EP0698039A1 - Peptides for suppression of myeloid progenitor cell proliferation and treatment of septic shock - Google Patents

Abstract

Peptides containing the sequence Ala-Lys-Pro-Arg have suppressive effects on the proliferation of a broad range of myelopoietic progenitor cells. In a preferred embodiment, the peptide has the sequence Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro. The peptides of the present invention are useful in the treatment of myelopoietic hyperproliferative disorders, and in protecting myeloid cells from damage induced by chemotherapy and radiation. Peptides containing the sequence Ala-Lys-Pro-Arg are also useful in the treatment of endotoxin-induced septic shock.

Description

94/28013

PEPTIDES FOR SUPPRESSION OF MYELOID PROGENITOR 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 molecules. The bio olecules 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 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 myelopoiesis. Erythropσietin (EPO), granulocyte-colony stimulating factor (G-CSF), and granulocyte macrophage- colony stimulating factor (GM-CSF) are known to be stimulatory to the production of myeloid cells, and have clinical utility in the treatment of chemotherapy- induced myelσsuppression.

Molecules which act to suppress myelopoiesis are also known, and have been reviewed by Broxmeyer

(1992) Am. J. Pediatr. Hematol. Oncol. 3,4:22. Both purified natural and recombinant molecules have been implicated as physiologically active suppressor molecules. Some of these molecules act directly at the progenitor cell level to decrease the proliferative capabilities of such cells, while others act indirectly to decrease production and/or release of stimulatory cytokines. 94/28013

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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 administered to mice. Both immature and more mature progenitor cells are responsive to inhibition by H ferritin. Macrophage inflammatory protein (MΙP)-lα has suppressive activity on more immature progenitor cells, including human and murine colonies forming units of granulocytes, erythrocytes, macrophages, and megakaryocytes (CFU-GEMM) and murine CFU-A. The interferons (IFNs), tumor necrosis factors (TNFs), prostaglandins PGEi and PGE₂, inhibin, and transforming growth factor (TGF)-β have also been implicated as suppressor factors for myeloid progenitor cells. The iron-binding protein lactoferrin exerts indirect suppressive activity by decreasing the production or release of colony stimulating factors or IL-1 from onocytes and macrophages.

Bone marrow and spleen cells from mice treated ith lactoferrin, H ferritin or PGE release a molecule in vitro with suppressive activity for early myeloid progenitors (Gentile et al., 1989, Blood 74: 228a; U.S. Patent No. 5,149,544 to Gentile et al.). The suppressor molecule has an apparent molecular weight of 8 kD, which is similar to MlP-lα, but it is apparently biochemically and immunologically distinct from MlP-lα.

Peptide suppressors of myelogenesis have also been reported. U.S. Patent No. 4,384,991 describes an inhibitor purified from granulocytes which inhibits the proliferation of normal and leukemic myeloid cells. The inhibitor is reported to have the amino acid composition 94/28013

^•3-

Tau₁Asx₁Ser₂Thr_3.Glx₃Gly₂Ala_i(P0₄)^~. Lu et al. (1989) Exp. Hematol. 17: 935 report the suppressive activity of the synthetic pentapeptide Glu-Glu-Asp-Cys-Lys. Moore et al. (1988) J. Immunol. 141:2699 report that a tuftsin analog, (ALA³-)-tuftsin, has an inhibitory effect on a restricted population of progenitor cells responsive to acrophage colony stimulating factor (M- CSF or CSF-1) and lipopolysaccharide (LPS).

Cytokines which have stimulatory effects on 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 selectively placing normal progenitors out of cycle and thus in a reversibly protected state from the effects of S-phase specific chemotherapeutic drugs. Accordingly, 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 intravascular 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 sepsis and septic shock.

In accordance with the present invention, novel peptides have been discovered which are capable of 94/28013

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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 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 at least five amino acids and further comprising the sequence 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 sequence Ala-Lys-Pro-Arg-Ala. In a preferred 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 present invention also encompasses pharmaceutical compositions containing 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 prevention 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 another aspect of the present invention provides a method of prevention of endotoxin-induced septic shock. 94/28013

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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. 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 acids Ala-Lys-Pro-Arg (SEQ ID NO:l). 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 yelosuppressive activity. 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 peptide comprises the sequence Lys-Ala-Lys-Pro-Arg (SEQ ID NO:2). In another embodiment the peptide comprises the sequence Ala-Lys-Pro-Arg-Ala (SEQ ID NO:3). In yet another embodiment the peptide comprises the sequence Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro (SEQ ID NO: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 NO:5). In still another preferred embodiment the peptide is Ala-Lys-Pro-Arg-Ala-Asn-Phe- Pro (SEQ ID NO:6) 94/28013

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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 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 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, 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 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 granuloσyte-macrophage progenitor cells (CFU-GM) acrophage progenitors (CFU-M), and granulocyte progenitors (CFU-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 suppressive activity of the present peptides. Further, the activity of the instant peptides is not species specific, and thus activity can be measured in assays using mammalian cells including, for example, human or mouse myeloid progenitor cells.

A typical assay measures granulocyte- acrophage colony and cluster formation and is described in U.S. Patent No. 5,149,544. 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 10^s cells/ml. Proliferation of CFU-GM is 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 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 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 ouse 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 progenitors (CFU-M) stimulated with murine CSF-1, or immature erythroid (BFU-E) or multipotential (CFU-GEMM) progenitors stimulated with recombinant human EPO can 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 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 immature BFU-E stimulated with recombinant human EPO in combination with either recombinant human IL-3 or recombinant human Steel Factor, or immature CFU-GEMM 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 ^wi^tn 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 spleen, and on nucleated cellularity in bone marrow, spleen and blood. For example, the effect of the peptides of the present invention on proliferation of 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 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 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 example, by Broxmeyer et al. (1987) J. Clin. Invest. J _. '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 progenitors or percentage of progenitors in S-phase (p < 0.05) relative to saline controls in the above-described or a similar assay.

The present invention is further directed to the pharmaceutically acceptable salts of the instant peptides. The salts include those prepared by standard 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 ethanesulfonic. The preferred salt is hydrochloride. It has been found in accordance with the present invention that peptides comprising the sequence 94/28013

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Ala-Lys-Pro-Arg are capable of inducing yelosuppression, i.e. suppressing the proliferation of myeloid progenitor cells, both in vitro and in vivo. Accordingly, the present peptides are useful in 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 of the present invention 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 comprising the sequence Ala-Lys-Pro-Arg to a patient. 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. In another embodiment the peptide comprises the sequence Ala-Lys-Pro-Arg-Ala or 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. In another embodiment the peptide has the formula Glu- Thr-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 proliferation of myeloid progenitor cells. The effectiveness of treatment can be assessed by analysis of peripheral blood counts or of the freguency 94/28013

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(colonies/cells plated) of hematopoietic progenitors (CFU-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 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 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 treatment. 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 or 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 cells 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 94/28013

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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 subsequent 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 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 Glu-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 sequence Ala-Lys- Pro-Arg-Ala or Lys-Ala-Lys-Pro-Arg. In yet another embodiment the peptide comprises the sequence Glu-Thr- al-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 by 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 øsubject peptides. Effects on proliferation can also be assessed by determining the percentage of progenitor cells in S-phase in bone marrow aspirates.

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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 Gram negative bactere ia 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. Induction of nitric oxide synthase (NOS) in the vessel wall by endotoxin and cytokines mediates enhanced vasodilator tone and endothelial damage. Current therapeutics in development target specific events in the cascade and include anti-endotoxin products, nitric oxide 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 septic 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 the 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 comprises the sequence Ala-Lys-Pro-Arg-Ala. In another embodiment the peptide comprises the sequence Lys-Ala- Lys-Pro-Arg. In yet another embodiment the peptide 4/28013

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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 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 at 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 lipopσlysaccharide (LPS), the major toxic component of Gram negative bacterial endotoxin. In this model, the dosage of LPS is 800 μg/mouse (40 g/kg) which is approximately 2LD₅₀, and the majority of deaths occur between 24 and 48 hours after injection of LPS. The preferred dosage of the peptide of the present invention is this model is about 10 μg. 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 present 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 sequence 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 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 another embodiment the peptide comprises the sequence Ala-Lys-Pro-Arg-Ala or 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 vasodilation, 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 present 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 be 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 hypotension. The present peptides may be administered to a host as a pharmaceutical composition in a therapeutically effective amount. The pharmaceutical 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 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 is 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 orally, 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 generally 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 range of from about 10 to about 500 mg/kg of body weight of treated mammal. When given parenterally, the compounds are administered 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 ranges from 0.5 to 10 mg/kg of body weight of treated mammal. 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 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 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., bioσompatible buffers), 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 antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid or thimerσsal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.

Production of sterile injectable solutions containing 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 obtain a sterile powder, the above solutions are vacuum- dried or freeze-dried as necessary. When the peptides are administered orally, the pharmaceutical compositions thereof containing an effective dosage of the peptide may also contain an inert diluent, an assimilable edible carrier and the 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.

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EXAMPLE 1

Lack of Cross-Reactivity Between Anti-Ferritin and Anti-14-mer Peptide

The 14-mer peptide Glu-Thr-Val-Ile-Met-Lys- 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, and the amino acid analysis performed by the Molecular 0 Biology Resource Facility, University of Tennessee, Knoxville was compatible with the molar ratios of the peptide. This was confirmed at Indiana University School of Medicine.

Antibodies to 14-mer peptide and rat liver 5 . . 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

(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 subsequent injections. Immunoglobulin G was prepared by 5 twice precipitation with 18% Na₂S0₄. 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-

3 ^J0ethyl-3-(3-dιmethylaminopropyl) carbodiimide hydrochloride. The peptide-gel was treated with 0.5M

35 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 0.5M ammonium acetate, pH 4.0 was dialyzed against PBS, filter sterilized and 5 stored at -20°C.

Competitive ELISAs for detecting cross- reactivity for the two IgG preparations were performed in 96-well I mulon 2 plates. Ferritin or peptide at 1.0 μg/50μl in 0.01M carbonate buffer, pH 9.6, were added to 0 each well and allowed to adsorb overnight at 5°C. Antibodies in 50 μl of PBS containing 0.05% Tween 20 were added to washed wells in the presence or absence of soluble ferritin or peptide and incubated on a shaker at 37^βC for 60 min. Following washing with PBS-Tween, 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 n using an Automated Microplate Reader. Western o 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 A. The proteins were transferred to Immobilon-P PVDF membranes using a _cBioTrans se idry 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 7.5, for 15 min., the membranes were blocked with 3% nonfat dry milk in TBS for 30 min. at _₀37°C 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-

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1 Tween, washed and incubated with HRP-protein G for 3 hr. at room temperature. Bound protein G was detected with 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.

I munoglobulin 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 of im uno-relatedness of the 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 obtained when ferritin and the 14-mer o 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 Figure 1, anti-peptide at 600 ng/ l final concentration was added

2 to 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.

30

35 1 EXAMPLE 2

This example demonstrates the suppressive effect of the 14-mer peptide against proliferation of murine CFU-GM.

5 The effects of the 14-mer peptide and a control scrambled peptide on colony formation by murine CFU-GM were assessed. 7.5 x 10" mouse BDF_α bone marrow cells/plate/ml were plated in the presence of 100 μ/ml rmuGM-CSF plus 50 ng/ml rmu Steel Factor and either the 0 14-mer peptide, scrambled peptide or control medium. Colonies were scored after seven days of incubation at 5% C0₂ and 5% 0₂.

As shown in Tables 1 and 2, the 14-mer peptide synthesized as in Example 1 and also synthesized at the 5 Indiana University School of Medicine, suppressed colony formation by immature subsets of mouse BDF_α bone marrow granulocyte-macrophage progenitor cells (CFU-GM) stimulated by the combination of recombinant (r) murine (mu) granulocyte-macrophage colony stimulating factor o (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

2514-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 _QUniversity School of Medicine) was not suppressive.

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TABLE 1

Effect of 14-Mer Peptide (NH₂-

ETVIMKAKPRANFP-COOH) on Colony Formation by Immature Subsets of Mouse Bone Marrow

Granulocyte-Macrophage Progenitor Cells (CFU-GM)'

^a Results are expressed as mean + 1 SEM for 3 plates/point. ^to Significant % change from control medium, p < 0.001 based on a two-tailed student's t-test; other values not significantly different from control, p > 0.05.

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EXAMPLE 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 (Amgen).

Interestingly, rmu macrophage inflammatory protein (MΙP)-lOα (R & D Systems) that has suppressive 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 MlP-lOα (Table 2).

As seen in Table 3, the 14-mer peptide had suppressive activity on human bone marrow immature CFU- GM stimulated with rhuGM-CSF (Immunex Corporation) plus rhuSLF (Immunex Corporation) mature CFU-GM stimulated by 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 stimulated by rhuEpo plus rhuSLF.

TAB 2 2

^*" Colony formation was assessed for 1 x 10* low density ( < 1.077 gm/cm³ after separation by Ficoll-Hypaque) cells/ml based an 3 plates/point after 14 days incubation at 5% C0_a and 5% 0„ . Results are given as mean _+ 1 3EM.

*^* % Change from control (McCoy' s 5A) aedium-

" Significant change from control, p < 0.01. p < 0.05.

TABLE 3

^~ Colony formation was assessed for 7.5 x 10 mouse BDF-_. bone marrow cells/ml based on 3 plates/point after 7 days of incubation at 5% C0₂ and 5% 0_... Results are given as mean + 1 SEM.

^~~ % change from control (McCoy's 5A) medium.

" Significant % change from control, p < 0.01. ^c3 p < 0.05.

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^"4""*^""'^" sorted bone marrow cells/ml/plate were incubated for 14 days in a humidified atmosphere adjusted to 5% C0₂ and lowered (5%) O- 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 1/1000 of the cells in a population is a progenitor cell (see e.g. Table 3 and 4).

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TABLE 4

" Results are based on the mean _+ 1 SEM of 3 plates/point.

-' Significant difference from control medium p < 0.001.

° p < 0.05

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-lα (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 individually assessed. The sequence of the 14-mer peptide is: NH₂-Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Prσ- Arg-Ala-Asn-Phe-Pro-COOH. The sequence of the scrambled 14-mer peptide is: NH₂-Glu-Ala-Thr-Lys-Val-Pro-Ile-Arg- Met-Lys-Phe-Ala-Asn-Pro-COOH (SEQ ID NO:7). The in 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 (CFU-GM), erythroid (BFU-E) and multipotential (CFU- GEMM) progenitor cells, and in the cycling rates (percentage of progenitors in S-phase of the cell cycle) of these cells. The specificity of the 14-mer peptide is demonstrated by the inability of the scrambled 14-mer peptide (also inactive in vitro; see Table 1) to suppress myeloid progenitor cells in vivo (Table 5).

TABLE 5

^~ Significantly different from control, p < 0.005 as determined by 2- tailed student's test. ^b p < 0.05.

EXAMPLE 6

Five week old female Balb/c mice were injected i.v. with a single dose of the 14-mer peptide Glu-Thr- Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro at a 5 dose ranging from 1 μg - 1.0 mg/mouse in 0.1 ml of PBS pH 7.0 immediately prior to i.p. injection of 800 μg 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 ° hours. The efficacious dose (10 μg) saved 50% of mice given LPS sufficient to kill 80 - 100% of controls.

5

0

5

0

5 EXAMPLE 7

Five week old female Balb/c mice were injected i.v. with a single 10 μg dose of the 14-mer peptide or carrier buffer immediately prior to i.p. injection of 100-800 μg 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.

/2801

-33-

TABLE 6

TABLE 7

4/28013

-34-

EXAMPLE 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 peptide (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 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 37°C in 5% C0₂ were enumerated. The results in Table 8 demonstrate that the 8-mer, 10-mer and 14-mer peptides were equivalently 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 NO in aqueous solution, has properties identical to LPS as a transitional cell stimulant. The peptides of the present invention inhibit stimulation of colony formation by transitional progenitors stimulated by either LPS or SNP, indicating that NO produced in response to LPS is responsible for stimulation of S- phase positive transitional progenitors and that the peptides interfere with the effects of NO. /28013

-35-

TABLE 8

^a 6 day colony response for triplicate cultures. ^to All inhibitors at 10^"9 M final concentration. ^e 750 U M-CSF/ml

^Λ Significantly less than LPS or SNP positive controls p < 0.05.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Research Corporation Technologies, Inc. 101 N. il ot Road, Suite 600 Tucson, Arizona 85711-3335 U.S.A.

(ii) TITLE OF INVENTION: MYELOPOIETIC PROGENITOR CELL

INHIBITOR PEPTIDES

(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 (E) COUNTRY: United States (F) ZIP: 11530

(v) COMPUTER 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 NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: DiGiglio, Frank S.

(B) REGISTRATION NUMBER: 31,346

(C) REFERENCE/DOCKET NUMBER: 8933 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (516) 742-4343

(B) TELEFAX: (516) 742-4366

(C) TELEX: 230 901 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 1 (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) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Lys Ala Lys Pro Arg

1 5 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide ^x (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Ala Lys Pro Arg Ala 1 5

-. (2) INFORMATION FOR SEQ ID NO:4: 5

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear 0 (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Glu Thr Val lie Met Lys Ala Lys Pro Arg Ala Asn Phe Pro 1 5 10

(2) INFORMATION FOR SEQ ID NO:5: 5

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear 0 (ϋ) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Glu Thr Val lie Met Lys Ala Lys Pro Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO:6: 5

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear 0 (ϋ) MOLECULE TYPE: peptide

5 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Ala Lys Pro Arg Ala Asn Phe Pro 1 5 NFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Glu Ala Thr Lys Val Pro lie Arg Met Lys Phe Ala Asn Pro 1 5 10

Claims

WHAT IS CLAIMED IS:

1. A peptide comprising at least five amino acids wherein said peptide comprises the sequence Ala-Lys-Pro-Arg.

2. The peptide of Claim 1 comprising the sequence Ala-Lys-Pro-Arg-Ala, Lys-Ala-Lys-Pro-Arg, or Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.

3. A peptide having the formula Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.

4. A peptide having the formula Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg.

5. A peptide having the formula Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.

6. A peptide capable of suppressing the proliferation of myeloid progenitor cells wherein said peptide comprises at least five amino acids and wherein said peptide comprises the seguence Ala-Lys-Pro-Arg.

7. The peptide of Claim 6 comprising the sequence Ala-Lys-Pro-Arg-Ala, Lys-Ala-Lys-Pro-Arg, or Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.

8. A peptide capable of reducing the susceptibility of a mammal to septic shock wherein said peptide comprises at least five amino acids and wherein said peptide comprises the sequence Ala-Lys-Pro-Arg.

9. The peptide of Claim 8 comprising the sequence Ala-Lys-Pro-Arg-Ala, Lys-Ala-Lys-Pro-Arg, or Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.

10. A pharmaceutical composition comprising a peptide comprising the sequence Ala-Lys-Pro-Arg and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition comprising the peptide of any one of Claims 1-10.

12. A method of treatment of a myelopoietic hyperproliferative disorder which comprises

administering a therapeutically effective amount of a peptide comprising the sequence Ala-Lys-Pro-Arg to a patient.

13. The method of Claim 12 wherein said myelopoietic hyperproliferative disorder is acute or chronic myelogenσus leukemia or polycythemia vera.

14. The method of Claims 12 or 13 wherein said peptide comprises the sequence Ala-Lys-Pro-Arg-Ala, Lys-Ala-Lys-Pro-Arg, or Glu-Thr-Val-Ile-Met-Cys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.

15. The method of Claims 12 or 13 wherein said peptide has the formula Glu-Thr-Val-Ile-Met-Cys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro, Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg, Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro, or Ala-Lys-Pro-Arg.

16. The method of Claims 12 or 13 wherein said therapeutically effective amount is from about 0.01 mg/kg to about 200 mg/kg body weight.

17. A method of reducing chemotherapy-induced or radiation-induced myelosuppression which comprises administering a therapeutically effective amount of a peptide comprising the sequence Ala-Lys-Pro-Arg to a patient prior to chemotherapy or radiation.

18. The method of Claim 17 wherein said peptide comprises the sequence Ala-Lys-Pro-Arg-Ala, Lys-Ala-Lys-Pro-Arg, or Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.

19. The method of Claim 17 wherein said peptide has the formula Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro, Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg, Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro or Ala-Lys-Pro-Arg.

20. The method of any one of Claims 17-19 wherein said therapeutically effective amount is from about 0.01 mg/kg to about 200 mg/kg body weight.

21. A method of prevention of septic shock which comprises administering a therapeutically

effective amount of a peptide comprising the sequence Ala-Lys-Pro-Arg to a patient at risk of septic shock.

22. The method of Claim 21 wherein said peptide comprises the sequence Ala-Lys-Pro-Arg-Ala, Lys-Ala-Lys-Pro-Arg, or Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.

23. The method of Claim 21 wherein said peptide has the formula Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro, Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg, Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro or Ala-Lys-Pro-Arg.

24. The method of any one of Claims 21-23 wherein said therapeutically effective amount is from about 0.01 mg/kg to about 200 mg/kg body weight.

25. A method of reducing nitric-oxide induced hypotension which comprises administering a

therapeutically effective amount of a peptide comprising the sequence Ala-Lys-Pro-Arg to a patient at risk of septic shock.