Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4703—Inhibitors; Suppressors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the proliferation and differentiation of hematopoietic progenitor cells are regulated by an interactive network of stimulatory and inhibitory molecules.
• the biomolecules have the capacity to directly stimulate the proliferation and/or
• cytokines are multifunctional, and may manifest stimulatory or suppressive activity depending upon the target cell or assay system.
• EPO Erythrop ⁇ ietin
• G-CSF granulocyte-colony stimulating factor
• GM-CSF granulocyte macrophage- colony stimulating factor
• H ferritin suppress growth factor induced clonal growth factor
• Macrophage inflammatory protein (M ⁇ P)-1 ⁇ has suppressive activity on more immature progenitor cells, including human and murine colonies forming units of granulocytes,
• CFU-GEMM erythrocytes, macrophages, and megakaryocytes
• IFNs interferons
• necrosis factors TNFs
• prostaglandins PGEi and PGE 2 TGFs
• inhibin TGF- ⁇
• TGF transforming growth factor
• 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.
• the suppressor molecule has an apparent molecular weight of 8 kD, whichis similar to MlP-1 ⁇ , but it is apparently biochemically and immunologically distinct from MlP-1 ⁇ .
• U.S. Patent No. 4,384,991 describes an inhibitor purified from granulocytes which inhibits theproliferation of normal and leukemic myeloid cells. The inhibitor is reported to have the amino acid composition Tau 1 Asx 1 Ser 2 Thr 3. Glx 3 Gly 2 Ala 1 (PO 4 )-. Lu et al. (1989) Exp. Hematol. 17: 935 report the suppressive activity of the synthetic pentapeptide Glu-Glu-Asp-Cys-Lys.
• M-CSF macrophage colony stimulating factor
• LPS lipopolysaccharide
• Cytokines which have stimulatory effects on myelopoiesis have clinical utility in the treatment of chemotherapy-induced myelosuppression.
• regulators are potentially useful in treatment of hematopoietic disorders by dampening blood cell
• 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
• novel peptides have been discovered which are capable of 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.
• the peptide comprises the sequence Lys-Ala-Lys-Pro-Arg.
• the peptide comprises the sequence
• the biologically active peptide is Glu-Thr- Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.
• the peptide is Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg.
• the peptide is Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.
• the present invention also encompasses pharmaceutical compositions containing a peptide comprising the
• FIG. 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
• 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:1).
• the residues Ala-Lys-Pro-Arg are referred to herein as the tetramer.
• the tetramer can be at any position in the peptide such that the peptide maintains myelosuppressive activity.
• 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.
• the peptide comprises the sequence Lys-Ala-Lys-Pro-Arg (SEQ ID NO: 2).
• the peptide comprises the sequence Ala-Lys-Pro-Arg-Ala (SEQ ID NO:3).
• the peptide comprises the sequence Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro (SEQ ID NO: 4).
• the peptide comprises the sequence Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro (SEQ ID NO: 4).
• the peptide comprises the sequence Glu-Thr-Val-Ile-Met-Lys-Ala-L
• biologically active peptide is Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.
• the peptide is Glu-Thr-Val-Ile-Met-Lys-Ala- Lys-Pro-Arg (SEQ ID NO:5).
• the peptide is Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro (SEQ ID NO: 6)
• the peptides of the present invention may be synthesized by methods known in the art.
• the peptides may be derived by chemical or enzymatic cleavage from proteins or polypeptides containing the subject peptides.
• the peptides are
• 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 granulo ⁇ yte-macrophage progenitor cells (CFU-GM) macrophage progenitors (CFU-M), and granulocyte
• CFU-G CFU-G progenitors
• BFU-E erythroid
• CFU-GEMM multipotential progenitors
• 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- macrophage 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 ⁇ 10 5 5ells/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 CO 2 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 mouse 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).
• CFU-M mature macrophageprogenitors
• BFU-E immature erythroid
• CFU-GEMM multipotential progenitors stimulated with recombinant human EPO
• Other appropriate assays include, but are not limited to, measurement 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.
• CFU-G mature granulocyte progenitor cells
• 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.
• a peptide is considered to have suppressor activity if it is capable of
• the myelosuppressive activity of the peptides of the present invention can also be assessed by
• the peptides of the present invention can be determined as described by Maze et al. (1992) J. Immunol. 149:1004.
• mice are injected intravenously
• 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.
• cycling rates percentage of cells in the S-phase of the cell cycle
• progenitors or percentage of progenitors in S-phase 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 instantpeptides.
• 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 andethanesulfonic.
• the preferred salt is hydrochloride.
• myelosuppression i.e. suppressing the proliferation of myeloid progenitor cells, both in vitro and in vivo.
• proliferative process has escaped regulation, such as leukemia and polycythemia vera.
• Another aspect of the present invention provides a method of treatment of a myelopoietic
• 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.
• the peptide has the formula Ala-Lys-Pro-Arg.
• the peptide has the formula Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.
• the peptide comprises the sequence Ala-Lys-Pro-Arg-Ala or Lys-Ala-Lys-Pro-Arg.
• 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
• a therapeutically effective amount is defined as an amount which results in suppression of
• 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
• hyperproliferative disorder is leukemia.
• the leukemia is acute or chronic myelogenous leukemia.
• 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 myeloidcells from the effects of chemotherapy and irradiation by placing the progenitors in a non-S phase portion of the cell cycle.
• treatment 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
• 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.
• the peptide has the formula Ala-Lys-Pro-Arg.
• 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 peptide comprises the sequence Ala-Lys-Pro-Arg-Ala or Lys-Ala-Lys-Pro-Arg.
• the peptide comprises the sequence Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.
• 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 assessedby analysis of peripheral blood counts or of the
• Endotoxin-induced septic shock is a disorder characterized by Gram 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.
• NOS nitric oxide synthase
• 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.
• the tetramer can be at any position in the peptide such that the peptide maintains the activity of reducing susceptibility to toxic shock.
• 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
• the peptide comprises the sequence Ala-Lys-Pro-Arg-Ala.
• the peptide comprises the sequence Lys-Ala-Lys-Pro-Arg.
• the peptide comprises the sequence Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.
• 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.
• 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 lipop ⁇ lysaccharide (LPS), the major toxic component of Gram negative bacterial endotoxin.
• LPS E. coli lipop ⁇ lysaccharide
• the dosage of LPS is 800 ⁇ g/mouse (40 mg/kg) which is approximately 2LD 50 , 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 ⁇ 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 thepresent invention.
• the present invention provides a method of prevention of endotoxin-induced septic shock which comprises administering a therapeutically
• the peptide has the formula Ala-Lys-Pro-Arg.
• the peptide has the formula Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro.
• the peptide comprises the sequence Ala-Lys-Pro-Arg-Ala or Lys-Ala-Lys-Pro-Arg.
• 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.
• nitric oxide (NO) synthase In sepsis, endotoxin and cytokines cause induction of nitric oxide (NO) synthase in the
• Enhanced NO synthesis has a
• the peptides of the present invention are capable of reducing susceptibility to endotoxin-induced septic shock.
• 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.
• NOS inducible nitric acid synthase
• 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
• compositions contain a therapeutically effective dosage of the peptides according to the present invention together with a pharmaceutically acceptable carrier.
• 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 therange of from about 10 to about 500 mg/kg of body weight of treated mammal.
• 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
• Typical carriers include a solvent or dispersion medium containing, for example, water buffered aqueous solutions (i.e., bio ⁇ ompatible 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 thimer ⁇ sal.
• isotonic agents such as sugars or sodium chloride may be
• 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 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
• the peptide was purified by G-10 Sephadex chromatography, and the amino acid analysis performed by the Molecular Biology Resource Facility, University of Tennessee, Knoxville was compatible with the molar ratios of the peptide. This was confirmed at Indiana University
• Antibodies to 14-mer peptide and rat liver ferritin were raised in female New Zealand white rabbits purchased from Myrtles Rabbitry, Thompson Station, TN.
• IgG was prepared by affinity purification over
• the peptide-gel was treated with 0.5M 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 stored at -20°C.
• Antibodies in 50 ⁇ l 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 ⁇ C for 60 min. Following washing with PBS-Tween, bound IgG was detected with horseradish peroxidase
• BioTrans 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 7.5, for 15 min., the membranes were blocked with 3% nonfat dry milk in TBS for 30 min. at37°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- Tween, washed and incubated with HRP-protein G for 3 hr. at room temperature. Bound protein G was detected with IBI Enzygraphic Web.
• Immunoglobulin G antibodies were prepared from sera of rabbits immunized with ferritin and the 14-mer 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 immuno-relatedness of the 14-mer and ferritin.
• soluble ferritin did not reduce the binding of anti-peptide to peptide affixed in ELISA wells although soluble peptide effectively competed for the antibodies.
• 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 ⁇ 10 4 mouse BDF 1 bone marrow cells/plate/ml were plated in the presence of 100 ⁇ /ml rmuGM-CSF plus 50 ng/ml rmu Steel Factor and either the 14-mer peptide, scrambled peptide or control medium. Colonies were scored after seven days of incubation at 5% C0 2 and 5% O 2 .
• CFU-GM bone marrow granulocyte-macrophage progenitor cells
• GM-CSF granulocyte-macrophage colony stimulating factor
• SSF rmu Steel Factor
• the specificity of the suppressive activity of the 14-mer peptide was substantiated by showing in Table 1 that a 14-mer peptide containing the same amino acids as the suppressive peptide except with a scrambled (random) sequence: NH 2 -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
• the 14-mer peptide (NH 2 -ETVIMKAKPRANFP-COOH) has a broad range of
• rmu macrophage inflammatory protein (M ⁇ P)-10 ⁇ R & D Systems
• M ⁇ P macrophage inflammatory protein
• 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.
• IL interleukin
• a progenitor cell see e.g. Table 3 and 4).
• 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-1 ⁇ (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
• the sequence of the 14-mer peptide is: NH 2 -Glu-Thr-Val-Ile-Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro-COOH.
• the sequence of the scrambled 14-mer peptide is: NH 2 -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
• mice 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 dose ranging from 1 ⁇ g - 1.0 mg/mouse in 0.1 ml of PBS pH 7.0 immediately prior to i.p. injection of 800 ⁇ g E. coli LPS (phenol-water extraction, Sigma).
• E. coli LPS phenol-water extraction, Sigma
• mice 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.
• the 14-mer peptide enhances resistance to lethal endotoxin shock.
• sodium nitroprusside (SNP) a generator of NO in aqueous solution, has properties identical to LPS as a transitional cell stimulant.
• SNP sodium nitroprusside
• the peptides of the present invention inhibit stimulation of colony
• 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.

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