. injection, mice are assessed for effects of
the peptide on cycling rates (percentage of cells in the
S-phase of the cell cycle) of femoral bone marrow and
10 splenic CFU-GM, BFU-E and CFU-GEMM, and also on absolute
numbers of progenitor cells and nucleated cells in bone
marrow and spleen. Methods for evaluating absolute
numbers and cycling status of progenitor cells are known
to the ordinarily skilled artisan and described, for
15 example, by Broxmeyer et al. (1987) J. Clin. Invest.
79:721. In accordance with the present invention, a
peptide is considered to have suppressive effects of
progenitor cell proliferation if the peptide is capable
of significantly inhibiting absolute numbers of
20progenitors or percentage of progenitors in S-phase (p <
0.0~) relative to saline controls in the above-described
or a similar assay.
The present invention is further directed to
the pharmaceutically acceptable salts o~ the instant
25Peptides. The salts include those prepared by st~n~Ard
methods with pharmaceutically acceptable inorganic acids
such as hydrochloric, hydrobromic, nitric and sulfuric
acids, and pharmaceutically acceptable organic acids
such as citric, tartaric, fumaric, methanesulfonic and
30ethanesulfonic. The preferred salt is hydrochloride.
It has been found in accordance with the
present invention that peptides comprising the se~uence
PCT~S91105773 ~
W094/~80~ 2 1 6 2 6 1 0
--10--
l Ala-Lys-Pro-Arg are capable of inducing
myelosuppression, i.e. suppressing the proliferation of
myeloid progenitor cells, both in vitro and in vivo.
Accordingly, the present peptides are useful in
5 decreasing the proliferation of myeloid cells in
hyperproliferative disease states in which the
proliferative process has escaped regulation, such as
leukemia and polycythemia vera.
Hence another aspect o~ the present invention
lO provides a method of treatment of a myelopoietic
hyperproliferative disorder responsive to a
therapeutically effective amount of a peptide of the
present invention which comprises administering a
therapeutically effective amount of at least one peptide
15comprising the se~uence Ala-Lys-Pro-Arg to a patient.
In a preferred PmhoAiment of the method of treatment,
the peptide has the formula Ala-Lys-Pro-Arg. In another
preferred embodiment the peptide has the formula Glu-
Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro. In
20another embodiment the peptide comprises the se~uence
Ala-Lys-Pro-Arg-Ala or Lys-Ala-Lys-Pro-Arg. In yet
another embodiment the peptide comprises the seguence
Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.
In another embodiment the peptide has the formula Glu-
25Thr-val-Ile-Met-Lys-Ala-Lys-pro-Arg~ In yet another
embodiment the peptide has the formula Ala-Lys-Pro-Arg-
Ala-Asn-Phe-Pro. In accordance with the present
invention, a therapeutically effective amount is defined
as an amount which results in suppression of
30proliferation of myeloid progenitor cells. The
effectiveness of treatment can be assessed by analysis
of peripheral blood counts or of the frequency
-
2 1 6 2 6 1 0 PCT~S94/05773
V094/280~
1 (colonies/cells plated3 of hematopoietic progenitors
ICFU-GEMM, BFU-E, CFU-GM) in bone marrow or absolute
- numbers of such cells (colonies/ml of blood) in
peripheral blood before and after treatment. Effects on
5 proliferation can also be assessed by determining the
percentage of cells in S-phase (i.e., cycling
progenitors) in bone marrow aspirates. A reduction in
absolute number of progenitor cells or the percentage of
cells in S-phase is correlated with efficacy of
10 treatment. In a preferred embodiment, the
hyperproliferative disorder is leukemia. In a more
preferred embodiment, the leukemia is acute or chronic
myelogenous leukemia. In another preferred embodiment,
the hyperproliferative disorder is polycythemia vera.
Chemotherapeutic agents and irradiation are
known to cause severe myelosuppression due to their
effects on rapidly proliferating cells. Chemotherapy-
induced myelosuppression is the most common dose-
limiting and potentially fatal complication of cancer
20treatment. Hematopoietic growth factors including EPo,
G-CSF and GM-CSF are currently used to stimulate
hematopoiesis in patients with chemotherapy-induced
myelosuppression. Treatment of patients with the
peptides of the present invention prior to chemotherapy
25or radiation and between courses of treatment can
reversibly suppress the cycling rates of myeloid
progenitors and thus reduce the population of cells
subject to chemotherapy-induced damage. Treatment with
the peptides of the present invention protects myeloid
30cells from the effects of chemotherapy and irradiation
by placing the progenitors in a non-S phase portion of
the cell cycle. In a preferred embodiment, treatment
2 1 2 PCT~S94/05773 ~
WO94/28013 ~ 6 1 0
-~2-
l with at least one peptide comprising the sequence Ala-
Lys-Pro-Arg prior to chemotherapy is used in conjunction
with treatment with colony stimulating factors
subse~uent to chemotherapy.
Accordingly, another aspect of the present
invention provides a method of reducing chemotherapy-
induced myelosuppression which comprises administering a
therapeutically effective amount of at least one peptide
comprising the sequence Ala-Lys-Pro-Arg to a patient
lO prior to chemotherapy. In a preferred embodiment of the
method of treatment, the peptide has the formula Ala-
Lys-Pro-Arg. In another preferred embodiment the
peptide has the formula Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-
Pro-Arg-Ala-Asn-Phe-Pro. Peptides having the formulas
15Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg and Ala-Lys-Pro-
Arg-Ala-Asn-Phe-Pro are also contemplated. In another
embodiment the peptide comprises the se~uence Ala-Lys-
Pro-Arg-Ala or Lys-Ala-Lys-Pro-Arg. In yet another
embodiment the peptide comprises the sequence Glu-Thr-
20Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro. In
accordance with the present invention, a therapeutically
effective amount is defined as an amount which results
in suppression of proliferation of myeloid progenitor
cells. The effectiveness of treatment can be assessed
25bY analysis of peripheral blood counts or of the
frequency (colonies/cells plated) of hematopoietic
progenitors ~CFU-GEMM, BFU-E, CFU-GM) in bone marrow, or
absolute number of such cells (colonies/ml of blood) in
peripheral blood before and after treatment with the
30subject peptides. Effects on proliferation can also be
assessed by determining the percentage of progenitor
cells in S-phase in bone marrow aspirates.
PCT~S94105773
~ 094128013 2 1 6 2 6 1 0
-13-
l The present peptides are also useful in the
treatment and prevention of endotoxin-induced septic
shock. Endotoxin-induced septic shock is a disorder
characterized by ~ram negative bacteremia and sepsis
accompanied by circulatory changes such as hypotension
and disseminated intravascular coagulation resulting in
multiple organ failure. Septic shock is characterized
by a cascade of physiological disturbances resulting in
hypotension and resistance to vasoconstrictors.
lOInduction of nitric oxide synthase (NOS) in the vessel
wall by endotoxin and cytokines medi~tes enhanced
vasodilator tone and endothelial damage. Current
therapeutics in development target specific events in
the cascade and include anti-endotoxin products, nitric
15oxide inhibitors and cytokine inhibitors.
In accordance with the present invention, it
has been found that peptides comprising the sequence
Ala-Lys-Pro-Arg are capable of reducing susceptibility
to endotoxin-induced septic shock in a mammal at risk of
20septic shock. The peptides contain from five to twenty
amino acids and can be synthesized by art recognized
methods, as discussed hereinabove. As will be evident
to the skilled artisan, the tetramer can be at any
position in the peptide such that the peptide maintains
25the activity of reducing susceptibility to toxic shock.
Similarly, the additional residues in the peptide can be
any amino acids so long as biological activity (i.e.
reduction of susceptibility to septic shock) is
maintained. In a preferred embodiment the peptide
30comprises the sequence Ala-Lys-Pro-Arg-Ala. In another
emboAimPnt the peptide comprises the sequence Lys-Ala-
Lys-Pro-Arg. In yet another embodiment the peptide
WO941~8013 2 ~ 6 2 6 ~ 0 PCT~S94/05773
l comprises the sequence Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-
Pro-Arg-Ala-Asn-Phe-Pro. In a preferred embodiment the
peptide has the formula Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-
Pro-Arg-Ala-Asn-Phe-Pro. Peptides having the formulas
5 Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg and Ala-Lys-Pro-
Arg-Ala-Asn-Phe-Pro are also contemplated.
The efficacy of the peptides of the present
invention in reducing susceptibility to septic shock can
be assessed in an in vivo model which mimics a patient
lOat risk of lethal endotoxin-induced septic shock.
Briefly, an experimental animal such as a mouse is
treated intravenously (i.v.) with a peptide of the
present invention immediately prior to intraperitoneal
(i.p.) injection of E. coli lipopolysaccharide (LPS),
15the major toxic component of Gram negative bacterial
endotoxin. In this model, the dosage of LPS is 800
~g/mouse (40 mg/kg) which is approximately 2LD~o, and
the majority of deaths occur between 24 and 48 hours
after injection of LPS. The preferred dosage of the
20peptide of the present invention is this model is a~out
lO ~g. Peptides which statistically significantly
reduce mortality in this model relative to saline
controls are considered to be effective in a method of
prevention of septic shock in accordance with the
25Present invention.
Accordingly, the present invention provides a
method of prevention of endotoxin-induced septic shock
which comprises administering a therapeutically
effective amount of at least one peptide comprising the
3osequence Ala-Lys-Pro-Arg to a patient at risk of septic
shock. In a preferred embodiment of a method for
prevention of septic shock, the peptide has the formula
~ 094/~80~ 2 1 6 2 6 1 0 PCT~S94/05773
-15-
l Ala-Lys-Pro-Arg. In another pre~erred embodiment the
peptide has the formula Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-
Pro-Ar~-Ala-Asn-Phe-Pro. In another embodiment the
peptide comprises the sequence Ala-Lys-Pro-Arg-Ala or
5 Lys-Ala-Lys-Pro-Arg. In yet another embodiment the
peptide comprises the sequence Glu-Thr-Val-Ile-Met-Lys-
Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro. Peptides having the
formulas Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg and
Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro are also contemplated.
In sepsis, endotoxin and cytokines cause
induction of nitric oxide (NO) synthase in the
endothelium and vascular smooth muscle and in other
cells and tissues. Enhanced NO synthesis has a
cytotoxic effect characterized by increased
15vasodilation, endothelial damage and damage to other
cells (see, for example, Palmer (1993) Arch. Surg.
128:396), and is thus contributory to cardiovascular
collapse associated with the lethal phase of septic
shock. As discussed hereinabove, the peptides of the
20present invention are capable of reducing susceptibility
to endotoxin-induced septic shock. In particular, as
demonstrated by Tables 6, 7 and 8, the peptides of the
invention interfere with the action of LPS, the major
toxic component of bacterial endotoxin LPS is known to
25be a potent stimulant of inducible nitric acid synthase
(NOS), the enzyme which catalyzes the synthesis of NO.
Accordingly, the peptides of the present invention
reduce NO-induced pathological effects of cytotoxic
shock, i.e. NO-induced hypotens~on.
The present peptides may be administered to a
host as a pharmaceutical composition in a
therapeutically effective amount. The pharmaceutical
W094/28013 2 ~ 6 2 6 1 0 PCT~S94/0~773 a
-16-
l compositions contain a therapeutically effective dosage
of the peptides according to the present invention
together with a pharmaceutically acceptable carrier.
The skilled artisan can determine the dosage
5 of the present therapeutic peptides and compositions
which will be most suitable and it will vary with the
form of administration and the particular peptide
chosen, and furthermore, it will vary with the
particular patient under treatment. Generally treatment
lOis initiated with small dosages, substantially less than
the optimum dose of the compound, and the dosage is
increased by small increments until the optimum effect
under the circumstances is reached. It will generally
be found that when the composition is administered
15orally, larger quantities of the active agent will be
required to produce the same effect as a smaller
quantity given parenterally. The peptides are useful in
the same manner as comparable therapeutic agents and the
dosage level is of the same order of magnitude as is
20generally employed with those other therapeutic agents.
When given orally, the therapeutic doses of
the peptides of the present invention are generally
effective, even in the nanomolar range, and these
compounds are effective in micromolar quantities in the
25range of from about 10 to about 500 mg/kg of body weight
of treated mammal. When given parenterally, the
compounds are ~m; nistered generally in dosage of, for
example 0.01 mg/kg to about 200 mg/kg, also depending
upon the host and effect desired. The preferred dosage
30ranges from 0.5 to 10 mg/kg of body weight of treated
mammal.
2162610
094n80~ PCT~S94/05773
-17-
1 The compositions can be administered by well-
known routes including oral, intravenous (if soluble),
intramuscular, intranasal, intradermal, subcutaneous,
parenteral, enteral and the like. Depending on the
5 route of administration, the pharmaceutical composition
may require protective coatings.
The pharmaceutical forms suitable for
injectionable use include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous
lO preparation of sterile injectable solutions or
dispersions. In all cases the ultimate solution form
must be sterile and fluid. Typical carriers include a
solvent or dispersion medium containing, for example,
water buffered aqueous solutions (i.e., biocompatible
15buffers), ethanol, polyol such as glycerol, propylene
glycol, polyethylene glycol, suitable mixtures thereof,
surfactants or vegetable oils. Sterilization can be
accomplished by an art-recognized technique, including
but not limited to, addition of antibacterial or
20antifungal agents, for example, paraben, chlorobutanol,
phenol, sorbic acid or thimerosal. Further, isotonic
agents such as sugars or sodium chloride may be
incorporated in the subject compositions.
Production of sterile injectable solutions
25cont~i ni ng the subject peptides is accomplished by
incorporating these peptides in the required amount in
the appropriate solvent with various ingredients
enumerated above, as required, followed by
sterilization, preferably filter sterilization. To
30obtain a sterile powder, the above solutions are vacuum-
dried or freeze-dried as necessary.
2 1 6261 0
WO94/28013 PCT~S91/05773
-18-
l When the peptides are A~m;nistered orally, the
pharmaceutical compositions thereof contA;ning an
effective dosage of the peptide may also contain an
inert diluent, an assimilable edible carrier and the
5 like, be in hard or soft shell gelatin capsules, be
compressed into tablets, or may be in an elixir,
suspension, syrup or the like.
The following examples further illustrate the
present invention.
3o
2 1 6~6 1 ~
PCT~S94/05773
094/~8013
-19-
EXAMPI,~ 1
Lack of Cross~Reactivity
Between Anti-Ferritin and Anti-14-mer Peptide
The 14-mer peptide Glu-Thr-Val-Ile-Met-Lys-
5 Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro was synthesized by solid
phase synthesis at the peptide facility of the
Department of Medicine, Veterans Administration Medical
Center, University of Tennessee, Memphis, TN. The
peptide was purified by G-10 Sephadex chromatography,
lO and the amino acid analysis performed by the Molecular
Biology Resource Facility, University of Tennessee,
Knoxville was compatible wlth the molar ratios of the
peptide. This was confirmed at Indiana University
School of Medicine.
Antibodies to 14-mer peptide and rat liver
ferritin were raised in female New Zealand white rabbits
purchased from Myrtles Rabbitry, Thompson Station, TN.
One mg of antigen in 0.01M phosphate buffered saline, pH
7.2, (PBS) was emulsified in an equal volume of Freund's
complete adjuvant. Four subcutaneous (s.c.) injections
(0.25 ml each) were given above each appendage, and, at
30 day intervals thereafter, subsequent identical
antigen injections in PBS were given. Serum was
obtained on the seventh day following the second and
subse~uent injections. Immunog~obulin G was prepared by
25twice precipitation with 18~ Na2SO~. Peptide-specific
IgG was prepared by affinity purification over
immobilized 14-mer peptide. Seven mg of peptide with
free amine groups blocked with citraconic anhydride were
coupled to 2 ml of diaminodipropylamine gel using 1-
3ethyl-3-(3-dimethylaminopropyl) carbodiimide
- hydrochloride. The peptide-gel was treated with 0.5M
W094/280~ 2 1 6 2 6 1 0 PCT~S94/05773
-20-
1 ammonium acetate, pH 4.0, to release the anhydride from
amine groups of the bound peptide. Peptide specific IgG
eluted from the peptide-gel with O.SM ammonium acetate,
pH 4.0 was dialyzed against PBS, filter sterilized and
5 stored at -20C.
Competitive ELISAs for detecting cross-
reactivity for the two IgG preparations were performed
in 96-well Immulon 2 plates. Ferritin or peptide at 1.0
~g/50~1 in O.OlM carbonate buffer, pH 9.6, were added to
10 each well and allowed to adsorb overnight at 5C.
Antibodies in 50 ~1 of PBS containing 0.05~ Tween 20
were added to washed wells ln the presence or absence of
soluble ferritin or pcptide and incubated on a shaker at
37C for 60 min. Following washing with PBS-Tween,
15 bound IgG was detected with horseradish peroxidase
(HRP)-conjugated recombinant Protein G using 2,2'-azino-
di[3-ethyl-benzthiazoline-6-sulfonic acid~ as substrate.
Absorbance of individual wells was measured at 403 nm
using an Automated Microplate Reader. western
20 immunoblots were made from SDS-PAGE of ferritin using a
Mini Protean II gel apparatus with precast 4-15%
gradient gels. Samples were electrophoresed for 90 min.
with constant amperage of 14 mA. The proteins were
transferred to Immobilon-P PVDF membranes using a
25BioTrans semidry electrophoretic transfer unit with
0.025 M Tris-buffer, pH 8.5, containing 0.19 M glycine
and 0.1 M SDS. Following washing in Tris-buffered
saline (TBS), pH 7.S, for 15 min., the membranes were
blocked with 3% nonfat dry milk in TBS for 30 min. at
3037C and washed again with TBS containing 0.05 Tween 20
(TBS-Tween). The membranes were incubated overnight at
room temperature with the appropriate antibody in TBS-
2 ~ 626 1 0
094/28013 PCT~S94/05773
-21-
1 Tween, washed and incubated with HRP-protein G for 3 hr.
at room temperature. Bound protein G was detected wi~h
IBI Enzygraphic Web.
All assays were readily reproducible, and
5 assays were performed in triplicate. Values given are
means + the standard error of the mean. Statistical
analyses were performed by the student t test.
Immunoglobulin G antibodies were prepared from
sera of rabbits immunized with ferritin and the 14-mer
10 peptide. The anti-peptide IgG was further purified by
affinity purification using immobilized peptide. The
antibodies showed minimal cross-reactivity between
ferritin and the 14-mer peptide when assessed by ELISA,
thus demonstrating the lack o~ immuno-relatedness of the
15 14-mer and ferritin. As shown in Figure 1, soluble
ferritin did not reduce the binding of anti-peptide to
peptide affixed in ELISA wells although soluble peptide
effectively competed for the antibodies. Similar
results were ob~ained when ferritin and the 14-mer
20 peptide were used as competitors in a ferritin/anti-
ferritin ELISA, i.e., the peptide did not compete while
ferritin significantly reduced antibody binding to
affixed antigen. For the ELISA depicted in F~gure 1,
anti-pept~de at 600 ng/ml final concentration was added
25to peptide coated wells simultaneously with soluble
ferritin and peptide at the given concentration. Values
shown are absorbance at 403 nm following detection of
bound rabbit IgG with HRP-conjugated protein G.
3o
W094/280~ 2 1 6 2 6 1 0 PCT~S94/05773
-22-
1 EXAMPLE 2
This example demonstrates the suppressive
effect of the 14-mer peptide against proliferation of
murine CFU-GM.
The effects of the 14-mer peptide and a
control scrambled peptide on colony formation by murine
CFU-GM were assessed. 7.5 x 104 mouse BDFl bone marrow
cells/plate/ml were plated in the presence of 100 ~/ml
rmuGM-CSF plus SO ng/ml rmu Steel Factor and either the
lO 14-mer peptide, scrambled peptide or control medium.
Colonies were scored after seven days of incubation at
5~ CO2 and 5~ 2-
As shown in Tables 1 and 2, the 14-mer peptide
synthesized as in Example 1 and also synthesized at the
15 Indiana University School of Medicine, suppressed colony
formation by immature subsets of mouse BDFl bone marrow
granulocyte-macrophage progenitor cells (CFU-GM)
stimulated by the combination of recombinant (r) murine
(mu) granulocyte-macrophage colony stimulating factor
20 (GM-CSF) (Immunex Corporation) and rmu Steel Factor
(SLF, Immunex Corporation; also called c-kit ligand,
stem cell factor, and mast cell growth factor). The
specificity of the suppressive activity of the 14-mer
peptide was substantiated by showing in Table 1 that a
25 14-mer peptide containing the same amino acids as the
suppressive peptide except with a scrambled (random)
sequence: NH2-Glu (E)-Ala (A)-Thr (T)-Lys (K)-Val (V)-
Pro (P)-Ile (I)-Arg (R)-Met (M)-Lys (K)-Phe (F)-Ala (A)-
Asn (N)-Pro (P)-COOH (synthesized at the Indiana
30University School of Medicine) was not suppressive.
W094/280~ 2 1 S 2 6 l~ PCT~S94/0~773
-23-
1 TABLE 1
Effect of 14-Mer Peptide (NH2-
ETVIMKAKPRANFP-COOH) on Colony Formation
by Immature Subsets o~ Mouse Bone Marrow
Granulocyte-Macrophage Progenitor Cells (CEU-GM)~
Colony Formation (%
Change from Control
Medium)
Control Medium 134 + 3
14-Mer Peptide
~made in Tennessee) ~10-1M) 78 + 1 (-42)~
14-Mer Peptide
(made in Indiana) (10-1M) 81 + 4 (-40)~
15 Scrambled 14-Mer Peptide
(made in Indiana) (10-1M) 134 + 7 (O)
~ Results are expressed as mean + 1 SEM for 3
plates/point.
~ Significant % change from control medium, p
20 < 0.001 based on a two-tailed student's t-test; other
values not significantly different from control, p >
0.05.
3o
-
W094/28013 2 ~ 6 2 6 l O PCT~S94/05773
1 EXAMPL~ 3
As shown in Tables 2 and 3, the 14-mer peptide
(NH2-ETVIMKAKPRANFP-COOH) has a broad range of
activities on different subsets of myeloid progenitor
cells, and this activity is apparent on both murine
(Table 2) and human (Table 3) progenitors. As seen in
Table 2, the 14-mer peptide suppressed proliferation of
murine bone marrow immature CFU-GM stimulated with
rmuGM-CSF, mature macrophage progenitors (CFU-M)
stimulated with muCSF-1 and immature erythroid (BFU-E)
and multipotential (CFU-GEMM) progenitors stimulated
with 1 U/ml rhu erythropoietin tAmgen).
Interestingly, rmu macrophage inflammatory
protein (MIP)-lOa (R & D Systems) that has suppressive
15 activity against immature CFU-GM, BFU-E and CFU-GEMM
(Broxmeyer et al. Blood 76:1110, 1990; J. Immunol.
147:2586, 1991; Mantel et al. Proc. Natl. Acad. Sci. USA
90:2232, 1993), did not have activity on the mature CFU-
M, thus distinguishing the 14-mer peptide from MIP-lOa
(Table 2).
As seen in Table 3, the 14-mer peptide had
suppressive activity on human bone marrow immature CFU-
GM stim~ ted with rhuGM-CSF (Immunex Corporation) plus
rhuSLF (Immunex Corporation) mature CFU-GM st;m~ ted by
25 rhuGM-CSF, mature granulocyte progenitor cells (CFU-G)
stimulated by rhu granulocyte colony stimulating factor
(G-CSF; Immunex Corporation), immature BFU-E stimulated
with rhuEpo plus either rhu interleukin (IL)-3 (Immunex
Corporation) or rhuSLF and immature CFU-GEMM st;~ ted
3 by rhuEpo plus rhuSLF.
~WO 94128013 2 1 6 2 6 1 0 PCT/US94/05773
--25--
t JJ ^ ^ ~
t~ r~ tn ~ tn -- o
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t~ ut ~ ~r~ t~ ct
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r,~ .~ tn -~ O ~i
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p ", ~ ~ v a~
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~ J l ~ 1 ~ 1 t l ; ~a
n~ ~ o 4 rrt ~D tn tn ~t r~ O
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t r' tr~ O
~a ~ ~n -~ --t --r r~l rt ~ y.
_ ~ I J l ~ I J
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rn â~ , x
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I n _t ~ s 8 æ
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P~ ~~ t ~ t,,cn,
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J C~ ~ O ~ O l O l O l O ~
t ~r --~ t ~ I r4
t .0 ~ 'D
WO 94/28013 2 1 6 2 6 1 0 PCT/US94/05773
~1
~'5 D 1~ U 'J
Y ~ O
~ O ~ O O
C
o ~ o o o ~ a
~ + 1 + 1 + 1 + 1 + 1 +
O ~ D~
.. o
~1 IJ~ ~ D D C
~Id U ~rl 1~ U U ~ j~
~ a\ u~
~ O
+l +l +l +l +l +l +l U~ ~
r o ~ ao a~ 6
tn O r~l ~ ~ r~l ,
r~ .c I
o u ~ c
~,c~ I I _ r~
0 ~ r~l ~ a~ h
C~ ~ ~ +1 +1 ~'1 +1 +1 +1+1 ,C~ U
O~ ~ O o a~ D r~l ~ o
~~ ~ o o~ r-
~ I m
o ~ a
0 a~ ^ -- u~
~ ô ~P ~I rl~l r~l r~ ~
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0 E~ rJ ~ ~ r~ ~ ~ r~
UO~ ~ ~ I I I +l +l I +lO u~ o
.~ o ~ r~l r~l ~n ~9 0 ~ ~ X C ~,1
r~l r~
. N E Q~
U U
tn m ~n rr~ r~ tn f~
c _ _ I I I I , ~u~
Ql ~ _1 0 _ rJ r~ ~ r~
~ 0 E m u ~t,
J ~ ~ + ~ + I + I 1 l+ I ~1 + I+1 1 2E Eo
aJ E u~ ~ o
o ~ ~ r~ n ~n ~D 0 r~
u
3 o ~ o
- o
-- o o o o o E t~
:~: ~ ~ ~ ~ ~ u~ o
a) a) al a
O ~ ~a, ~, .,, ._,
~ s.~ , o
~ a) c) s~ Sa~ ~ u ~
-- ~, ~ ~ ' ~ ~ D '~ ~
2162~1~
WO94/28013 PCT~S94/05773
-27-
1 EXAMPLE 4
The effect of the 14-mer peptide on highly
purified normal human bone marrow progenitor cells was
examined. Five hundred non-adherent low density T-
lymphocyte depleted CD34'++ sorted bone marrow
cells/ml/plate were incubated for 14 days in a
humidified atmosphere adjusted to 5% COz and lowered
(5%) 2 tension in the presence of 1 U/ml rhu EPO, 50
ng/ml rhu Steel factor, 200 U/ml rhu GM-CSF and 200 U/ml
rhu IL-3. The results in Table 4 indicate 14-mer
peptide exerts suppressive effects directly on the
myeloid progenitor cells, since greater than 50% of the
cells in the population are progenitor cells. This
inhibition is similar to that noted when less than
15 1/1000 of the cells in a population is a progenitor cell
(see e.g. Table 3 and 4).
3o
2 1 6 2 6 1 0 PCT~US94/05773
W O 94/~80~
-28-
1 TABLE 4
Colony Formation (% Inhibition)'
Colonie~ Col nn; es + BFU-~ CFU-GEMH
Cluster Colonies Colonie~
Control57 + 10 126 + 10 60 + 10 17 + 5
14-Her Peptide9 + 1 28 + 4 25 + 4 7 + 2
(lo-lo M) (-84)~ (-78)~ (-58)~ (-59)
" Results are based on the mean + 1 SEM of 3
plates/point.
Significant difference from control medium p < 0.001.
~ p < 0.05
3o
2 1 6 2 6 1 ~ PCT/USg4/05773
094/~8013
-29-
1 EXAMPLE 5
Mice were injected i.v. with 0.2 ml of sterile
pyrogen-free saline, or 0.2 ml (containing 2 ng) of the
14-mer peptide or a scrambled 14-mer peptide. Mice were
sacrificed 24 hours later. These studies were done
exactly as reported for the in vivo action of rmuMIP-la
(Maze et al. J. Immunol. 149:1004, 1992). Results shown
are the means + 1 SEM for one representative experiment
in which a total of 4 mice per group were each
lO individually assessed. The se~uence of the 14-mer
peptide is: NH2-Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-
Arg-Ala-Asn-Phe-Pro-COOH. The se~uence of the scrambled
14-mer peptide is: NH~-Glu-Ala-Thr-Lys-Val-Pro-Ile-Arg-
Met-Lys-Phe-Ala-Asn-Pro-COOH (SEQ ID NO:7).
The _ vivo activity of the 14-mer peptide is
demonstrated by the results in Table 5, which shows that
24 hours after an iv injection of 2 ng 14-mer peptide,
there is a significant decrease in the absolute numbers
of femoral marrow and splenic granulocyte-macrophage
20 (CFU-GM), erythroid (BFU-E) and multipotential (CFU-
GEMM) progenitor cells, and in the cyclin~ rates
(percentage of progenitors in S-phase of the cell cycle)
of these cells. The specificity of the 14-mer peptide
is demonstrated ~y the inability of the scrambled 14-mer
25 peptide (also inactive in vitro; see Table 1) to
suppress myeloid progenitor cells in vivo (Table 5).
3o
WO 94/28013 2 1 6 2 6 1 0 PCT/US94/05773
--30--
+l +l l l +l+l +
O ~D t~l r l
.
o ~ ~ ~D er O a
~ rt ~0 U')
-
:1
0 ~ N
+l +l +l +l +l +l
i~ ~ ~ O O r1 r-~~
U ~ o
R .,
_~ O rt t~ O _t
o o o o o or~
15 ~ ~ +l +l +l +l +l +
N 1~
r
O O O O O O
N ~ ~ 0 N O~
~ OOO OOO~
P I I I I + I r_
~D O ~ ~ N ISl ¢l
2 0 N --t N N ~~1 IYi Ir7
o
t~ N O 1~ ~ ~D
... ... ~/
O O t O O O
+l +l +l +l +l +l
t O ~ r 0
a~ m r~
a
C ~1 ~ d' E
~ O o ~ o co tr~ a~ o
'--~ ooo oooU~
O +l ~ 1 t l +1 +1 +1 -~
~-t ~) ~ r~ o0 r~ )
t X ~ t rt 0`1
:~ '
a~ ~
.1 ,-
r ~ rt
u ~ ~ r .~
a~ ~C ~ -- ~ ~ ~
h ~ ~ ~ Q~ o
rt .a) rt rt
3 5 ~ o s, -- ~
u~ ~ 1 u. Q,
u c
t C. m ~. tU'~
~ 094/~013 2 1 6 2 6 ~ ~ PCT~594/05773
1 EXAMPLE 6
Five week old female Balb/c mice were injected
i.v. with a single dose of the l4-mer peptide Glu-Thr-
Val-Ile-Me~-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro at a
dose ranging from l ~g - l.0 mg/mouse in O.l ml of PBS
pH 7.0 immediately prior to i.p. injection of 800 ~g E.
coli LPS (phenol-water extraction, Sigma). The
influence of the peptide on survival is shown in Table
6. Lethality represents deaths occurring within 72
lO hours. The efficacious dose (lO ~g) saved 50% of mice
given LPS sufficient to kill 80 - lO0~ of controls,
WO94/28013 2 1 6 2 6 1 0 PCT~S94/05773
-32-
1 EXAMPLE 7
Five week old female Balb/c mice were injected
i.v. with a single 10 ~g dose of the 14-mer peptide or
carrier buffer immediately prior to i.p. injection of
100-800 ~g E. coli LPS. The influence of the peptide on
survival is shown in Table 7. Lethality represents
deaths occurring within 72 hours. As demonstrated by
the data in Table 7, the 14-mer peptide enhances
resistance to lethal endotoxin shock.
3o
2 1 6 2 6 1 0 PCT~US94/05773
WO94/28013
-33-
1 TABLE 6
Peptide dose/mouse (mg/kg) Dead/Total % Survival
1.0 mg 50 5/5 0
100 ~g 5 20/25 20
10 ~g 0.5 8/19 58
1 ~g 0.05 6/8 25
1 o o 7/8 12
TABLE 7
LPS Dose Peptide Dead/Total
100 ~g _ 0/6
100 ~g + 0/6
200 ~g - 0/6
200 ~g + 0/6
400 ~g 3/6
400 ~g + 1/6
800 ~g - 6/6
800 ~g + 3/6
3o
2 1 6 2 6 1 0 PCT/USg1/05773
W094/28013
-34-
1 EXANPLE 8
The inhibitory influence of the 14-mer peptide
(Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-
Pro), a 10-mer pep~ide (Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-
Pro-Arg) and an 8-mer peptide (Ala-Lys-Pro-Arg-Ala-Asn-
Phe-Pro) on colony formation by murine marrow
progenitors was examined. Soft-agar culture containing
5 x 104 adherent cell-depleted nucleated marrow cells
were stimulated with M-CSF at 750 U/ml and either 0.1
lO ng/ml LPS or 0.6 mM sodium nitroprusside (SNP) added to
stimulate additional colony formation by S-phase
positive transitional progenitors. Colonies formed
after 6 day incubation at 37C in 5% C02 were
enumerated. The results in Table 8 demonstrate that the
15 8-mer, lO-mer and 14-mer peptides were e~uivalently
inhibitory for colony formation by the S-phase positive
transitional progenitors. None of the peptides were
inhibitory for the basal colony response stimulated
solely by M-CSF.
As demonstrated by the results presented in
Table 8, sodium nitroprusside (SNP~, a generator of N0
in a~ueous solution, has properties identical to LPS as
a transitional cell stimulant. The peptides of the
present invention inhibit stimulation of colony
25 formation by transitional progenitors stimulated by
either LPS or SNP, indicating that N0 produced in
response to LPS is responsible for stimulation of s-
phase positive transitional progenitors and that the
peptides interfere with the effects of N0.
3o
2~6261()
PCT~S94/05773
W094/280
-35-
1 TABLE 8
Colonie~ + S~5 x 10~ Nucleated Marrow Cells-
Tnh; hi tor~
St~mulant None 14-merlO-mer 8-mer 4-mer
CSF~ 188 + 4~ 192 + 2~ 185 + 6~ 183 + 4~ 190 + 4
CSF + 0.1 233 + 4 194 + 5~ 186 + 12~ 183 + 4~ 188 + 4
lO ng~ml LPS
CSF+0.06 235 + 3 204 + 3~ 194 + 6~ 188 + 5~ 185 + 6
mM SNP
' 6 day colony response for triplicate cultures.
~ All inhibitors at 10-9 M final concentration.
c 750 U M-CSF/ml
d Significantly less than LPS or SNP positive controls at
p < 0.05.
3o
2 ~ 6 2 6 l 0 PCT~S94105773 ~
W094/280~
-36-
u~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Research Corporation Technologies, Inc.
101 N. Wilmot Road, Suite 600
Tucson, Ariæona 85711-3335
U.S.A.
(ii) TITLE OF INVENTION: MYELOPOIETIC PROGENITOR CELL
INHIBITOR ~ll~ES
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Scully, Scott, Murphy & Presser
(B~ STREET: 400 Garden City Plaza
(C) CITY: Garden City
(D) STATE: New York
1 (E) COUNTRY: United States
(F) ZIP: 11530
(V) ~oIl~l~K READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version ~1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NU.~K:
(B) FILING DATB:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: DiGiglio, Frank S.
(B) REGISTRATION NUMBER: 31,346
(C) REFERENCE/DOCKET NUMBER: 8933
3o
2~62610
W094/280~ PCT~S94/05773
-37-
1 (ix) TELECGI~luNICATION INFORMATION:
(A) TELEPHONE: (516) 742-4343
(B) TELEFAX: (516) 742-4366
(C) TELEX: 23û 901 SANS UR
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ala Lys Pro Arg
15 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
~xi) ~ CE DESCRIPTION: SEQ ID NO:2:
Lys Ala Lys Pro Arg
1 5
25 ~2) INFORMATION FOR SEQ ID NO:3:
~NCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: l;ne~r
(ii) MOLECULE TYPE: peptide
PCT~S94/05773 ~
W094/280~ 2 1 6 2 6 1 0
-38-
1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Lys Pro Arg Ala
1 5
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Glu Thr Val Ile Met Lys Ala Lys Pro Arg Ala Asn Phe Pro
1 5 10
15 (2) INFORMATION FOR SEQ ID NO:5:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Glu Thr Val Ile Met Lys Ala Lys Pro Arg
1 5 10
25 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
tD) TOPOLOGY: linear
3o (ii) MOLECULE TYPE: peptide
2162610
PCT~S94tO5773
WO941~8013
-39-
l (xi) SEQUENCE DBSCRIPTION: SEQ ID NO:6:
Ala Lys Pro Arg Ala Asn Phe Pro
l 5
. 5 (2) INFORMATION FOR SEQ ID No:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Glu Ala Thr Lys Val Pro Ile Arg Met Lys Phe Ala Asn Pro
1 0
3o
