ZA200401574B - Casein derived peptides and uses thereof in therapy - Google Patents

Casein derived peptides and uses thereof in therapy Download PDF

Info

Publication number
ZA200401574B
ZA200401574B ZA200401574A ZA200401574A ZA200401574B ZA 200401574 B ZA200401574 B ZA 200401574B ZA 200401574 A ZA200401574 A ZA 200401574A ZA 200401574 A ZA200401574 A ZA 200401574A ZA 200401574 B ZA200401574 B ZA 200401574B
Authority
ZA
South Africa
Prior art keywords
peptide
casein
pharmaceutical composition
derived
terminus portion
Prior art date
Application number
ZA200401574A
Inventor
Sidelman Zvi
Original Assignee
Peptera Pharmaceuticals Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/942,121 external-priority patent/US20020147144A1/en
Application filed by Peptera Pharmaceuticals Ltd filed Critical Peptera Pharmaceuticals Ltd
Publication of ZA200401574B publication Critical patent/ZA200401574B/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4732Casein
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/06Antihyperlipidemics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/08Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • A61P31/18Antivirals for RNA viruses for HIV
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/02Antineoplastic agents specific for leukemia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00General protective or antinoxious agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00Drugs for disorders of the blood or the extracellular fluid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06Antianaemics

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Oncology (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Epidemiology (AREA)
  • Communicable Diseases (AREA)
  • Obesity (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • AIDS & HIV (AREA)
  • Endocrinology (AREA)
  • Emergency Medicine (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Description

CASEIN DERIVED PEPTIDES AND USES THEREOF IN THERAPY
. FIELD OF THE INVENTION:
The present invention relates to biologically active peptides that are ) s derived from or are similar to sequences identical with the N-terminus of the a
S1 fraction of milk casein. These peptides are capable of stimulating and enhancing immune response, protecting against viral infection, normalizing serum cholesterol levels, and stimulating hematopoiesis. The casein-derived peptides are non-toxic and can be used to treat and prevent immune pathologies, hypercholesterolemia, hematological disorders and viral-related diseases.
BACKGROUND OF THE INVENTION
Bioactive molecules from nutrients:
In addition to the nutritional value of many foods, certain fractions and products of digestive pathways possess the ability to influence physiological processes. Some of these “extranutritional” constituents are present in their active form in the whole nutriment, such as the immunoglobulins in mother’s milk and colostrums, phytoestrogens found in soy-based foods, polyphenolic antioxidants from fruits and vitamins. Others are encrypted within nutrient molecules, and are rcleased in an active form during digestion or food processing, for example antihypertensive peptides from lactoglobin [Kitts, D.
D. (1999), Can. J. Physiol. Pharmacol. 72:4; 423-434}.
Biological activity in milk proteins:
Casein, the predominant milk protein, has been traditionally defined as composed of three fractions, a, B and y, according to their electrophoretic mobility [N. J. Hipp, et al. (1952), Dairy Sci., 35:272]. Today casein is defined according to the amino acid sequences of each of the subgroups aS1, aS2, f and x [W. N. Engel et al. (1984), }. Dairy Sci. 67: 1599].
In the course of digestion, the cascin proteins are subjected to proteolytic cleavage by acid proteases such as chymosin (rennin), trypsin and pepsin. producing shorter peptides and causing curdling and calcium sequestration by - the resultant protein fragments. A few studies with milk compounds 5s demonstrated casein-related bacteriocidal activity. U.S. Patent No. 3.764.670 : discloses proteolytic casein digests possessing antibiotic properties against microorganisms. Isracl Patent No. 42863 describes a casein-derived peptide consisting of 23 amino acids of the N-terminus of cascin. possessing anti-bacterial activity. In addition, other physiologically active properties. such as opioid and growth factor-like activities have been proposed for casein or its derivatives [Kitts, D. D., (1999), ibid ].
Immune modulating activity has also been observed in casein peptides. [Coste et al. (1992), Immun. Lett. 33: 41-46)] observed enhancement of rat lymphocyte proliferation following treatment with a peptide derived from the
C-terminus of j cascin. However. none of these studies have determined the specific sequences in these cascin peptides which confer their “extranutritional” properties.
Hematopoiesis in cancer therapy:
Following high-dose chemotherapy, especially following myeloablative 50 doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT), patients are at high risk duc 10 pancytopenia.
Granulocytopenia may lead to development of serious, occasionally fatal infectious complications from common bacterial, viral, fungal and parasitic agents in the immediate post transplant period. Similarly, thrombocytopenia frequently results in bleeding tendency and occasionally, in long lasting platelet dependence. Whenever resistance to platelets develops, blecding episodes can be life threatening and hemorrhagic complications are frequently lethal. The risk due to granulocytopenia can be partially overcome by supportive measures 50 and most effectively by administration of recombinant human cytokines that can enhance reconstitution of granulocytes, particularly granulocyte colony stimulation factor (G-CSF) and granulocyte macrophage colony stimulating . factor (GM-CSF). These agents are extremely expensive (approximately $200-400/day/patient) and infrequently cause side effects due to ’ 5 hypersensitivity reactions, fever, bone pain and occasionally vascular leak syndromes, including pericarditis and pleuritis. Some of the side effects may be due to other cytokines that may be intrinsically released by these hematopoietic growth factors. Moreover, the use of these hematopoietic growth factors may be prohibitive in patients with tumor cells bearing G-CSF or GM-CSF receptors such as in acute and chronic myeloid leukemias and in myelodysplastic syndromes. Whereas major progress in treating patients at risk of pancytopenia has deen achieved from the use of hematopoietic cytokines, no progress has been made in the treatment of thrombocytopenia. Following high dose chemotherapy and especially following ASCT, patients are at risk for thrombocytopenia which may last for many months even up to 3 years and some thromboctyopenic patients may never recover. Many patients previously treated with multiple blood products become platelet resistant and hence thrombocytopenia may be impossible to overcome, even transiently, despite intensive and frequent platelet transfusions from a single donor. Resistance to platelets and protracted thrombocytopenia represent a common cause of death at ASCT centers worldwide.
Currently, several new recombinant cytokines such as recombinant human interleukin-3 (rhIL3) and recombinant human interleukin-6 (rhIL6) are being investigated as potential agents for enhancing megakaryocytopoiesis and platelet reconstitution. Unfortunately, preliminary clinical trials showed that although rhIL3 and rhIL6 may enhance platelet reconstitution, such effects are by no means dramatic and may take considerable time.
Clearly, protracted thrombocytopenia represents a major problem in clinical Bone Marrow Transplant centers today, for which no satisfactory solution has yet been found.
There is thus a widely recognized need for, and it would be highly advantageous to have a safe, inexpensive, rapidly effective and well-defined stimulator of hematopoiesis, and specifically megakaryocytopoiesis, devoid of the above limitations.
Thrombopoetin (TPO) in regulation of hematopoiesis and platelet y function:
TPO appears to be the major regulator of platelet production m vivo, although increase in the kidncy- and liver-derived growth factor in platelet deficiencies is not caused by adaptation of TPQ biosynthesis in these organs.
Rather. a “feed-back loop” secms to exist in which the number of circulating platelets determines how much of the circulating TPO is available to the bone marrow for platelet production. In addition, it has been demonstrated that TPO is an carly acting cytokine with important multilineage cffects: TPO alone, or in combination with other early acting cytokines, can (i) promote viability and
Is suppress apoptosis in progenitor cells: (11) regulate hematopoietic stem ccll production and function; (iii) trigger cell division of dormant multipotent cells: (iv) induce multilineage differentiation and (v) enhance formation of multilineage colonies containing granulocytes, erythrocytes, macrophages. and megakaryocytes (MK, CFU-GEMM). Moreover, TPO stimulates the production of more limited progenitors for granulocyte/monocyte, megakaryocyte and erythroid colonics, and stimulates adhesion of primitive human bone marrow and megakaryocytic cells to fibronectin and fibrinogen.
Thus. TPO is an important cytokine for clinical hematologists/transplanters: for the mobilization, amplification and ex vivo expansion of stem cells and 2s committed precursor cells for autologous and allogeneic transplantation [von dem Borne, A.E.GKr, et al, (1998) Thrombopoietin: it’s role in platelet disorders and as a new drug in clinical medicine. In Bailliers Clin. Hematol.
June:11(2), 427-45).
In addition to TPO effects in hematopoiesis, this potent growth factor 350 primes platelets for various agonists and modulates platelet-extracellular matrix
: interactions. Although it does not itself cause platelet aggregation, TPO upregulates ADP-induced aggregation, especially on the second wave of v aggregation, upregulates granule (ADP, ATP, serotonin, etc.) release and production of thromboxane B2, increases platelet attachment to collagen and 5 potentiates shear-induced platelet aggregation. TPO also stimulates PMN activation, inducing IL-8 release and priming oxygen metabolite production, likely enhancing antimicrobial defense.
Clinical studies suggest TPO’s value in understanding and treating a variety of hematological conditions. In patients with idiopathic aplastic anemia (AA), elevated TPO levels persist even in remission following immunosupressive therapy, indicating a hematopoietic defect. TPO is clevated in other forms of aplastic thrombocytopenia as well, but not in conditions of
N increased platelet destruction. Apparently, the reactive increase in TPO production is insufficient in cases of destructive thrombocytopenia. Thus, TPO is not only a therapeutic option for aplastic, but also for destructive thrombocytopenia.
Thrombopoietic agents are of great clinical interest, for prevention and/or treatment of pathological or treatment-induced thrombocytopenia, and as a substitute for platelet transfusions. Of the cytokines evaluated, all but the marginally potent IL-11 have been deemed unacceptable for clinical use. TPO is widely believed to become the cytokine of choice for throbocytopenia treatment. Recombinant human TPO (Genentech) has recently become available, enabling accurate pharmacokinetic determinations and clinical trials.
Thus, TPO’s potential applications encompass the rcalms of supportive care (post chemo/radio-therapy, bone marrow and stem cell transplantation), hematological discase (AA, myelodysplasia, congenital and acquired thrombocytopenia), liver diseases, transfusion (expansion, harvest, mobilization and storage of platelets) and surgery (including liver transplantation). Of particular interest is the potential use of TPO/EPO/G-CSF cocktail for 50 myelodysplasia, G-CSF and TPO combination for peripheral stem cell mobilization and TPO in harvesting CD 34+ cells and ex vivo expansion of megakaryocytes for superior platclet reconstitution. However. similar to other hematopoietic agents under consideration for clinical application, TPO is costly and potentially antigenic at therapeutically effective levels. Thus, it would be advantageous to have a safe, inexpensive and readily available stimulator of : thrombopoiesis capable of augmenting TPO activity.
The aS1 fraction of casein:
The aS] fraction of casein can bc obtained from milk proteins by various methods [D. G. Schmidth and T. A. J. Paynes (1963), Biochim, i0 Biophys. Acta, 78:492; M. P. Thompson and C. A. Kiddy (1964), J. Dairy Sci., 47-626; J. C. Mercier, et al. (1968), Bull. Soc. Chim. Biol. 50:521}, and the complete amino acid sequence of the aS1 fraction of casein was determined by
J. C. Mercier et al. (1971) (Eur. J. Biochem. 23:41). The genomic and coding sequences of bovine aSl fraction of casein have also been cloned and is sequenced employing recombinant DNA techniques [D. Koczan, ef al. (1991).
Nucl. Acids Res. 19(20): 3391; McKnight, R. A., er @l (1989). 1. Dairy Sci 72:2464-73]. Proteolytic cleavage and identification of N-terminal fragments from the aS1 fraction of casein has been documented [J. C. Mercier, er al. (1970), Eur. J. Biochem. 16:439: P. L. H. McSweeney et al. (1993), J. Dairy
Res.. 60:401], as has the intestinal absorption and appearance of this fragment in mammalian plasma following ingestion of whole milk proteins [Fiat, AM.. et al. (1998) Biochimie, 80(2):2155-65]. Meisel, H. and Bockelmann, W. [(1999), Antonie Van Leeuwenhock, 76:207-15] detected amino acid sequences of immunopeptidcs, casokinins and casomorphins in peptides liberated by lactic acid bacteria digests of o and B casein fractions. Of particular interest 1s the anti-aggregating and thrombolytic activity demonstrated for C-terminal portions of the o- and k-casein fractions [Chabance, B. er al. (1997). Biochem. Mol.
Biol. Int. 42(1) 77-84; Fiat AM. et al. (1993), J. Dairy Sci. 76(1): 301-310].
Previous studies documented potential bioactive peptides encrypted In the N-terminal aS1 amino acid sequence, but no mention was made of use of these protein fragments, specific sequences or defined synthetic peptides to ‘ enhance hematopoiesis, prevent viral infection or modulate the development of autoimmune diseases.
SUMMARY OF THE INVENTION
According to the present invention there is provided a method of preventing or treating an autoimmune disease, the method effected by administering to a subject in nced thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating a virai disease, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS] casein.
Further according to the present invention there is provided a method of preventing viral infection, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of inducing hematopoiesis, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS] casein.
Further according to the present invention there is provided a method of inducing hematopoietic stem cells proliferation, the method effected by 5 administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS] casein.
Further according to the present invention there is provided a method of inducing hematopoietic stem cells proliferation and differentiation, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of inducing megakaryocytopoiesis, the method effected by administering to a s subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of inducing erythropoiesis, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N jo terminus portion of aS! casein.
Further according to the present invention there is provided a method ot inducing leukocytopoiesis, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein. is Further according to the present invention there is provided a method of inducing thrombocytopoiesis. the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an
N terminus portion of aS] casein.
Further according to the present invention there is provided a method of inducing plasma cell proliferation, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of inducing dendritic cell proliferation, the method effected by administering to a 2s subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS] casein.
Further according to the present invention there is provided a method of inducing macrophage proliferation, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein. . Further according to the present invention there is provided a method of preventing or treating thrombocytopenia, the method effected by administering ' 5 10 a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating pancytopenia, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS! casein.
Further according to the present invention there is provided a method of preventing or treating gganulocytopenia, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating hyperlipidemia, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating cholesteremia, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating glucosuria, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived a from an N terminus portion of aS1 casein. » Further according to the present invention there is provided a method of preventing or treating diabetes, the method effected by administering to a subject in need thercof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of’ preventing or treating AIDS, the method cffected by administering to a subject s in necd thereof a therapeutically effective amount of a peptide derived trom an .
N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating infection by HIV, the method effected by administering to a subject in need thercof a therapeutically effective amount of a peptide 10 derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating conditions associated with myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT). the method effected by administering to a subject in need thereot a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of treating an erythropoietin treatable condition, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived trom an N terminus portion of aS1 casein.
Further according to the present invention there 1s provided a method of augmenting the cffect of erythropoietin, the method cffected by administering to a subject in need thereof a therapeutically effective amount of a peptide »s derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of - treating a thrombopoietin treatable condition, the method effected by } administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Bi
Further according to the present invention there is provided a method of augmenting the effect of thrombopoietin, the method effected by administering , to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS| casein.
Further according to the present invention there is provided a method of enhancing peripheral stem cell mobilization, the method effected by administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising effective amounts of thrombopoietin and a peptide derived from an N terminus portion of a Si casein.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating an autoimmunc disease, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating a viral disease, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing viral infection, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing hematopoiesis, the pharmaceutical h composition comprising, as an active ingredient, a peptide derived from an N
R terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing hematopoietic stem cells proliferation, the pharmaceutical composition comprising. as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing hematopoietic stem cells proliferation and differentiation, the pharmaceutical composition comprising. as an active ingredient, a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing megakaryocytopoiesis, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a 1s pharmaceutical composition for inducing erythropoiesis, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of uS1 cascin and a pharmaceutically acceptable CATTICT.
Further according to the present invention there is provided a pharmaceutical composition for inducing leukocytopoiesis, the pharmaccutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing thrombocytopoiesis, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there 1s provided a pharmaceutical composition for inducing plasma cell proliferation, the pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier. . Further according to the present invention there is provided a pharmaceutical composition for inducing dendritic cell proliferation, the ’ 5 pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing macrophage proliferation, the pharmaceutical composition comprising a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating thrombocytopenia, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating pancytopenia, the pharmaceutical composition comprising, as an active ingredient, a peptide 50 derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating granulocytopenia, the pharmaceutical composition comprising, as an active ingredient, a peptide 2s derived from an N terminus portion of aSI casein and a pharmaceutically ) acceptable carrier. . Further according to the present invention there is provided a pharmaceutical composition for preventing or treating hyperlipidemia, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating cholesteremia. the s pharmaceutical composition comprising, as an active ingredient, a peptide . derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating glucosuria, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating diabetes, the is pharmaceutical composition comprising. as an active ingredient. a peptide derived from an N terminus portion of aS! casein and a pharmaceuncally acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating AIDS, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSI casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating infection by HIV. the pharmaceutical composition comprising, as an active ingredient. a peptide derived from an N terminus portion of aSl casein and a pharmaceutically ~ acceptable carrier. .
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating conditions associated with myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic . bone marrow transplantation (BMT), the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus - 5 portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for treating a thrombopotetin treatable condition, the pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceuticai composition for augmenting the effect of tnrombopoietin, the pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for enhancing peripheral stem cell mobilization, the pharmaceutical composition comprising, as active ingredients thrombopoietin and a peptide derived from an N terminus portion of a SI casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing hematopoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier. ” Further according to the present invention there is provided a . pharmaceutical composition for inducing hematopoietic stem cells proliferation, the pharmaceutical composition comprising, as active ingredients,
thrombopoietin and a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing hematopoietic stem cells proliferation s and diffcrentiation, the pharmaceutical composition comprising, as active ingredients. thrombopoietin and a peptide derived from an N terminus portion of aS1 cascin and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing megakaryocytopoiesis. the to pharmaceutical composition comprising, as active ingredients, thrombopoictin and a peptide derived from an N terminus portion of oaS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing erythropoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSi casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing leukocytopoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing thrombocytopoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of oS casein and a ) pharmaceutically acceptable carrier. .
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating thrombocytopenia, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl cascin and a . pharmaceutically acceptable carrier.
Further according to the present invention there is provided a ’ s pharmaceutical composition for preventing or treating pancytopenia, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating granulocytopenia, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a 15s pharmaceutical composition for treating or preventing an indication selected from the group consisting of autoimmune disease or condition, viral disease, viral infection, hematological disease, hematological deficiencics, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, glucosuria, hyperglycemia, diabetes, AIDS, HIV-1, helper T-cell disorders, dendrite cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, human immune system disorders resulting from treatment of diseases of »s immune deficiency and bacterial infections, the pharmaceutical composition ) comprising, as an active ingredient, a peptide derived from an N terminus . portion of aS1 cascin and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for treating or preventing an indication selected from the group consisting of hematological disease, hematological deficiencies. thrombocytopenia, pancytopenia. granulocytopenta, dendrite cell deficiencies. macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions. s leukemic conditions, myelodysplastic syndrome, aplastic anemia and bone . marrow insufficiency. the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of «S1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a method of enhancing colonization of donated blood stem cclls in a myeloablated recipient. the method effected by treating a donor of said donated blood stem cells with a peptide derived from an N terminus portion of aS! casein prior to donation and : implanting the donated blood stem cells in the recipient.
Further according to the present invention there is provided a method of is enhancing colonization of donated blood stem cells in a myeloablated recipient. the method eftected by treating suid donated blood stem cells with a peptide derived from an N terminus portion of aS1 casein prior to implanting the donated blood stem cells in the recipient.
Further according to the present invention there is provided a method of enhancing colonization of blood stem cells in a myeloablated recipient. the method effected by treating said blood stem cells with a peptide derived from an N terminus portion of aS1 casein prior to implanting the blood stem cells in the recipient.
Further according to the present invention there is provided a method of 2s enhancing colonization of donated blood stem cells in a myeloablated recipient. the method effected by treating a donor of said donated blood stem cells with a - peptide derived from an N terminus portion of aS1 casein and thrombopoietin . prior to donation and implanting the donated blood stem cells in the recipient. -
Further according to the present invention there is provided a method of enhancing colonization of donated blood stem cells in a myeloablated recipient, ] the method effected by treating said donated blood stem cells with a peptide derived from an N terminus portion of aS1 casein and thrombopoietin prior to ) 5 implanting the donated blood stem cells in the recipient.
Further according to the present invention there is provided a method of enhancing colonization of blood stem ceils in a myeloablated recipient, the method effected by treating said blood stem cells with a peptide derived from an N terminus portion of aS1 casein and thrombopoietin prior to implanting the blood stem cells in the recipient.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for the preparation of a medicament for preventing or treating an autoimmune disease.
Further according to the present invention there is disclosed the use of a ts peptide derived from an N terminus portion of aS1 casein for the preparation of a medicament for preventing or treating a viral disease.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for the preparation of a medicament for preventing viral infection.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS} casein for the preparation of a medicament for inducing hematopoiesis.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for the preparation of a medicament for inducing hematopoietic stem cells proliferation. } Further according to the present invention there is disclosed the use of a
A peptide derived from an N terminus portion of aS! casein for the preparation of a medicament for inducing hematopoietic stem cells proliferation and differentiation.
Further according to the present invention there is disclosed the use of a peptide derived trom an N terminus portion of aS1 casein for the preparation of a medicament for inducing megakaryocytopoiesis.
Further according to the present invention there is disclosed the use of a s peptide derived from an N terminus portion of aS1 casein for the preparation of - a medicament for inducing erythropoicsis.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for the preparation of a medicament for inducing lcukocytopoiesis.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for the preparation of a medicament for inducing thrombocytopoiesis.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS! casein for inducing plasma 's cell proliferation.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS! casein for the preparation of a medicament for inducing dendritic cell proliferation.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for the preparation of a medicament for inducing macrophage proliferation.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for the preparation of a medicament for preventing or treating thrombocytopenia.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for the preparation of - a medicament for preventing or treating pancytopenia. . h—
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for the preparation of . a medicament for preventing or treating granulocytopenia.
Further according to the present invention there is disclosed the use of a ) 5 peptide derived from an N terminus portion of aS1 casein for the preparation of a medicament for preventing or treating hyperlipidemia.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS} casein for the preparation of a medicament for preventing or treating cholesteremia.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS] casein for the preparation of a medicament for preventing or treating giucosuria.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS| casein for the preparation of a medicament for preventing or treating diabetes.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS] casein for the preparation of a medicament for preventing or treating AIDS.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS] casein for the preparation of a medicament for preventing or treating infection by HIV.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS casein for the preparation of a medicament for preventing or treating conditions associated with myeloablative doses of chemoradiotherapy supported by autologous bone i marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic . bone marrow transplantation (BMT).
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of a$1 casein for the preparation of a medicament for treating a thrombopoictin treatable condition.
Further according to the present invention there is disclosed the use of a 5s peptide derived from an N terminus portion of aS1 casein for the preparation of . a medicament for augmenting the ctfect of thrombopoictin.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS] cascin for the preparation of a medicament for enhancing peripheral stem cell mobilization.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS] casein for the preparation of a medicament for enhancing colonization of donated blood stem cells in a myeloablated recipient.
Further according to the present invention there is disclosed the use of a is peptide derived from an N terminus portion of aS1 casein for the preparation ot a medicament for enhancing colonization of blood stem cells in a myeloablated recipient.
Further according to the present invention there is disclosed the usc of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for preventing or treating an autoimmune disease.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of oS! casein, and a pharmaceutically 2s acceptable carrier for preventing or treating a viral disease.
Further according to the present invention there is disclosed the use of a - pharmaceutical composition comprising, as an active ingredient, a peptide i derived from an N terminus portion of aS} casein, and a pharmaceutically acceptable carrier for preventing or treating a viral infection. a
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide . derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for inducing hematopoiesis. ‘ 5 Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cell proliferation.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSI casein, and a pharmaceutically acceptabie carrier for inducing hematopoietic stem cells proliferation and differentiation.
Further according to the present invention there is disclosed the use of a 1s pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for inducing megakaryocytopoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for inducing erythropoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for inducing leukocytopoiesis. ) Further according to the present invention there is disclosed the use of a . pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for inducing thrombocytopoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSI casein, and a pharmaceutically acceptable carrier for inducing plasma cell proliferation.
Further according to the present invention there is disclosed the use of a } pharmaceutical composition comprising, as an active ingredient, a peptide derived trom an N terminus portion of aS] casein, and a pharmaccutically acceptable carrier for inducing dendritic cell proliferation.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for inducing macrophage proliferation.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide 15s derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for presenting or treating thrombocytopenia.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient. a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for preventing or treating pancytopenia.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide - derived from an N terminus portion of aS] casein, and a pharmaceutically } acceptable carrier for preventing or treating hyperlipidemia. j-
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating cholesteremia. ‘ 5 Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSI casein, and a pharmaceutically acceptable carrier for preventing or treating glucosuria.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating diabetes.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for preventing or treating AIDS.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for preventing or treating infection by HIV.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for preventing or treating conditions associated with 25s myeloablative doses of chemoradiotherapy supported by autologous bone i marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT).
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for treating a thrombopoietin treatable condition.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide s derived from an N terminus portion of aS1 casein, and a pharmaceutically : acceptable carrier for augmenting the effect of thrombopoietin.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier peptide derived from an N terminus portion of aS1 casein for enhancing colonization of donated blood stem cells in a myeloablated recipient.
Further according to the present invention there is discloscd the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaccutically is acceptable carrier enhancing colonization of blood stem cells tn a mycloablated recipient.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein. and a pharmaceutically acceptable carrier for enhancing peripheral stem cell mobilization.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for inducing hematopoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin ] and a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cells proliferation. ] Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin ’ 5s and a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cells proliferation and differentiation.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl1 casein, and a pharmaceutically acceptable carrier for inducing megakaryocytopoiesis.
Further according to the present invention there is disciosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of «Sl casein, and a ts pharmaceutically acceptable carrier for inducing erythropoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for inducing leukocytopoiesis.
Further according to the present invenuon there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for inducing thrombocytopoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin ) and a peptide derived from an N terminus portion of aS] casein, and a . pharmaceutically acceptable carrier for preventing or treating thrombocytopenia.
Further according to the present invention there is disclosed the use ot a pharmaceutical composition comprising, as active ingredients. thrombopotetin and a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating pancytopenia.
Further according to the present invention there is disclosed the use of a . pharmaceutical composition comprising, as active ingredients. thrombopoictin and a peptide derived from an N terminus portion of aS casein. and a pharmaccutically acceptable carrier for preventing or treating granulocytopenia.
Further according to the present invention there is disclosed the use ot a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS} casein, and a pharmaceutically acceptable carrier for preventing or treating an indication sclected from the group consisting of autoimmune disease or condition, viral disease. viral infection, hematological discase, hematological deficiencies, thrombocytopenia. pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, glucosuria, hyperglycenna, diabetes, AIDS, HIV-1, helper T-cell disorders, dendrite cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, human immune system disorders resulting from treatment of diseases of immune deficiency and bacterial infections.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for preventing or treating an indication - selected from the group consisting of hematological disease. hematological deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, dendrite cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders -
including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, leukemic conditions, myelodysplastic syndrome, } aplastic anemia and bone marrow insufficiency.
Further according to the present invention there is disclosed the use of a ’ s pharmaceutical composition comprising, as active ingredients, a peptide derived from an N terminus portion of aS! casein and thrombopoietin, and a pharmaceutically acceptable carrier for enhancing colonization of donated blood stem cells in a myeloablated recipient.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, a peptide derived from an N terminus portion of aS1 casein and thrombopoietin, and a pharmaceuticaily acceptable carrier for enhancing colonization of blood stem cells in a myeloablated recipient.
According to further features in preferred embodiments of the invention 1s described below, the peptide is a fragment derived by fragmentation of aS] casein.
According to yet further features in the described preferred embodiments the peptide is a synthetic peptide.
According to still further features in the described preferred embodiments the peptide has a sequence as set forth in one of SEQ ID
NOs:1-25.
Further according to the present invention there is provided a purified peptide having an amino acid sequence selected from the group consisting of
SEQ ID NOs:1-25.
Further according to the present invention there is provided a i pharmaceutical composition comprising a purified peptide having an amino } acid sequence selected from the group consisting of SEQ ID NOs:1-25 and a pharmaceutically acceptable carrier.
Further according to thc present invention there is provided a pharmaceutical composition comprising thrombopoictin and a purified peptide having an amino acid sequence selected from the group consisting of SEQ 1D
NOs: 1-23 and a pharmaceutically acceptable carrier.
The present invention successfully addresses the shortcomings of the . presently known configurations by providing peptides for the treatment of human disease. which peptides are derived from the N terminus portion of aS] casein and posses no detectable toxicity and high therapeutic efficacy.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred is embodiments of the present invention only, and are presented 1n the cause of providing what is belicved to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
In the drawings:
FIG. 1depicts the stimulation of Natural Killer (NK) cell activity in cultured murine bone marrow cells by peptides derived from natural casein.
Lysis of 3 labeled YAC target cells by cultured murine bone marrow cells incubated in the presence or absence of 100 pg per ml peptides derived from - natural casein is expressed as the fraction of total radioactivity released from the YAC cells into the culture supernatant ( % Release 33). Figure 1 represents NK activity at an effector:target cell ratio of 25:1 and 50:1. -
FIGs. 2a and 2b depict the stimulation of Natural Killer (NK) ccll activity in cultured human Peripheral Blood Stem Cells (PBSC) by peptides ) derived from natural casein. Lysis of 33S labeled K 562 target cells by cultured human PBSC from Granulocyte Colony Stimulating Factor (G-CSF) treated ’ 5 donors incubated without (0 pg) or with increasing concentrations (5- 500 pg per ml) of peptides derived from natural casein is expressed as the fraction of total radioactivity released from the K562 cells into the culture supernatant (%
Release >°S). Figure 2a represents NK activity of two blood samples from the same patient, incubated at different effector:target cell ratios (1:25 and 1:50).
Figure 2b represents NK activity of blood samples from normal and affected donors incubated at the same effector:target cell ratio. Squares represent an effector:target cell ratio of 100:i, diamonds represent an effector:target cell ratio of 50:1.
FIGs. 3a-3c¢ depict the stimulation of proliferation of Natural Killer (NK) ts and T-lymphocyte (T) cells from cultured human Peripheral Blood Stem Cells (PBSC) by peptides derived from natural casein. NK and T cell proliferation in cultured PBSC from Granulocyte Colony Stimulating Factor treated donors incubated with or without peptides derived from natural casein is expressed as the percentage (%) of cells binding the anti-CDy/FITC fluorescent anti-T cell antibody UCHT,, or the anti CDs/RPE fluorescent anti-NK cell antibody : MOC-1 (DAKO A/S Denmark). Controls are FITC and RPE-conjugated anti-mouse IgG antibody. Figure 3a represents the percentage of cultured human PBSC binding fluorescent antibody CDs (5 independent samples) after 10 days incubation with (peptides) or without (control) 100 pg per ml peptides derived from natural casein. Figure 3b represents the percentage of cultured : human PBSC cells binding fluorescent anti-CDj; (T cell) antibody, following 14 . days of incubation with (peptides) or without (control) 100 pg per ml peptides derived from natural casein. Figure 3c represents the percentage of cultured human PBSC cells binding fluorescent anti-CDj (1 cell) antibody and cells binding both CD; and CDs (T and NK-like cells) antibodies after 28 days incubation with (peptides) or without (control) 100 pg per mi peptides derived from natural casein.
FIG. 4 depicts the stimulation of Natural Killer (NK) cell activity in cultured human Peripheral Blood Stem Cells (PBSC) by synthetic peptides - derived from casein. Lysis of S labeled K562 target cells by cultured human
PBSC (from a breast cancer paticnt) incubated without (0 pg) or with increasing concentrations (10 - 500 pg per ml) of synthetic peptides derived from casein is expressed as the fraction of total radioactivity released from the
K562 cells into the culture supernatant (% Release). Peptides represent
N-terminal sequences of 1-10 (1a, diamonds), 1-11 (2a, squares) and 1-12 (3a, triangles) first amino acids of the N terminus portion of «S1 casein (sec Table 3 below for sequences of synthetic peptides).
FIGs. Sa-5c depict the stimulation of proliferation of cultured human cells of diverse origin by peptides derived from natural casein. Proliferation of the culturcd human ceils after 14-21 days incubation with increasing concentrations of the peptides derived from natural casein is expressed as the amount of [*H]-thymidine incorporated into each sample. Figure 5a represents the incorporation of label into two samples (PBSC 1, squares, 15 days incubation; and PBSC 2, diamonds, 20 days incubation) of human Peripheral
Blood Stem Cells incubated with or without (ctrl) 50 - 600 ug per mi peptides derived from natural casein. Figure Sb represents the incorporation of [’H)-thymidine into cultured human bone marrow cells after 21 days incubation with or without (ctrl) 50 - 600 ng per ml peptides derived from natural cascin.
Bone marrow was donated by cancer patients in remission (BM Auto. closed squares, BM 1, triangles, and BM 2.-open squares-) or healthy volunteers (BM ) normal, diamonds). Figure 5c represents incorporation of [*H]-thymidine into i cultured human Cord Blood cells after 14 days incubation with or without (ctrl) _
50- 1000 pg per ml peptides derived from natural casein. Cord blood cells were donated by two separate donors (C.B. 1, triangles, C.B. 2, squares). . FIG. 6 shows a Table depicting the proliferation of blood cell progenitors from human bone marrow and cord blood in response to incubation ’ 5 with peptides derived from natural casein. The relative cell number x 10* per ml, reflecting the proliferation of cultured cells, was determined by counting cells as described in the Examples section that follows. Bone marrow from healthy volunteers (Bone Marrow) and Cord Blood from normal births (Cord
Blood) was incubated for 13 (Cord Blood) or 14 (Bone Marrow) days in the presence of growth factors and AB serum, with or without increasing concentrations of peptides derived from natural casein (25-500 pg/ml).
FIG. 7 shows a tabie depicting the effect of in-vitro incubation with
Synthetic peptides derived from casein on the relative distribution of
Megakaryocyte, Erythroid, Plasma and Dendritic cells (differential count) in 1s CFU-GEMM colonies from murine bone marrow progenitor cells. Cells were scored in the macroscopic colonies grown from murine bone marrow cells prepared similarly to the CFU-GEMM colonies previously described. Cells were incubated with hematopoietic factors, and 25 pg or more of Synthetic peptides derived from casein, for 14 days. The differential count is expressed as the percentage of total cells represented by individual cell types. FIG. 8 depicts the stimulation of peripheral white blood cell reconstitution in myeloablated, bone marrow transplanted mice in response to treatment with peptides derived from natural casein. Cell counts represent the number of white blood cells (x 10° per ml, as counted in a haemocytometer). The mice (n = 6 per group) received sub-lethal irradiation and syngeneic bone marrow : transplantation (10° cells per mouse) on the following day, and intravenous administration of 1 mg per recipient peptides derived from natural casein (peptides: squares) or 1 mg per recipient human scrum albumin (CONTROL: diamonds) one day later.
FIG. 9 depicts the stimulation of platelet reconstitution in myeloablated, bone marrow transplanted mice in response to treatment with peptides derived from natural casein. Platelet (PLT) counts represent the number of thrombocytes (x 10° per ml, as counted in a haemocytometer). The mice (n = 5s 60 per group) received lethal irradiation and syngeneic bone marrow i transplantation (10° cells per mouse) on day 1. and intravenous administration of 1 mg per recipient peptides derived from natural casein (Peptides, diamonds) or 1 mg per recipient human serum albumin (control. squares).
FIGs. 10a-10f depict the penetration and nuclear uptake of
FITC-conjugated peptides derived from natural casein in cultured human
T-lymphocyte cells, as recorded by fluorescent microscopy. Fl and F2 are identical fractions of the FITC-conjugated peptides derived from natural cascin.
Sup-T1 cells were incubated with 100 pg per ml FITC-conjugated peptides derived from natural casein as described in the Examples section that follows. 1s At the indicated times, the cells were washed of free label, fixed in formalin and prepared for viewing and recording by Laser Scanning Confocal
Microscopy. Figures 10a through 10f are sclected images of cells from consecutive incubation times, demonstrating FITC-conjugated peptides derived from natural casein penetrating the Sup-T} ceil membrane (Figures 10a, 10b)- - and concentrating in the nucleus (Figures 10c- 10f).
FIG. 11 shows a Table depicting the stimulation of Sup-T] Lymphocyte cell proliferation in response to incubation with peptides derived from natural casein. Sup-Ty cells (5000 per well) were incubated with increasing concentrations (50 - 1000 pg per mi) of peptides derived from natural casein, counted in their wells at the indicated times post culture and pulsed with [> H]-thymidine for 18 hours. Proliferation index is the ratio of the average of - the incorporation of (>H]-thymidine into cells cultured with peptides derived from natural casein (triplicate samples) divided by the incorporation into cells cultured without peptides derived from natural casein (control). -
FIG. 12 shows a Table depicting inhibition of HIV-1 infection of CEM lymphocytes by peptides derived from natural casein. CEM cells were either } contacted with HIV-1 virus preincubated 3 hours with peptides derived from natural casein (3 hours), or preincubated themselves with increasing ’ 5 concentrations (50 - 1000 pg per ml) of peptides derived from natural casein for the indicated number of hours (24 and 48 hours) before contact with HIV-1 virus, as described in the Examples section that follows. On day 15 post infection, cells were counted for cell numbers and assayed for severity of
HIV-1 infection by the P** antigen assay, as described in the Examples section that follows. Control cultures were IF: CEM cells contacted with HIV-1 virus without pretreatment with peptides derived from natural casein, and UIF: CEM cells cultured under identical conditions without peptides derived from natural cascin and without contact with HIV-1 virus.
FIG. 13 shows a Table depicting inhibition of HIV-1 infection of CEM 1s lymphocytes by Synthetic peptides derived from casein. CEM cells were contacted with HIV-1 virus which had been preincubated with various concentrations (10- 500 ug per ml) of synthetic peptides derived from natural casein (IP, 3P and 4P) for 3 hours (in the presence of the peptides), as described in the Examples section that follows. On day 7 post infection, cells were counted for cell numbers and assayed for severity of HIV-1 infection by the P** antigen assay, as described in the Examples section that follows.
Control cultures (IF) were CEM cells contacted with HIV-1 virus without pretreatment with peptides derived from natural casein.
F1G. 14 depicts the prevention by peptides derived from natural casein of
Type 1 (IDDM) Diabetes in female Non Obese Diabetic (NOD) mice. : Glucosuria was monitored at intervals during 365 days post treatment in female } NOD mice receiving a once (triangles and squares) or twice weekly injection of 100 pg peptides derived from natural casein for 5 weeks (5 or 10 injections total) and untreated controls. All the controls developed glucosuria and subsequently died.
FIG. 15 depicts the reduction by Synthetic peptides derived from casein of” diet-induced hypercholesterol/hyperlipidemnia in female C57 Bl/6 mice.
Total cholesterol (TC), High Density (HDL) and l.ow Density Lipoproteins } (LDL) were assayed in pooled blood of two (2) mice per sample from hypercholesterol/hyperlipidemic mice receiving (IP) casein-derived peptides B,
C, 2a or 3P, or no treatment (control). “Normal” samples represent control mice not fed the atherogenic diet.
FIG. 16 shows a Table depicting the stimulation of hematopoiesis in cancer patients in response to injections of peptides derived from natural cascin. Peripheral blood from five female cancer patients either receiving or having received chemotherapy, as described above, was counted for total White
Blood Cells (WBC, x 10°), Platelets (PLT, x 10%), Erythrocytes (RBC, x 10%) 1s and Hemoglobin (gm per dl) before (n) and after (n +...) intramuscular injections with peptides derived from natural casein. as described above.
Patient 1 relates to G.T.. patient 2 relates to E.C.; patient 3 relates to E.S.. patient 4 rclates to J.R. and patient 5 relates to D.M. © FIG. 17 depicts the stimulation by peptides derived from natural casein of thrombocytopoiesis in a platelet-resistant patient with Acute Myeloid
Leukemia (M-1). Thrombocyte reconstitution was expressed as the change 1n platelet content of peripheral blood (PLT, x 10° per ml), counted as described above at the indicated intervals following intramuscular injection (as described in the Examples section that follows) of 100 ug peptides derived from natural casein.
FIG. 18 depicts the stimulation by peptides derived from natural casein : of thrombocytopoiesis in a platelet-resistant patient with Acute Myeloid
Leukemia (M-2). Thrombocyte reconstitution was expressed as the change in platelet content of peripheral blood (PLT, x 10° per ml), counted as described above at the indicated intervals following intramuscular injection (as described —
in the Examples section that follows) of 100 mg peptides derived from natural casein.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
. 5 The present invention is of biologically active peptides that are derived from or are similar to sequences identical with the N-terminus of the aSl fraction of milk casein, compositions containing same and methods of utilizing same in, for example, stimulating and enhancing immune response, protecting against viral infection, normalizing serum cholesterol levels, and stimulating to hematopoiesis. The casein-derived peptides are non-toxic and can be used to treat and prevent, for example, immune pathologies, hypercholesterolemia, hematological disorders and vira!l-related diseases. ~The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions,
Is Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
As used herein, the term “treating” includes substantially inhibiting, slowing or reversing the progression of a disease, substantially ameliorating clinical symptoms of a disease.
As used herein, the term “preventing” includes substantially preventing - the appearance of clinical symptoms of a discase.
As used herein the term "peptide" includes native peptides (either ’ degradation products, synthetically synthesized peptides or recombinant peptides) and peptido-mimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which arc peptide analogs, which may have,
for example. modifications rendering the peptides more stable while in a body.
Such modifications include, but are not limited to. cyclization. N terminus modification, C terminus modification, peptide bond modification, including. but not limited to, CH»-NH, CH3>-S, CH»-S=0, O=C-NH. CH2-0O. CH>-CH»~.
S=C-NH, CH=CH or CF=CH. backbone modification and residue . modification. Methods for preparing peptido-mimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design.
C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992). which is incorporated by reference as if fully set forth herein. Further detail in this respect are provided hereinunder.
Thus, a peptide according to the present invention can be a cyclic peptide. Cyclization can be obtained, for example, through amide bond formation. c.g., by incorporating Glu. Asp, Lys, Om, di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at various positions in the chain (-CO-NH or -NH-CO bonds). Backbone to backbone cyclization can also be obtained through incorporation of wmadified amino acids of the formulas
H-N((CH»)p-COOH)-C(R)H-COOH or H-N((CH7)n-COOH)-C(R)H-NH>. wherein n = 1-4, and further wherein R is any natural or non-natural side chain of an amino acid.
Cyclization via formation of S-S bonds through incorporation of two
Cys residues is also possible. Additional side-chain to side chain cyclization can be obtained via formation of an interaction bond of the formula -(-CH9-)n-S-CH)-C-, wherein n = 1 or 2, which is possible, for example, through incorporation of Cys or homoCys and reaction of its free SH group with, e.g., bromoacetylated Lys, Orn, Dab or Dap.
Peptide bonds (-CO-NH-) within the peptide may be substituted, for . example, by N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-0-0-C(R)-N-), ketomethylen bonds (-CO-CHjy-), a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g, methyl, carba bonds 50 (-CH»-NH-), hydroxyethylene bonds (-CH(OH)-CHj-), thicamide bonds -
(-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)-CH»-CO-), wherein R is the "normal" side chain, } naturally presented on the carbon atom.
These modifications can occur at any of the bonds along the peptide ’ s chain and even at several (2-3) at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
Tables 1-2 below list all the naturally occurring amino acids (Table 1) and non-conventional or modified amino acids (Table 2).
Table 1
Three-Letter Abbreviation One-letter Symbol
CI LO [I
Eo LS LI
EC LR LE
EC CN LA
EC LC LA
Table 2
Non-conventional Code Non-conventional amino acid Code
O-aminobutyric acid Abu L-N-muthyvhalanine Nmala ! . 1.-N-methylaspartic acid 1.-N-methvlcysteine | Nmcys
L-N-methvlgiutamine Nmgein
EE —— =
D-aspartic acid Dasp L-N-methyvimethionine Nmmet
D-glutamine Dgln L-N-methylnorvaline Nmnva !
D-glutamic acid © | pel | 1.-N-methylomithine Nor —
Dine Dhis | }.-N-methylphenylalanine | Nmphe [ D-isoleucine L-N-methylproline I Nmpro :
L-N-methylserine Nmeer —
D-at-methy;tryptophan , Omip \ N-cyciohexyigiycine Nchex
CET a a ha
L-t-butylglycine N-(thiomethyl)glycine | Neys
Etg Penicillamine i Pen
I.-u-methylglutamate Men
L-a-methylthistidine Mhphe
N-methyl-y-aminobutyrate Nmgabu
N-methylcyciohexylalanine D-N-methyimethionine | Dnmmaet 1 D-N-methvlomithine Dnmom t N-methylcyclopentylalunine | Nmepen
N-methylglycine Nala D-N-methyiphenvialanine Dnmphe
N-methylaminoisobuny rate Nmaib D-N-mieihy lproline Dnmpro — -
EC I x oc NO 1-carboxy- 1-(2,2-diphenyl Nmbc
A peptide according to the present invention can be used in a self standing form or be a part of moieties such as proteins and display moieties such as display bacteria and phages. The peptides of the invention can also be chemically modified to give active dimers or multimers, in one polypeptide chain or covalently crosslinked chains.
Additionally, a peptide according to the present invention includes at least two, optionally at least three, optionally at least four, optionally at least five, optionally at least six, optionally at least seven, optionally at least eight, optionally at least nine, optionally at least ten, optionally at least eleven, optionally at least twelve, optionally at least thirteen, optionally at least fourteen, optionally at least fifteen, optionally at least sixteen, optionally at least seventeen, optionally at least eighteen, optionally at least nineteen, optionally at least twenty, optionally at least twenty-one, optionally at least
Is twenty-two, optionally at least twenty-three, optionally at least twenty-four, . optionally at least twenty-five, optionally at least twenty-six, optionally between twenty-seven and sixty, or more amino acid residues (also referred to - herein interchangeably as amino acids).
Accordingly, as used herein the term "amino acid" or "amino acids" is 50 understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example. hydroxyproline, phosphoserine and phosphothreconine; and other unusual amino acids including, but not limited to. 2-aminoadipic acid. hydroxylysine, isodesmosine, nor-valine, nor-leucine and omithine. Furthermore, the term "amino acid" includes both D- and L-amino acids. :
As uscd herein the phrase "derived from an N terminus portion of aS} casein” refers to peptides as this term is defined herein, c.g., cleavage products of aS1 casein (referred to herein as peptides derived from natural casein). synthetic peptides chemically synthesized to correspond to the amino acid sequence of an N terminus portion of aS1 casein (referred to herein as synthetic peptides derived from casein), peptides similar (homologous) to an N terminus portion of aSI casein, for example, peptides characterized by one or more amino acid substitutions, such as, but not limited to, permissible substitutions, provided that at least 70 %, preferably at least 80 %, more preferably at least 90 1s % similarity is maintained, and functional homologues thereof. The terms “homologues” and "functional homologues” as used herein mean peptides with any insertions, deletions and substitutions which do not affect the biological activity of the peptide.
As used herein the term "aS1 casein” refers to aS1 casein of a mammal. including, but not limited to, livestock mammals (e.g., cow, sheep, goat, mare, camel, deer and buffalo) human beings and marine mammals. The following provides a list of aS] caseins having a known amino acid sequence, identified by their GenBank (NCBI) Accession Nos. and source: CAA26982 (Ovis aries (sheep)), CAAS51022 (Capra hircus (goat)), CAA42516 (Bos taurus (bovine}),
CAASS185 (Homo sapiens), CAA38717 (Sus scrofa (pig)), PO9115 (rabbit) and 097943 (Camelus dromedurius (camel)). ’
As used herein the term "N terminus portion" refers to M amino acids of aS]! casein derived from the first 60 amino acids of aS casein, wherein M is —
4s any of the integers between 2 and 60 (including the integers 2 and 60).
Preferably, the term refers to the first M amino acids of aS1 casein. . The peptides of the invention can be obtained by extraction from milk as previously described, or by solid phase peptide synthesis, which is a standard ’ 5s method known to the man skilled in the art. Purification of the peptides of the invention is performed by standard techniques, known to the man skilled in the art, such as high performance liquid chromatography (HPLC). Milk casein fragmentation to obtain the peptides of the invention may be effected using various enzymatic and/or chemical means.
As is further detailed hereinunder and exemplified in the Examples section that follows, the peptides of the present invention have a variety of therapeutic effects. In the Exampies scction there are provided numerous assays with which one of ordinary skills in the art can test a specific peptide designed in accordance with the teachings of the present invention for a is specific therapeutic effect. Any of the peptides described herein can be administered per se or be formulated into a pharmaceutical composition which can be used for treating or preventing a disease. Such a composition includes as an active ingredient any of the peptides described herein and a pharmaceutically acceptable carrier.
As used herein a "pharmaceutical composition” refers to a preparation of one or more of the peptides described herein, with other chemical components such as pharmaceutically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Hereinafter, the term "pharmaceutically acceptable carrier” refers to a : carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Examples, without limitations, of carriers are: propylene glycol, saline, emulsions and mixtures of organic solvents with water. Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation. of excipients include calcium carbonate, calcium phosphate. various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in . “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition.
Suitable routes of administration may, for example, include oral. rectal. transmucosal, transdermal, intestinal or parenteral delivery. including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal. or intraocular injections.
Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, eg. by means of 15s conventional mixing, dissolving, granulating, dragee-making. levigating, emulsifying, encapsulating, entrapping or Ivophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active peptides into preparations which. can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the peptides of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as
Hank's solution, Ringer’s solution, or physiological saline buffer with or without organic solvents such as propylene glycol, polyethylene glycol. For : transmucosal administration, penetrants are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the peptides can be formulated readily by combining the active peptides with pharmaceutically acceptable carriers well — -
known in the art. Such carriers enable the peptides of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, ] suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally s grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked poiyvinyi pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active ingredient doses.
Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, : stabilizers. In soft capsules, the active peptides may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of 50 administration.
For buccal administration, the compositions may take the form of tablets or lozenges tormulated in conventional manner.
For administration by inhalation, the peptides according to the present invention are convenicntly delivered in the form of an aerosol spray s presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane. dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized acrosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The peptides described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be prescnted in unit dosage form, e.g, in ampoules or in 1s multidose containers with optionally. an added preservative. The compositions may be suspensions. solutions or emulsions in oily or aqueous vehicles. and may contain formulatory agents such as suspending, stabilizing and:or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form.
Additionally, suspensions of the active peptides may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl olcate. triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethy! cellulose, sorbitol or dextran. Optionally, the suspension may . also contain suitable stabilizers or agents which increase the solubility of the peptides to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. -
The peptides of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, c.g, } conventional suppository bases such as cocoa butter or other glycerides.
The pharmaceutical compositions herein described may also comprise : 5 suitable solid of gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.
Persons ordinarily skilled in the art can easily determine optimum dosages and dosing methodology for any of the peptides of the invention.
For any peptide used in accordance with the teachings of the present invention, a therapeutically effective amount, alsc referred io as a therapeutically effective dose, which can be estimated initially from cell culture assays or in vivo animal assays. For example, a dose can be formulated in 1s animal models to achieve a circulating concentration range that includes the
IC, or the IC, as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be estimated from in vivo data. Using these initial guidelines one having ordinary skill in the art could determine an effective dosage in humans.
Moreover, toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LDs, and the EDs.
The dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LDso and EDs. Peptides which exhibit high therapeutic indices are preferred. The data obtained from these cell ~ cultures assays and animal studies can be used in tormulating a dosage range that is not toxic for use in human. The dosage of such peptides lies preferably ] within a range of circulating concentrations that include the EDs, with little or no toxicity. The dosage may vary within this range depending upon the dosage form emploved and the route of administration utilized. The exact formulation,
S0 » »ute of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl et al. 1975. In: The
Pharmacological Basis of Therapeutics, chapter 1. page 1).
Dosage amount and interval may be adjusted individually to provide 5s plasma levels of the active ingredient which are sufficient to maintain . therapeutic effect. Usual patient dosages for oral administration range trom about 1-1000 mg’kg/administration, commonly from about 10-300 mg/kg/administration, preferably from about 20-300 mg/kg/administration and most preferably from about 50-200 mg/kg/administration. In some cases. therapeutically effective serum levels will be achieved by administering multiple doses each day. In cases of local administration or selective uptake. the effective local concentration of the drug may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
Depending on the scverity and responsiveness of the condition to be treated. dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state ts achieved.
The amount of a composition to be administered will, of course. be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The g pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the
U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a peptide of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an - 5 appropriate container, and labeled for treatment or prevention of an indicated condition or induction of a desired event. Suitable indica on the label may include treatment and/or prevention of an autoimmune disease or condition, viral disease, viral infection, bacterial infection, hematological disease, hematological deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, glucosuria, hyperglycemia, diabetes,
AIDS, infection with HIV-1, helper T-cell disorders, dendrite cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, hematopoietic stem cell proliferation, hematopoietic stem cell proliferation and differentiation, 1s pre-leukemic conditions, leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, and human immune system disorders resulting from treatment of diseases of immune deficiency.
The pharmaceutical compositions according to the invention may be useful in maintaining and/or restoring blood system constituents, in balancing blood cell counts, in balancing levels of metabolites in the blood including sugar, cholesterol, calcium, uric acid, urea and enzymes such as alkaline phosphatase. Further, the pharmaceutical compositions of the invention may be useful in inducing blood cell proliferation, modulating white and/or red blood cell counts, particularly increasing white and/or red blood cell counts, elevating 2s haemoglobin blood level and in modulating platelet counts. - The term “balancing” as used hercin with relation to levels of certain physiological parameters, means changing the levels of referred parameters and ) bringing them closer to normal values.
The term “normal values” as used herein with relation to physiological parameters, means values which are in the range ot values of healthy humans or animals.
In specifically preferred embodiments, the peptides of the invention balance counts of red blood cells, white blood cells, platelets and haemoglobin } level. The pharmaceutical compositions of the invention may be used for activating blood cell proliferation.
In addition, the pharmaceutical compositions may be used for the treatment and/or prevention of hemopoietic stem cell disorders, including platelet, lymphocyte, plasma cell and neutrophil disorders, as well as deficiency and malfunction in pre-leukemic and leukemic conditions and thrombocytopenia.
Further, the pharmaceutical compositions may be used for the treatment and’or prevention of cell proliferative diseases. In this connection. it is worth i5 noting that the pharmaceutical compositions of the invention arc advantageous in the stimulation of the immune response during chemotherapy or radiation treatments, in alleviating the negative effects, reducing chemotherapy and irradiation-induced vomiting and promoting a faster recovery.
Still further, the pharmaceutical compositions of the invention may be used for the stimulation of human immune response during treatment of diseases associated with immune deficiency, for example HIV and autoimmune diseases.
The compositions of the invention may also be intended for veterinary use.
The pharmaceutical compositions of the invention may be used in the treatment and/or prevention of, for example, disorders involving abnormal - levels of blood cells, disorders involving hemopoietic stem cells production and differentiation, treatment of platelet, lymphocyte and/or neutrophil disorders, ) for the treatment of pre-leukemic and leukemic conditions and for the treatment of thrombocytopenia. The pharmaceutical compositions of the invention may
I also be uscd in the treatment of cell proliferative diseases and diseases involving immune deficiency, such as HIV, and of autoimmune diseases.
Further, the pharmaceutical compositions of the invention may be used for modulating the immunc response during chemotherapy or radiation treatments, : 5 for example for reducing chemotherapy-associated vomiting.
While reducing the present invention to practice, it was surprisingly observed that the peptides of the invention exert a synergistic effect on human hematopoietic stem cell proliferation and differentiation with addition of other hematopoietic growth factors. Of notable significance was the potentiation of erythropoictin-mediated stimulation of erythroid colony formation and the dose-dependent enhancement of thrombopoietin (TPO) induction of megakaryocyte proliferation by peptides of the present invention. Recombinant human (rth) EPO is currently an approved therapy for indications such as renal anemia, anemia of prematurity, cancer- and AIDS-associated anemia, and for pre-elective surgical treatment (Sowade, B et al Int J Mol Med 1998;1:305).
Thus, according to the present invention there is provided a method of treating an erythropoietin treatable condition, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS! casein.
Further according to the present invention there is provided a method of augmenting the effect of erythropoietin, the method effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Thrombopoietin is an early acting cytokine with important multilineage 2s effects: TPO alone, or in combination with other early acting cytokines, can (i) : promote viability and suppress apoptosis in progenitor cells; (ii) regulate hematopoietic stem cell production and function; (iii) trigger cell division of ] dormant multipotent cells; (iv) induce multilineage differentiation and (v) enhance formation of multilineage colonies containing granulocytes,
ervthrocytes. macrophages, and megakaryocytes (MK, CIFU-GEMM).
Moreover. TPO stimulates the production of more hmited progenitors for granulocyte/monocyte, megakaryocyte and erythroid colonies, stimulates adhesion of primitive human bone marrow and megakaryocytic cells to s fibronectin and fibrinogen. Thus, TPO is an important cytokine for clinical hematologists/transplanters: for the mobilization, amplification and ex vivo expansion of stem cells and committed precursor cells for autologous and allogeneic transplantation. In addition, administration of TPO to healthy platelet donors has been employed to enhance pheresis yields. However. clinical application of TPO therapy is complicated by, among other considerations, relatively high costs of the recombinant human cytokine rhTPO, © + and the potential antigenicity of TPO with repeated administration.
Combined treatment with TPO and the peptide of the present invention. either together in a pharmaceutical composition comprising both, or separately, can provide inexpensive, proven non-toxic augmentation of TPOs effects on target cell proliferation and function. In such a combination. the peptide of the present invention may be applied to the treatment of, in addition to the abovementioned conditions, disorders such as myclodysplastic syndrome (MDS), aplastic anemia and complications of liver failure. Pre-treatment of platelet donors with the peptide of the present invention, alone or in combination with TPO, may even further enhance the efficiency of pheresis yields.
Thus, according to the present invention there is provided a method of treating a thrombopoietin treatable condition, the method cffected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS] casein. ‘
Further according to the present invention there is provided a method of augmenting the effect of thrombopoietin, the method effected by administering to a subject in necd thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of enhancing peripheral stem cell mobilization, the method effected by ) 5s administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising effective amounts of thrombopoietin and a peptide derived from an N terminus portion of a S1 casein.
Further according to the present invention there is provided a pharmaceutical composition for treating a thrombopoictin treatable condition, the pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aSI1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for augmenting the effect of thrombopoictin, the pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for enhancing peripheral stem cell mobilization, the pharmaceutical composition comprising, as active ingredients thrombopoietin and a peptide derived from an N terminus portion of a Si casein and a pharmaceutically acceptable carrier.
Further according to the present invention there 1s provided a pharmaceutical composition for inducing hematopoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide ’ derived from an N terminus portion of aSI casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing hematopoietic stem cells proliferation, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a 5s pharmaceutical composition for inducing hematopoietic stem cells proliferation and differentiation, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there 1s provided a pharmaceutical composition for inducing megakaryocytopoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS casein and a pharmaceutically acceptable carrier.
Further according to the present invention there 1s provided a pharmaceutical composition for inducing erythropoiesis, the pharmaceuticai composition comprising, as active ingredients thrombopoicetin and a pepude derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing leukocytopoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing thrombocytopoiesis. the pharmaceutical composition comprising, as active ingredients, thrombopoietin ) and a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating thrombocytopenia, the ) pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein and a : 5s pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating pancytopenia, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceuticai composition for preventing or treating granuiocytopenia, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein and a 1s pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for treating or preventing an indication selected from the group consisting of hematological disease, hematological deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, dendrite cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including plaielet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, leukemic conditions, myelodysplastic syndrome, aplastic anemia and bone marrow insufficiency, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion 2s of aS1 casein and a pharmaceutically acceptable carrier. ) Further according to the present invention there is provided a pharmaceutical composition comprising thrombopoietin and a purified peptide having an amino acid sequence selected from the group consisting of SEQ ID
NOs: 1-25 and a pharmaceutically acceptablc carrier.
Further according to the present invention there is provided a method of enhancing colonization of donated blood stem cells in a myeloablated recipient. the method effected by treating a donor of the donated blood stem cells with a peptide derived from an N terminus portion of oS1 casein and thrombopoietin prior to donation and implanting the donated blood stem cells in the recipient. :
Further according to the present invention there is provided a method of enhancing colonization of donated blood stem cells in a myeloablated recipient. the method effected by treating the donated blood stem cells with a peptide derived from an N terminus portion of aS1 casein and thrombopoietin prior to implanting the donated blood stem cells in the recipient.
Further according to the present invention there is provided a method of enhancing colonization of blood stem cells in a myeloablated recipient, the method comprising treating the blood stem cells with a peptide derived trom an
N terminus portion of aS! casein and thrombopoietin prior to implanting the 15s blood stem cells in the recipient.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising. as an active ingredient. a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for treating a thrombopoietin treatable condition.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for augmenting the effect of thrombopoictin.
Further according to the present invention there is disclosed the usc of a 2s pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically ) acceptable carrier peptide derived from an N terminus portion of aS1 casein for enhancing colonization of donated blood stem cells in a myeloablated recipient.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide i derived from an N terminus portion of oS! casein, and a pharmaceutically acceptable carrier enhancing colonization of blood stem cells in a myeloablated ) 5 recipient.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for enhancing peripheral stem cell mobilization.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for inducing hematopoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of «Sl casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cells proliferation.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cells proliferation and differentiation.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for inducing megakaryocytopoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients. thrombopoietin and a peptide derived from an N terminus portion of aS! casein. and a pharmaceutically acceptable carrier for inducing erythropoiesis.
Further according to the present invention there is disclosed the use of a : pharmaceutical composition comprising. as active ingredients, thrombopoictin and a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carricr for inducing leukocytopoiesis.
Further according to the present invention there is disclosed the usc of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable camer for inducing thrombocytopoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopuietin 1s and a peptide derived from an N terminus portion of aSl casein. and a pharmaceutically ~~ acceptabie currict for preventing or treating thrombocytopenia.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating pancytopenia.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating an indication selected from the group consisting of autoimmune disease or condition, viral disease, viral infection, hematological disease, hematological deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, glucosuria, hyperglycemia, diabetes, AIDS, HIV-1, helper T-cell disorders, s dendrite cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and ncutrophil disorders, pre-leukemic conditions, leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, human immune system disorders resulting from treatment of diseases of immune deficiency and bacterial infections.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating an indication selected from the group consisting of hematological disease, hematological deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, dendrite cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, leukemic conditions, myelodysplastic syndrome, aplastic anemia and bone marrow insufficiency.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, a peptide derived from an N terminus portion of aS1 casein and thrombopoietin, and a pharmaceutically acceptable carrier for enhancing colonization of donated blood stem cells in a myeloablated recipient.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as active ingredients, a peptide derived from an N terminus portion of aS! casein and thrombopoietin, and a pharmaccutically acceptable carrier for enhancing colonization of blood stem cells in a myeloablated recipient.
The invention further relates to anti-bacterial pharmaceutical compositions comprising as active ingredient at least one peptide of the invention and to the usc of the peptides of the invention as anti-bacterial agents.
As detailed in the Examples section hereinbelow, peptides of the invention, and pharmaceutical compositons comprising as an active ingredient a peptide of the invention, can be used in the treatment and prevention of blood cell disorders, cell proliferative diseases, diseases involving immune deficiency and autoimmune diseases.
Thus, according to the present invention there is provided a method of preventing or treating an autoimmunc disease, the method is effected by administering to a subject in need thercof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein. 18 Further according to the present invention there is provided a method of preventing or treating a viral disease. the method 1s effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS] casein.
Further according to the present invention there is provided a method of preventing viral infection. the method is effected by administering to a subject in necd thereof a therapeutically effective amount of a peptide derived from an
N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of inducing hematopoiesis, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of inducing hematopoietic stem cells proliferation, the method is effected by :
administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein. } Further according to the present invention there is provided a method of inducing hematopoietic stem cells proliferation and differentiation, the method : 5 is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS] casein.
Further according to the present invention there is provided a method of inducing megakaryocytopoiesis, the method is cffected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of inducing erythropoiesis, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS] casein.
Further according to the present invention there is provided a method of inducing leukocytopoiesis, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an
N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of inducing thrombocytopoiesis, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of 2s inducing plasma cell proliferation, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived } from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of inducing dendritic cell proliferation, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of inducing macrophage cell proliferation, the method is effected by administering to a subject in need thereof a therapeutically cffective amount of a peptide : derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method off preventing or treating thrombocytopenia, the method is cttected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS} casein.
Further according to the present invention there is provided a method of preventing or treating pancytopenia, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein. 13 Further according to the present invention there is provided a method of preventing or treating granulocytopenta, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating hyperlipidemia, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating hypercholesterolemia, the method is etfected by 2s administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating glucosuria, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein. . Further according to the present invention there is provided a method of preventing or treating diabetes, the method is effected by administering to a ) s subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating AIDS, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a method of preventing or treating infection by HIV, the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS} casein.
Further according to the present invention there is provided a method of preventing or treating conditions associated with myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT), the method is effected by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating an autoimmune disease, the pharmaceutical composition comprising, as an active ingredient, a peptide 2s derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating a viral disease, the pharmaceutical composition comprising, as an active ingredicnt, a peptide derived from an N terminus portion of oS1 casein and a pharmaceutically acceptable carrer.
Further according to the present invention there is provided a pharmaceutical composition for preventing viral infection, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N - terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there 1s provided a pharmaceutical composition for inducing hematopoiesis, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing hematopoictic stem cells proliferation, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein and u pharmaceutically acceptable carrier.
Further according to the present invention there 1s provided a pharmaceutical composition for inducing hematopoietic stem cells proliferation and differentiation, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing megakaryocytopoiesis. the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing erythropoiesis, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing leukocytopoiesis, the pharmaceutical . composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier. } 5 Further according to the present invention there is provided a pharmaceutical composition for inducing thrombocytopoiesis, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing plasma cell proliferation, the pharmaceuticai composition comprising, as an active ingredient a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier. 1S Further according to the present invention there is provided a pharmaceutical composition for inducing dendritic cell proliferation, the pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aSl casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for inducing macrophage proliferation, the pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating thrombocytopenia, the . pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating pancytopenia. the pharmaceutical composition comprising, as an active ingredient. a peptide derived from an N terminus portion of aS} casein and a pharmaceutically 5s acceptable carrier. .
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating granulocytopenia. the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating hyperlipidemia, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating hypercholesterolemia. the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating glucosuria. the pharmaceutical composition comprising, as an active ingredient, 4 peptide derived from an N terminus portion of aS1 casein and a pharmaceutically 25s acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating diabetes, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier. . Further according to the present invention there is provided a pharmaceutical composition for preventing or treating AIDS, the ) s pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating infection by HIV, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein and a pharmaceutically acceptabie carrier.
Further according to the present invention there is provided a pharmaceutical composition for preventing or treating conditions associated with myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT), the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS! casein for preventing or treating an autoimmune disease.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS! casein for preventing or treating a viral disease.
Further according to the present invention there is disclosed the use of a . peptide derived from an N terminus portion of aS1 casein for preventing viral infection.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for inducing hematopoiesis.
Further according to the present invention there is disclosed the use of a s peptide derived trom an N terminus portion of aS1 casein for inducing : hematopoietic stem cells proliferation.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aSl casein for inducing hematopoietic stem cells proliferation and differentiation.
Further according to the present invention there is disclosed the usc of a peptide derived from an N terminus portion of aS1 casein for inducing megakaryocylopoiesis.
Further according to the present invention there is disclosed the usc of a peptide derived from an N terminus portion of aSIl casein for inducing is enthropoiesis. turther according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aSl casein for inducing leukocytopoiesis.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aSl casein for inducing thrombocytopoiesis.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS! cascin for inducing plasma cell proliferation.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS casein for inducing dendritic cell proliferation. )
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aSl casein for inducing } macrophage proliferation.
Further according to the present invention there is disclosed the use of a ) 5 peptide derived from an N terminus portion of aSI casein for preventing or treating thrombocytopenia.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for preventing or treating pancytopenia.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for preventing or treating granuiocytopenia.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aSl1 casein for preventing or treating hyperlipidemia.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aSI casein for preventing or treating cholesteremia.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aSl casein for preventing or treating glucosuria.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS! casein for preventing or treating diabetes.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS] casein for preventing or . treating AIDS.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of a1 casein for preventing or treating infection by HIV.
Further according to the present invention there is disclosed the use of a s peptide derived from an N terminus portion of aS1 casein for preventing or : treating conditions associated with myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT).
Further according to the present invention there is disclosed the use ot a peptide derived from an N terminus portion of aSl casein for treating a thrombopoietin treatable condition.
Further according to the present invention there 1s disclosed the usc of a peptide derived from an N terminus portion of aS1 casein for augmenting the effect of thrombopoietin. 1S Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS! casein for enhancing peripheral stem cell mobilization.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aSI casein for enhancing colonization of donated blood stem cells in a myeloablated recipient.
Further according to the present invention there is disclosed the use of a peptide derived from an N terminus portion of aS1 casein for enhancing colonization of blood stem cells in a mycloablated recipient.
Further according to the present invention therc is disclosed the use of a 2s pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl1 casein, and a pharmaceutically acceptable carrier for preventing or treating an autoimmune disease. )
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating a viral disease. ; Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide ) 5s derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating a viral infection.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for inducing hematopoiesis.
Further according to the present invention there is disclosed the use of a pharmaceuticai composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cell proliferation.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cells proliferation and differentiation.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for inducing megakaryocytopoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically . acceptable carrier for inducing erythropoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaccutically acceptable carrier for inducing leukocytopoiesis.
Further according to the present invention there is disclosed the use of a pharmaccutical composition comprising, as an active ingredient. a peptide s derived from an N terminus portion of aS1 cascin, and a pharmaceutically : acceptable carrier for inducing thrombocytopoiesis.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient. a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for inducing plasma cell proliferation.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for inducing dendritic cell proliferation. 13 Further according to the present invention there is disclosed the use of a phannaceutical composition comprising. as an active ingredient. a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for inducing macrophage proliferation.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for preventing or treating thrombocytopenia.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating pancytopenia.
Further according to the present invention there is disclosed the use of a } pharmaceutical composition comprising, as an active ingredient, a peptide
SR derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia. . Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide ’ 5 derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating hyperlipidemia.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for preventing or treating cholesteremia.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for preventing or treating glucosuria.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating diabetes.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for preventing or treating AIDS.
Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating infection by HIV. , Further according to the present invention there is disclosed the use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for preventing or treating conditions associated with mycloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT).
Further according to the present invention there is provided a purified : peptide having an amino acid sequence selected from the group consisting of
SEQ ID NOs:1-25.
Further according to the present invention there is provided a pharmaceutical composition comprising a purified peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:1-25 and a pharmaceutically acceptable carrier.
The present invention successfully addresses the shortcomings of the presently known configurations by providing peptides for the treatment of human disease, which peptides are derived from the N terminus portion of aS] cascin and posses no detectable toxicity and high therapeutic efficacy.
Additional objects. advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting.
Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion. ‘
MATERIALS AND EXPERIMENTAL METHODS
Preparation of peptides derived from natural casein: The casein fraction of cow’s milk was isolated as described by Hipp et al. (1952), ibid.,
and subjected to exhaustive proteolytic digestion with chymosin (also known as rennin) (20 ng per ml) at 30 °C. Upon completion of the reaction, the solution : was heated to inactivate the enzyme, and the digest was precipitated as paracaseinate by acidification with an organic acid, acetic or trichloracetic acid.
Paracaseinate was separated by centrifugation, and the supernatant fraction, containing the peptide fragments of interest, was re-precipitated as caseicidin by higher acid concentrations. The resulting caseicidin, following re-suspension, dialysis and neutralization was lyophilized. The resulting powdered preparation was assayed for biological activity as described below, and separated by HPLC for peptide analysis.
HPLC analysis of peptides derived from natural casein: Peptides derived from natura! casein as described above were analyzed by HPLC in two stages. Initially, the lyophilized casein digests were separated using a C18 reversed phase with a 0.1 % water triflouroacetic acid (w/w)-acetonitrile gradient. Detection was according to UV absorption at 214 nm. Following this the samples were analyzed by HPLC-Mass Spectroscopy (MS) equipped with an electrospray source. Mass calculations represent the mass of the ionized peptide samples, as derived from the retention times. Following separation, the amino acid composition of the peptides was determined with a gas-phase microsequencer (Applied Biosystems 470A).
The following data is representative: Eight peptide peaks were typically observed of which 3 were major peaks having Rt values of 17.79, 19.7, 23.02 and 5 were minor peaks having Rt values of 12.68, 14.96, 16.50, 21.9 and 25.1, which Rt values represent molecular mass of 2764, 1697, 1880, 2616, 3217, 2333, 1677 and 1669 Da, respectively. At Rt of 17.79 (corresponding to 2,764 ) Da) a major peak of a peptide of 23 amino acids representing amino acids 1-23 . of aS1 casein, having the sequence RPKHPIKHQGLPQEVLNENLLRF (SEQ
ID NO:22, see McSweeny et al., 1993, ibid., for the complete sequence of aS casein). Other peptides were from positions 208-224 of B casein, positions
16-37 of aS] cascin and positions 197-222 of aS2 like casein precursor. Other peptides were also present.
Synthetic peptides derived from casein: Peptides of increasing lengths corresponding to the N-terminal 2-26 amino acids of aS! casein were s synthesized by NoVetide Ltd., Haifa, Isracl. with purity of >95 % (HP1.C). -
Quality Control included: HPLC, Mass Spectrometry (EI), Amino acid analysis and Peptide Content. Table 3 below provides the sequence of these peptides:
TABLE 3
Identification Sequence (N terminus - C terminus) No. of amino acids SEQ ID NO: 74 RP 2 }
IP RPK 3 2 2P RPKH 4 3 3p RPKHP 5 4 ap RPKHPI 6 5 5p RPKHPIK 7 6
Y RPKHPIKH 8 7
X RPKHPIKHQ 9 R la RPKIHPIKHQG 10 y 2a RPKHPIKHQGL 1 10 3 RPKHPIKHQGL.P 12 H
A RPKHPIKHQGL.PQ 13 12
B RPKHPIKHQGLPQE 14 1?
C RPKHPIKHQGLPQEV 15 14 b) RPKHPIKHQGL.PQEVL 16 15
E RPKHPIKHQGLPQEVLN 17 16
F RPKHPIKHQG!.PQEVLNE 18 17
G RPKHPIKHQGLPQEVI.NEN 19 18
H RPKHPIKHQGLPQEVI.NENL 20 19
RPKHPIKHQGLPQEVLNENLL 21 20 ) RPKHPIKHQGLPQEVI.NENLLR 2 21
K RPKHPIKHQGLPQEVLNENLLRF 23 22
L. RPKHPIKHQGLPQEVLNENLLRFF 24 23
M RPKHPIKHQGLPQEVLNENLLRFFV 25 24
N RPKHPIKHQGLPQEVLNENLLRFFVA 26 25
Juvenile (Type I, IDDM) diabetes in Non-Obese Diabetic (NOD) mice:
Peptides derived from natural casein: NOD mice are a commonly used model for research of autoimmune disease and human Juvenile Diabetes. Six week old female NOD mice received either one or two injections per week of 100 pg of peptides derived from natural casein, for a total of 5 or 10 treatments. } Control mice received no treatment. The severity of disease was determined according to glucosuria, which was measured using Combi test sticks (Gross, ’ 5 DJ. er al. (1994), Diabetology, 37:1195]. Results were expressed as the percent of glucosuria-free mice in each sample over a 365-day period.
Synthetic peptides derived from casein: In another experiment, 6 week old female NOD mice received one injection per week of 100 pg of Synthetic peptides derived from casein for a total of 5 treatments. Control mice received no treatment. Results were expressed as the number of healthy mice in the various treated groups.
Intraperitoneal Glucose Tolerance Test (IPGTT): The glucose tolerance test is the definitive method for investigating glucose metabolism and diabetic tendencies in mammals. Twenty five (25) weeks after receiving
Synthetic peptides derived from casein, response to a glucose load was assessed with an intraperitoneal glucose tolerance test. Glucose injection consisted of lg/kg body weight. Glycemic values were determined from blood drawn prior to test (0 minutes) and 60 minutes after loading. Plasma glucose levels were determined with a Glucose Analyzer 2 (Beckman Instruments, Fullerton, CA) and expressed as mmol/L. Normal values do not exceed 140 mmol/L.
Stimulation of proliferation of Natural Killer (NK) cells:
From human Peripheral Blood Stem Cells (PBSC): PBSC of G-CSF treated subjects were separated on a FICOLL gradient, washed twice with
RPMI-1640 medium and seeded into 1.5 ml wells with or without peptides 2s derived from natural casein or synthetic peptides derived from casein, as ) indicated, (0-500 ng per ml). Following two days incubation the cells were assayed for Natural Killer activity by measuring radioactivity released from 35G-labeled K562 target cells (NEG-709A, 185.00 MBq, 2.00 mCi
EASY TAGth Methionine, L-[35S] 43.48 TBq per mmol, 1175.0 Ci per mmol,
0.488 mi, Boston USA). Two concentrations of effector cells (2.5 x 103 and 5 x 109 cells per well) were incubated with 5 x 103 target cells per well (effector:target cell ratios of 50:1 and 100:1, respectively) in U-bottomed 96 well tissue culture plates. The cells were incubated for 5S hours at 37 °C in 5 % s CO,, 95 % air and precipitated by 5 minutes centrifugation at 1000 rpm. 35S rclcase was measured in 50 pl samples of the supernatant liquid.
From murine Bone Marrow (BM) cells: Bone marrow was collected from 4 untreated BALB/c and C57Bl/6 mice. Bonc marrow was harvested from the long bones of front and hind limbs of the mice by injection of medium using a 25 Gauge needle. Aspirated cells were washed with RPMI 1640, counted in a hacmocytometer and vital-stained (20 ul of cells in 380 pl acetic acid/trypan blue), then seeded in culture bottles at 2-5 x 106 cells per ml in
RPMI-1640 containing 10 % Fetal Calf Serum, antibiotics and glutamine with or without 100 pg per ml peptides derived from natural casein. The cell cultures were incubated in S % CO,, 95 % air for 12-15 days at 37 °C. harvested by 10 minutes centrifugation at 1500 rpm. counted. and seeded in
U-bottom wells with Scr (Chromium-31, 740 MBq, 2.00 mCi activity) or 5S (NEG-709A, 185.00 MBq, 2.00 mCi EASYTAGth Methionine, L-[35S] 43.48
TBq per mmol, 1175.0 Ci per mmol, 0.488 ml, Boston USA) labeled murine lymphoma (YAC) cells at either 25:1 or 50:1 effector:target cell ratio. NK activity is expressed as the percent radioactivity in the cell-free supernatants.
Proliferation of human cells in culture: Peripheral blood (PB) was collected from healthy or affected patients. Affected patients received no treatment other than G-CSF supplementation prior to plasmapheresis. Bone marrow (BM) cells were collected from consenting healthy patients or affected patients in remission following chemotherapy by aspiration. Umbilical cord blood was collected during normal births. Human cells of the various origins were separated on a FICOLL gradient, washed twice with RPMI-1640 medium, and seeded into 0.2 ml flat bottom tissue culture wells at the indicated concentrations with or without peptides derived from natural casein or with or without synthetic peptides derived from casein, as indicated. All treatments, including controls, were repeated in triplicate. Cell proliferation was measured by ‘HT incorporation: radioactive thymidine was added [thymidine . 5 (methyl-[’H]) Specific activity 5 Ci per ml 37 MBq per ml, ICN Corp.] following incubation for the indicated number of days. Cells were then incubated 16-20 hours with the label, harvested and washed with medium.
Incorporated radioactivity was measured in a p scintillation counter.
Proliferation of K562 leukemia and colon cancer cell lines: Colon and
K562 are established lines of cancer cells grown in culture. Both cell lines were grown in culture bottles in 5 % CO,, 95 % air at 37 °C, harvested and washed with medium before seeding in tissue culture wells at 4 x 105 cells (K562) or 3 x 103 cells (Colon) per well. Peptides derived from natural casein were added to the wells, at the indicated concentrations, and after 9 (K562) or 3 1s (Colon) days of incubation labeled thymidine was added as described above.
Harvesting and measurement of radioactive uptake was as described above.
Fluorescent antibody detection of NK and T Cell proliferation in human Peripheral Blood Stem Cells (PBSC):
Peripheral Blood Stem cells (PBSC) from human subjects receiving
G-CSF treatment were collected by plasmapheresis, separated on a FICOLL gradient, washed twice with RPMI-1640 medium containing 10 % Fetal Calf
Serum and incubated in culture bottles at 37 °C in 5 % CO,, 95 % air with or without peptides derived from natural casein at the indicated concentrations.
Following 10, 14 or 28 days incubation with peptides derived from natural casein, the presence of T cells (CD; surface antigen) and NK cells (CDs, . surface antigen) was detected by direct immunofluorescence using anti-CD3 fluorescent antibody (CD,/FITC clone UHCT)), anti-CDs5g fluorescent antibody (CDs¢/RPE clone MOC-1) (DAKO A/S, Denmark) and mouse
IgG1/RPE and IgGI/FITC antibodies as a control. Detection of fluorescently tagged cells was performed using fluorescence activated cell sorting (FACS).
Stimulation of hematopoiesis from Bone Marrow (BM) Cells in culture:
Proliferation of megakaryocytes in multipotential colonies (CFU-GEMM) from murine Bone Marrow cells: Primary bone marrow cells s (1 x 103 per ml) from 8-12 week-old C3H/HeJ mice were grown in serum-free methyl cellulose-IMDM medium for 8-9 days at 5 % CO,, 95 % air, at 37 °C.
The medium, appropriate for thc growth of mulupotential colonies (CFU-GEMM), contained 1 % BSA (Sigma). 10-4 M thioglycerol (Sigma). 2.8 x 10-4 M human transferrin (TF, Biological industries, Israel), 10 % WEHI-CM as a source of I.-3 and 2 units per ml erythropoietin (thEPO, R & D Systems,
Minneapolis). Colonies were scored after 8-9 days using an Olympus dark field microscope. They were picked with a micropipette, cytocentrifuged and stained with May-Grunwald-Giemsa tor ditterential counts. At least 700 cells were counted for cach preparation. is Proliferation of Dendritic cells in CFU-GEMM: Multipotent (CFU-GEMM) colonies grown {rom primary bone marrow cells as described for the assay of megakaryocyte proliferation above were collected. stained and counted for dendritic cells. At least 700 cells were counted for each preparation.
Proliferation of Plasma cells in CFU-GEMM: Multipotent (CFU-GEMM) colonies grown from primary bone marrow cells as described for the assay of megakaryocyte proliferation above were collected, stained and counted for plasma cells. At least 700 cells were counted for each preparation.
Proliferation of Macrophage cells in CFU-GEMM: Multipotent 2s (CFU-GEMM) colonies grown from primary bone marrow cells as described for the assay of megakaryocyte proliferation above were collected, stained and counted for macrophage cclls. At least 700 cells were counted for each preparation.
Proliferation of Red Blood Cells in CFU-GEMM: Multipotent (CFU-GEMM) colonies grown from primary bone marrow cells as described for the assay of megakaryocyte proliferation above were collected, stained and counted for red blood cells. At least 700 cells were counted for each preparation.
Proliferation of Polymorphonuclear Cells (PMN) in CFU-GEMM: . 5 Multipotent (CFU-GEMM) colonies grown from primary bone marrow cells as described for the assay of megakaryocyte proliferation above were collected, stained and counted for polymorphonuclear cells. At least 700 cells were counted for each preparation.
Proliferation of megakaryocyte- and erythroid forming cells from human bone marrow and cord blood cells: A sample of bone marrow from an apparently healthy human being was processed by density gradient separation using Histopaque-107 (Sigma Diagnostics) to obtain a purified population of mononuclear cells (MNC). Colony assays were performed in a plating medium containing final concentrations of 0.92 % methyl cellulose (4000 centripasc 1s powder, Sigma Diagnostic), rehydrated in Iscoves modified Dulbecco’s medium containing 36 mM sodium bicarbonate (Gibco), 30 % fetal bovine serum (FBS) (Hyclone), 0.292 mg/ml glutamine, 100 units per ml penicillin and 0.01 mg per ml streptomycin (Biological Industries, Beit Haemek). Cord blood from normal births was collected and prepared as mentioned above.
Colony assay medium containing 10° MNC per ml was plated in triplicate wells within a 24 well tissue culture plate (Greiner), 0.33 ml per well.
The cultures were incubated at 37 °C in 5 % CO,, 95 % air and 55 % relative humidity with or without peptides derived from natural casein or synthetic peptides derived from casein, at the indicated concentrations. Plates were 25s scored after 14 days for colonies containing more than 50 cells. . Megakaryocytes were identified by indirect immunofluorescence using a highly specific rabbit antibody recognizing human platelet glycoproteins, and an ) FITC-conjugated goat anti-rabbit 1gGG. Added growth factors included 15 ng per ml leucomax (GM-CSF) (Sandoz Pharma), and 5 % vol. per vol. human phyto-hemagglutinin-m (Difco Lab)-induced conditioned medium (CM) to induce development of granulocyte-monocyte colonies (CFU-GM).
Erythropoietin (EPO) 2 units/ml was used to induce formation of erythroid colonies (burst-forming unit-erythroid-BFU-E).
Altematively, human bone marrow cells from consenting volunteer donors or patients undergoing autologous bone marrow transplantation were precultured in medium containing 10-1000 pg per ml peptides derived from natural casein, grown in semi-solid agar, and scored for granulocyte-macrophage hematopoietic colonies (GM-CFU) at 7 or 14 davs post treatment.
Megakaryocytopoiesis was measured in normal bone marrow cells from healthy consenting human donors by either scoring of the number of megakaryocytes in samples of liquid culture (RPMI-1640 plus 10 % human AB serum, glutamine and antibiotics) with or without 100 pg per ml peptides derived from natural casein, or in a methylcellulose assay for assessing colony 1s formation. 2 x 105 bonc marrow cells were seeded in the presence of’ a standard growth factor combination with or without peptides derived from natural casein. In the methylcellulose assay megakaryocytes were counted with an inverted microscope on days 12-14 after seeding.
Clinical trials using peptides derived from natural casein: In one series of trials, a single dose containing 50 mg peptides derived from natural casein was administered intra-muscular to human subjects in 3 depots, over a period of 2 hours. Clinical parameters were monitored at the indicated intervals. In other trials, patients at various stages of treatment for and/or remission from cancer and metastatic discase received peptides derived from natural casein once or twice, and were monitored for changes in the cell count of peripheral blood.
Inhibition of in vitro HIV infection of human lymphocyte cells:
Peptides: Peptides (either peptides derived from natural casein or synthetic peptides derived from casein (2-26 amino acids in length, see table 3)
supplied as lyophilized powder were resuspended in RPMI complete medium and added to cell cultures at a final concentrations of 50 to 1000 pg per ml.
Cells: Several types of freshly isolated human cells (primary cells) and cell lines are known to be susceptible to in vitro HIV-1 infection, although : 5 essentially any cell displaying even low surface levels of the CD 4molecule can be considered a potential target for HIV-1 infection. Two commonly used human cell lines which are highly sensitive for HIV-1 infection were chosen,
CEM and Sup-T1.
CEM is a human T4-lymphoblastoid cell line initially derived by G. E.
Foley et al. [(1965), Cancer 18:522] from peripheral blood buffy coat of a 4-year old caucasian female with acute lymphoblastic leukemia. These cells were continuously maintained in suspension in medium, and have been used widely for analysis of infectivity, antiviral agents and neutralizing antibodies.
Sup-T1 is a human T-lymphoblastoid cell line isolated from a pleural cffusion of an 8-year old male with Non-Hodgkin’s T-cell lymphoma {Smith, S.
D. et al. [(1984) Cancer Research 44:5657]. This cell expresses high levels of surface CD4and is useful in studies of cell fusion, cytopathic effect and infectivity of HIV-1. Sup-T1 cells are grown in suspension in enriched medium.
Medium: Cells were grown in RPMI-1640 complete medium enriched with 10 % Fetal bovine serum, 2 mM glutamine and 2 mM penicillin- streptomycin (GIBCO).
Virus: The HIV virus strain employed was HIV-1I1IB, originally designated HTLV-IIIB. Concentrated culture fluids of peripheral blood from several patients with AIDS or related diseases were used to establish a : permanent productive infection in H-9 cells. This subtype B virus has high capacity to replicate in human T-cell lines. Viral titer was 5.38 ng per ml in stock solution.
FITC-labeled peptides: FITC F-1300 (Fluorescein isothiocyanate, isomer I, Sigma (F250-2) St. Louis, MI, USA) having excitation/ecmission maxima ot about 494/520 nm, respectively, was employed. The amine-reactive fluorescein derivative is probably the most common fluorescent derivatization reagent for covalently labeling proteins. FITC-conjugated peptides derived from natural casein were prepared by covalent binding of FITC to the amine groups of lysine.
HIV-1 P* antigen capture assay: An HIV-1 P** Anti gen capture assay kit employed was designed to quantitate the HIV-1 P** core antigen. which 1s proportionally related to the degree of viral production in cells. This Kit was purchased from the AIDS Vaccine program of the SAIC-NCI-Frederick Cancer
Research Institute, P.O. Box B, Frederick, M.D 21702, USA and included 96 well plates coated with monoclonal antibody to HIV-1 P**, primary antibody-rabbit anti-HIV P?* serum, secondary antibody-Goat anti-rabbit-1gG (H+L) peroxidase conjugated antibody, TMB peroxidase substrate system and lysed HIV-1 P* standard. The HIV-1 P** antigen capture assay was analyzed 18 by Organon-Technica ELISA reader at 450 nm with a reference at 650 nm.
HIV-1 P* antigen capture ELISA: HIV infection was measured with an indirect enzyme immunoassay which detects HIV-1 P** core antigens in tissue culture media. Tissue culture supernatant was reacted with primary rabbit anti-HIV-1 P** antigen and visualized by peroxidase conjugated goat anti rabbit IgG. The reaction was terminated by adding 4N H2SOy4, wherein the intensity of the color developed is proportional to the amount of HIV-1] antigen present in the tissue culture supernatant.
Biological hazard level 3 (BL-3) laboratory: All virus production isolation and infection, tissue culture of HIV-1 infected cells, P24 antigen containing supernatant harvesting andP*' antigen capture ELISA, were performed in BL-3 facility of the Hebrew University, Hadassah Medical School and were in accordance with the bio safety practices set by the NIH and CDC (USA).
Flow cytometry: A FACSort cell sorter (Becton & Dickinson, San Jose,
CA. USA) was used to (i) determine the percentage of CD4 positive CEM and sup-T1 cells batches before infection with HIV-1 in order to assure the same degree of infection in each experiment; and (ii) detect T cells that harbor FITC conjugated peptides derived from natural casein in their cytoplasm and nuclei.
CO incubator: For viral culture production cells with HIV-1, cells and - 5 virus pretreated with peptides derived from natural casein and cells which were further incubated with HIV-1, were all kept in humidified CO, incubator for the duration of the experiment.
HIV infection of human cultured CD4 cells: For longer incubations, the cells (CEM, Sup-Tl) were preincubated with several increasing concentrations of peptides derived from natural casein (50-1000 pg per ml) or synthetic peptides derived from casein (10-500 pg per ml) for 24 (for synthetic and natural peptides) and 48 (only for natural pegtides) hours and HIV-111IB (45 pg per ml final concentration) was added to each well thereafter. For the shorter incubations (3 hours), HIV-11IIB was preincubated with the peptides for 3 hours and then added to cells (5000 cells/ well) in tissue culture plates.
Controls were IF (Infected, cells cultured with HIV-1 and without peptides),
UIF (Uninfected, cells cultured without HIV-1 and without peptides) and ULF +
Ch (Uninfected + peptides derived from natural casein, cells cultured in the presence of peptides derived from natural casein {50-1000 ug per mi}) to test the effect of peptides derived from natural casein and synthetic peptides derived from casein on cell viability and growth. Cells were counted for viability and proliferation rate on day 7, 10 and day 14 post infection (the day of P24 antigen culture supernatant harvest). Cells and tissue culture supernatants (media) were harvested and lysed immediately in 1/10 volume of 10 % Triton X-100. These samples were further incubated at 37 °C for 1 hour and kept at -80 °C until : tested for P** antigen.
Confocal microscopy: A Zeiss LSM 410 confocal laser scanning system attached to TW Zeiss Axiovert 135M inverted microscope, employing the laser scanning confocal microscopy technique, was used to detect penctration of FITC conjugated peptides into cclls. T cells were incubated with
FITC conjugated peptides derived from natural casein ina 5 % CO. 95 %o air. 37 °C incubator. after which the cells were washed 3 times with phosphate buffer saline (PBS) to remove unbound FITC-peptides. Cells were fixed with 5s 3.8 % formalin for 10 minutes, washed twice with PBS and resuspended in 50-100 pi PBS before viewing the cells under the microscope. Selected images of cells from different time points of incubation (15 minutes. 30 minutes. hour, 1.5 hour and 3 hours) displaying various amounts of FITC-peptides derived from natural casein in their cytoplasms and nuclei were stored on 3.5"
Zip drive (230 MB) and processed for pictures using Photoshop software. [ ‘HJ-thymidine incorporation test: In order to test the effect of peptides derived from natural casein on T cell proliferation, several concentrations of peptides derived from natural casein (10 mg/ml stock in RPMI) were addcd to
Sup-T1 cell cultures in 96 flat bottom microwell plate (5000 cells/well). as is described for HIV-1 infection in Sup-T1 cells. Cells were counted and their viability was determined by trypan blue dve exclusion. They were pulsed with [’H]-thymidine at each time point (3, 7, 10 and 14 days) for 18 hours (over night) and harvested on glass fiber filters for radioactivity rcading (Incorporation of [’H]-thymidine into cellular DNA is proportional to degree of cell proliferation).
Toxicity of peptides derived from natural casein in normal, myeloablated and transplant recipient mice and guinea pigs: Intramuscular, or intravenous injections of up to 5,000 mg peptides derived from natural casein per kg animal were administered in a single dose, or in three doses to os normal animals. A variety of strains were employed, including BALB/c,
C3H/HeJ and Non-Obese Diabetic (NOD) mice. The mice were either : monitored for 10 months before sacrifice and post-mortem examination (toxicity assay) or observed for 200 days (survival rate). Guinea pigs received a single intramuscular injection of 20 mg peptides derived from natural casein per animal. Fifteen days later they were sacrificed and examined for pathology.
Leukocyte and platelet reconstitution in bone marrow transplant recipient mice: BALB/c mice were sub-lethally irradiated at a source to skin ) distance of 70 cm, dosage of 50 cGy per minute, for a total of 600 ¢Gy. The irradiated mice were reconstituted with syngeneic bone marrow as described ; s above and injected intravenously 24 hours later with | mg per animal peptides derived from natural casein, synthetic peptides derived from casein (13-26 amino acids, see Table 3 above), or human serum albumin (controls), following a double-blinded protocol. Leukocyte reconstitution was determined according cell count in peripheral blood collected at indicated intervals from 6 to 12 10 days post treatment. Platelet reconstitution was determined by cell count in blood collected from the retro orbital plexus, into EDTA-containing vials, at indicated intervals from day © to day 15 post treatment.
In an additional series of experiments, CBA mice were lethally irradiated (900 cGy), reconstituted with BM cells and treated with peptides derived from natural casein or human serum albumin as described above. Platelet reconstitution was assayed as mentioned above.
In a third series of experiments, the mice were irradiated (800 cGy), reconstituted and injected intraperitoneally with 100 pg synthetic peptides derived from casein (peptides 3a and 4P, representing the first 6 and 12 amino acids of the N terminus of aS1 casein, respectively - see Table 3 above) daily, on days 4, 5, 6 and 7 post-transplantation. Platelet reconstitution was assayed at 10 and 12 days post-transplantation.
Reconstitution of bone marrow transplant recipient mice:
C57Bl/6 mice were lethally irradiated at a source to skin distance of 70 cm, dosage of 50 cGy per minute, for a total of 900 cGy. The irradiated mice . were reconstituted with syngeneic bone marrow cells from mice which were either treated a day prior to bone marrow collection with 1 mg per animal peptides derived from natural casein or with saline (controls), following a double-blinded protocol. In one experiment mice survival was monitored for
18 days. In another cxperiment mice were sacrificed after 8 days and spleen colonization monitored.
Synthetic peptides derived from casein significantly reduce Cholestrol levels:
The ability of synthetic caesin derived peptides to reduce cholesterol levels in 7-wcek old female C57Bl/6) mice was assessed after feeding an atherogenic diet. The mice were divided into groups of 8. One control group was fed a normal diet. A second control group was fed the modified Thomas
Hartroft diet containing cholate (#1D 88051: Teklad, Madison, WI) [Gerber, D.
W. et al, Journal of Lipid Research. 42, 2001]. The remaining experimental groups were all fed the modified Thomas Hartroft diet. After onc week on the diet, serum cholesterol values increased significantly and the synthetic peptides derived from casein were injected intraperitoneally, 1 mg per mouse, followed by a second injection of 0.1 mg one week later.
Cholestrol blood levels were determined according to Roche Cholesterol
Assay based on Roeschlou & Allin enzymatic method (Roche, Inc.. Germany).
EXPERIMENTAL RESULTS
Peptides derived from natural casein: Originating from the observation that curdled milk occasionally failed to support bacterial growth, a casein fragment possessing bacteriocidal properties was isolated from milk proteins (United States Patent No. 3,764,670 to Katzirkatchalsky, er al). Crude peptides derived by proteolysis of natural casein were prepared by acid precipitation of the soluble fraction of the casein proteolytic digest, dialysis and lyophilization. When tested for biological activity after extended storage, it was noted that this crude preparation, when lyophilized and stored at 4 °C. remained active (in vitro and in vivo) for at least 24 months.
In order to identify the active peptides contained in the peptides derived from natural casein the lyophilized crude preparation was fractionated using high performance liquid chromatography (HPLC), as described hereinabove.
All of the lyophilized samples analyzed demonstrated similar retention time profiles, with contents as described above.
. Thus, a major component of the crude peptides derived from natural casein preparation is the N-terminal fragment of aS1 casein.
’ 5 Peptides derived from natural casein are non-toxic in rodents and humans: Extensive investigation of the short and long term effects of high doses of peptides derived from natural casein on mice, rats, guinea pigs and human volunteers confirmed the absence of toxicity, teratogenicity or adverse side effects of the preparation.
In one series of tests, single doses representing
7,000 times the estimated effective dose of peptides derived from natural casein were administered intra muscularly to mice.
Standard post-mortem pathology examination of the mice at 14 days post treatment revealed no toxic effects on internal organs or other abnormalities.
Similar toxicity tests in guinea pigs revealed no abnormalities two weeks after single 20 mg intra-muscular doses of
1s peptides derived from natural casein.
In another series of experiments, high doses of peptides derived from natural casein administered to healthy mice had no effect on several hematological parameters measured two weeks later, including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), electrolytes, glucose and others.
A third series of experiments tested repeated
20 high doses of 100 mg per kg body weight in mice and rats for two weeks, revealing no allergic, delayed cutaneous or anaphylactic responses and no pathological effects upon post-mortem examination.
When peptides derived from natural casein were tested for their effect on the long-term survival of irradiated, bone marrow reconstituted BALB/c and C3H/HelJ mice, survival of
25 the treated mice (18 of 27 BALB/c and C3H/Hel; 66 %) clearly exceeded the
- survival rates of the albumin-treated controls (4 of 26 BALB/c and C3H/Hel; %). Standard teratogenicity tests [for details see, for example, Drug Safety in Pregnancy, Folb and Dakes, p. 336, Elsevicr; Amsterdam, NewYork, Oxford (1990)] in mice treated with peptides derived from natural casein revealed no
50 cffect of the peptides on any developmental parameters.
Similar to its lack of toxicity or side effects when tested in rodents. peptides derived from natural casein were safe when administered to humans as well. Comparison of blood and urine samples from seven healthy human volunteers before, during and 7 days after intramuscular injection of peptides derived from natural casein revealed no changes in any of the clinical paramcters. No other negative effects were observed.
Thus, high dosc and extended treatment of rodents with peptides derived from natural casein revealed no apparent toxic, pathological, hypersensitivity, teratogenic, serological or any other negative effects. Moreover, peptides derived from natural casein administration to irradiated mice, at risk for short-and long-term complications, conferred a significant survival advantage over 200-300 days. These, and the absence of any undesirable effects in healthy human volunteers receiving peptides derived from natural casein via injections clearly demonstrate the peptide’s safety in parenteral administration.
Reconstitution of bone marrow in transplant recipient mice: When
C57BY6 mice were lethally irradiated and reconstituted with syngeneic bone marrow from mice that were either treated a day prior to bone marrow collection with 1 mg per animal peptides derived from natural casein or not so treated, survival of irradiated mice that received bone marrow from treated mice far exceeded that of irradiated mice that received bone marrow from non treated mice (survival of irradiated mice that reccived bone marrow from treated mice was 15 out of 18, 10 days post irradiation; whereas survival of irradiated mice that received bone marrow cells from saline-treated control mice was 4 out of 17, 10 days post irradiation). Spleens derived from irradiated mice that received bone marrow from treated mice included about twice to three times as many colonies per spleen, as compared to spleens of irradiated mice that received bone marrow cells from saline-treated control mice (1-5 colonies as compared to 0-3 colonies).
Peptides derived from natural casein stimulate the proliferation of lymphocytes: Natural killer (NK) and cytotoxic T cells are crucial to the immune system’s ability to protect against invasion by both infectious pathogens and cancer cells, by both active cytotoxicity and the secretion of . immunoregulatory lymphokines. Immune compromise, such as in AIDS or following chemotherapy, results in abnormal, weakened T or NK cell activity. : 5 When normal murine bone marrow cells from BALB/c and C57BY/6 mice were cultured in the presence of 100 pg per ml peptides derived from natural casein, a clear increase in NK activity was observed in both effector:target cell ratio groups. Moreover, comparison between the two groups revealed a clear dose response relationship. At the 1:25 effector:target cell ratio the average NK activity was elevated from 13.93 % to 30.77 % and at the 1:50 effector:target cell ratio the average NK activity was elevated from 13.68 % to 44.05 % (Figure !). Similar experiments using human Peripheral Blood Stem Cells from Granulocyte Colony Stimulating Factor-treated donors demonstrated an even more significant, concentration- dependent stimulation of target cell lysis by peptides derived from natural casein.
In the first set of experiments (Figure 2a), NK activity was measured in blood samples taken from one patient and incubated at two effector:target cell ratios with increasing peptides derived from natural casein concentration. Only 4 % >S release was measured in the control, untreated PBSC culture. Almost the same percent radioactivity (4 %) was found at the lowest peptide concentration (Sug per ml). However, at higher peptide concentrations, in the range of 10ug per ml up to 100pg per mi, a release of 10.8-14.9 % »S was measured for effector:target cell ratios of 100:1 and 8.3-14.5 % 38 for effector target cell ratios of 50:1 (Figure 2a).
When PBS cells from normal (patient 1) and affected (patients 2-6) ) human donors were incubated with increasing concentrations of the peptides derived from natural casein, a significant enhancement of affected patients” NK cell activity could be measured. Thus, while the peptides derived from natural cascin had a minimal effect on the normal patient’s NK activity (increased from
13- 15 % *°S release, patient 1), PBS cells from both breast cancer and
Non-Hodgkins Lymphoma patients (patients 3 and 4, for example) exhibited dramatic. dose-dependent increases in NK activity (3.5 to 10.8 % *°S: 12.2 to 19.1 % *°S, respectively) (Figure. 2b).
S Peptides derived from natural casein stimulate the proliferation of :
CD56 surface antigen positive (NK) cells: In another series of experiments
Peripheral Blood Stem Cells (PBSC) from 5 human donors receiving GCSF treatment were incubated with peptides derived from natural casein for 10. 14. or 28 days, then assayed for presence of the CD 56 antigen. A sometimes dramatic increase in CD 56 antigen detection was observed in the peptide-treated cells from all the donors but one (patient 1). A representative response is depicted in Figure 3a: Following 10 days of incubation with or without peptides derived from natural casein. the presence ofCDS56 surfacc antigen-positive (NK) cells was detected by direct immunofluorescent staining.
Overall, incubation with peptides derived from natural casein increased the mean percentage of the cells positively stained for CD36 from 0.64 “6 in the : control group to 2.0 % following treatment (Figure 3a).
Peptides derived from natural casein stimulate the proliferation of
CD3 surface antigen-positive (T) cells: The effect of peptides derived from natural casein on the proliferation of CD3 surface antigen-positive (T) cells in
PBS cells from 5 subjects was assayed by direct immunofluorescence. In all but one patient (patient 4), 14 days incubation with peptides derived from natural casein significantly increased T-cell proliferation, up to more than 5 fold in some. Taken together, the mean percentage of the cells positively stained for CD3 increased from 19.45 % in the control group to 35.54 % in the treated group (Figure 3b).
Peptides derived from natural casein stimulate the proliferation of -
CD56 and CD3 (NK/ T-cells) positive cells: In an additional experiment
PBSCs from 7 patients were incubated with peptides derived from natural 50 casein for 28 days, and the effect on proliferation of NK/T cells (CD56 and
CD3 surface antigen-positive) was detected by direct immunofluoresence.
Incubation with peptides derived from natural casein stimulated proliferation of 3 T-cell greater than S fold in some cases (patient 6), while the mean percentage of the CD3- positive (T-) cells increased from 2.08 % in the control group to ’ 5 6.49 % in the treated group. The number of both CD56 and CD3 surface antigen-positive (NK/T) cells was increased from 1.1 % in the control to 4.3 % in the treated group (Figure 3c). Thus, peptides derived from natural casein stimulate the proliferation of both T-lymphocytes and Natural Killer cells from normal murine and human blood cell progenitors. Significantly, the greatest immune-stimulatory effect of the peptides derived from natural casein was noted in human donors having initially low T- and NK cell levels (Figures. 3a-c).
Synthetic peptides derived from casein stimulate human lymphocyte proliferation in vitro: When synthetic peptides derived from casein is representing the first 3 to 26 residues of aS1 casein were incubated with human
PBSC cells from healthy and cancer patients (see below), a significant increase in NK cell activity was observed. Target cell lysis was greatest (from 3 to greater than 5 fold that of controls) in Non-Hodgkin’s Lymphoma and Breast
Cancer patient’s PBSC cultures after two days incubation with as little as 10 pg per ml of peptides containing the first 9 or more residues of aS1 casein (Figure 4). Under identical conditions, none of the peptides tested had a significant effect on NK activity in PBSC cultures from healthy human donors. Thus, even low concentrations of peptides containing the first 10 residues of the N-terminal sequence of aSI casein are capable of selectively stimulating in vitro 2s lymphocyte proliferation in cells from cancer patients. : Similar stimulation of NK cell activity was observed when PBS cells from human donors with hematopoietic disease were incubated with Synthetic peptides derived from casein representing the first 3 amino acid residues of aS] casein. Incubation of the PBS cells with the peptides increased target cell lysis from 2- to greater then 8- fold that of the untreated controls. Of the 5 patients tested, three (3) responded to 25 pg/ml peptide concentration. one (1) responded to 100 pg/ml peptide concentration and one (1) to 250 pg/ml. Three out of the tive (3) patients responded at 25 pug/ml.. No significant effect on NK activity in PBSC cultures from healthy human donors treated with the svnthetic : peptide representing the first 3 amino acids of aS1 casein. was observed. confirming the selective nature of the human lymphocyte-stimulating properties of casein-derived peptides.
Stimulation of hematopoiesis in human blood cell progenitors:
Blood cell progenitors differentiate into a variety of blood cells: macrophages, monocytes, granulocytes, lymphocytes, erythrocytes and megakaryocytes. Progenitor cells are abundant in bone marrow, but arc also found in peripheral blood after Granulocyte Colony Stimulating Factor treatment (PBSC cells), and fresh Cord Blood. When increasing concentrations (50-600 pg per ml) of peptides derived from natural casein were added to cultures of human Bone Marrow, PBSC and Cord Blood, an increase in cell proliferation, as measured by [’H)-thvmidine incorporation was noted (Figures 5a-5¢). Human PBSC proliferation was most greatly effected by 300 pg per ml (Figure Sa) after 15 days in culture. An even greater effect was noted for Cord
Blood cells in culture (3 to 4 fold increase in [*H}-thymidine incorporation) after 14 days incubation (but not after 7 days) with peptides derived from natural casein (600 pg per ml, Figure Sc). Cultured human bone marrow cells from three out of four donors also reacted strongly (3 to 5 fold increase in incorporation) to peptides derived from natural casein (300 pg per ml) after 21 2s days incubation (Figure 5b). Thus, peptides derived from natural casein stimulate proliferation of human blood cell progenitors from bone marrow as well as other sources. Interestingly, incubation of cultured human K562 (Chronic Myeloid Leukemia) and Colon (Colon cancer) cell lines with high concentrations (up to 500 pg per ml) of peptides derived from natural casein under similar conditions had no effect on [’H]-thymidine incorporation. Thus, peptides derived from natural casein stimulate proliferation of human blood cell . progenitors but not growth of cancerous cells in vitro.
Stimulation of megakaryocytopoiesis by peptides derived from casein: ’ 5 Peptides derived from natural casein stimulate megakaryocyte progenitor proliferation in cultured murine bone marrow cells:
Multinucleated megakaryocytes develop in the bone marrow from primitive stem cells, mature to giant cells and give rise to thousands of thrombocytes per megakaryocyte. Thrombocytes are crucial for clot formation and thrombocytopenia is a major concern in myeloablative conditions (following chemotherapy or radiotherapy).
Primary bone marrow cell cultures can be induced to form CFU-GM (Granulocyte and Monocyte) colonies, and CFU-GEMM (Granulocyte,
Erythroid, Macrophage and Megakaryocyte) colonies, containing additional blood cell types. Colony counts reflect expansion of specific progenitors, cell numbers reflect proliferation rates and differential cell counts reflect which specific cell lineages have developed [Patenkin, D. ef al. (1990), Mol. Cel.
Biol. 10, 6046-50]. In cultured murine bone marrow cells incubated with erythropoietin and IL-3, addition of 25 pg per ml peptides derived from natural casein for 8 days increased the number of CFU-GEMM two and one half fold over controls, stimulating a three fold increase in relative cell numbers per colony in the CFU-GEMM. In a similar series of experiments, addition of peptides derived from natural casein to bone marrow cells incubated with erythropoietin and conditioned medium (sce Materials and Experimental
Methods) stimulated a concentration-dependent increase in the percentage of : early and late megakaryocytes (15 % megakaryocytes without peptides, to 50 % with 500 pg per ml peptides derived from natural casein). Thus, 8 days treatment with peptides derived from natural casein stimulated a significant increase in megakaryocyte formation and development in primary murine bone marrow cultures.
Synthetic peptides derived from casein stimulate megakaryocyte progenitor proliferation in cultured murine bone marrow cells:
Similar to the above and under similar experimental conditions, synthetic peptides derived from casein representing the first 5 to 24 amino acids ol aSlcasein increasc the percentage of early and late megakaryocytes from 15 : % without the synthetic peptide to more than 40 % with 25 pg per ml of synthetic peptides (Figure 7). Thus. 8 days treatment with synthetic casein derived peptides representing the first 5, 6, 11, 12, 17, 18. 19. 20. 21 and 24 amino acids stimulated a significant increase in megakaryocyte formation and development in primary murine bone marrow culture... Somewhat milder, yet appreciable, stimulation was observed with the other synthetic peptides derived from casein.
Peptides derived from natural casein stimulate Megakaryocytopoiesis in cultured human bone marrow cells: When 100 ug per mi peptides derived from natural casein were added under similar conditions to human bone marrow ccll cultures from healthy donors. CFU-GM colony formation was increased with or without additional stimulating factors (GM-CSF. CM).
Peptides derived from natural casein also stimulated erythroid cell forming colonies in the presence of erythropoietin. Treatment of the human bone marrow cells with thrombopoietin (TPO) stimulates megakaryocyte (MK) colony formation. Addition of 300 pg per ml peptides derived from natural casein to TPO-treated cells stimulates a more than twofold increase (16 colonies per 2 x 107 cells without peptides, 35 colonies per 2 X 105 with peptides derived from natural casein) in MK colony proliferation.
In the presence of additional hematopoietic factors, such as erythropoietin, human IL-3, hSCF and AB serum, 14 days incubation with : peptides derived from natural casein stimulated a nearly three fold increase in
CFU-GEMM colonies from human bone marrow cells (158 colonies with 500 pu g per ml peptides derived from natural casein, 68 colonies with the factors alone), but had a smaller (one and one half fold) effect on cultured cord blood
CFU-GEMM formation. The relative cell number counts in the cultured human v bone marrow and cord blood colonies reflect megakaryocyte cell proliferation in response to addition of 25 pg per ml peptides derived from natural casein s (see Table shown in Figure 6). Thus, incubation of cultured human primary bone marrow and cord blood cells with peptides derived from natural casein stimulates the development and proliferation of both committed megakaryocyte and erythroid cell colonies. Significantly, the synergy observed between TPO and peptides derived from natural casein in stimulating megakaryocytopoiesis io indicates a probable role for this potent hematopoietic growth factor in the mechanism of peptides derived from casein’s stimulatory properties, and further suggests the likelihood of similar augmentation of a wide range of
TPO-mediated effects by peptides derived from natural casein.
Peptides derived from natural casein and synthetic peptides derived is from natural casein potentiate the effect of Erythropoietin (EPO) in cultured human bone marrow cells: The effect of natural and synthetic peptides derived from casein on erythroid cell proliferation in cultured human bone marrow cells was assessed under the same conditions outlined hereinabove for megakaryocytopoiesis. When added in the presence of EPO, 50 -300 pg/ml peptides derived from natural casein, or 100 pg/ml Synthetic peptides derived from casein (F, Table 3, SEQ ID NO:18) stimulated a one and one-half (synthetic peptide) to four-fold proliferation of erythroid cell precursors (appearance of BFU-E colonies) compared to the bone marrow cells treated with EPO alone. Thus, peptides derived from natural casein and synthetic 2s derivatives thereof act to potentiate the erythropoietic-stimulating effects of . EPQ, and as such can be used to augment of a wide range of clinically important EPO-mediated effects.
Synthetic peptides derived from casein stimulate Dendritic cells proliferation in murine CFU-GEMM: The effect of Synthetic peptides derived from casein on dendritic cell proliferation in murine primary bone marrow cells was assessed under the same conditions outlined for the stimulation of megakaryocytes. Synthetic peptides derived from cascin representing the first: 2.3, 5,6, 7,9, 11, 12, 16, 23, 24 and 26 amino acids of a
S1 casein stimulated the proliferation of dendritic cells, from 2.2 % and up to s 23 % of total cells compared with 0.1 — 0.2 % dendritic cells in the cell samples incubated without Synthetic peptides derived from casein (Figure 7).
Synthetic peptides derived from casein stimulate Plasma cell proliferation in murine CFU-GEMM: The effect of Synthetic peptides derived from casein on plasma cell proliferation in murine primary bone marrow cells was demonstrated under the same conditions outlined for the stimulation of megakaryocytes. Synthetic peptides derived from casein representing the first: 2,3, 5,7, 11, 16, 17, 18, 19,20, 21, 22, 23 and 24 and 26 amino acids of aS1 casein, significantly stimulated the proliferation of plasma cells, from 1.5 % and up 12.3 % of total cell count, compared with 03 9 of total without Synthetic peptides derived from casein (Figure 7).
Synthetic peptides derived from casein stimulate Macrophage proliferation in CFU-GEMM: The cffect of Synthetic peptides derived from casein on macrophage proliferation in murine primary bonc marrow cells was demonstrated under the same conditions outlined for the stimulation of megakaryocytes. Incubation of cells with synthetic peptides derived from casein representing the first: 7, 9, 16, and 23 amino acids of aSlcasein significantly stimulated the proliferation of macrophages, from approximately 17 % of total cell count in controls, to nearly 30 % of total in cells incubated with Synthetic peptides derived from casein (Figure 7).
Synthetic peptides derived from casein stimulate Red Blood Cells proliferation in CFU-GEMM: The effect of Synthetic peptides derived from . casein on red blood cell proliferation in murine primary bone marrow cells was demonstrated under the same conditions outlined for the stimulation of megakaryocytes. Incubation of cells with Synthetic peptides derived from casein representing the first 4 amino acids from the N terminus of aSlcasein significantly stimulated the proliferation of red blood cells, from 53 % of total
N cell count in controls, 10 71 % of total in cells incubated with the synthetic peptide derived from casein (Figure 7). ’ 5 Synthetic peptides derived from casein stimulate Polymorphonuclear (PMN) cell proliferation in CFU-GEMM: The effect of Synthetic peptides : derived from casein on the proliferation of polymorphonuclear (PMN) cells in murine primary bone marrow cells was demonstrated under the same conditions outlined for the stimulation of megakaryocytes. Incubation of cells with
Synthetic peptides derived from casein representing the first: 3, 6, 7, 9, 16 and more, up to and including 26 amino acids of aS1 casein significantly stimulated the proliferation of PMNs, from 1.6 % cf total cell count in unincubated controls, to between 2.9 % and 14.9 % of total in cells incubated with Synthetic peptides derived from casein (Figure 7).
Peptides derived from natural casein stimulate hematopoiesis in vivo following irradiation and bone marrow transplant: Mpyeloablative therapy may lead to life-threatening reduction in thrombocytes and leukocytes, which may persist despite administration of blood cells and growth factors. The following demonstrates the effect of peptides derived from natural casein following irradiation and bone marrow transplantation.
Peptides derived from natural casein enhance leukocyte and platelet reconstitution following syngeneic bone marrow transplantation in mice:
When sub-lethally irradiated (600 cGy), minimally bone marrow-reconstituted,
BALB/c mice (n = 12) received 1 mg per mouse peptides derived from natural casein via intravenous injection one day after bone marrow cell reconstitution, . significant increases in peripheral white blood cell counts on days 4, 6 and 15 post-treatment were noted, compared to controls receiving human serum albumin (Figure 8). Platelet counts in the peripheral blood of both the treated and control irradiated. bone marrow transplanted mice were cqually depressed up to 8 days post treatment. However, by the thirteenth day a clear advantage i was noted for the mice treated with the peptides derived from natural cascin. demonstrating a significant increase over the human serum albumin-treated controls which became even more pronounced by day 15 (Figure 9). Thus, peptides derived from natural cascin enhance platelet and leukocvie s reconstitution following transplantation with limiting numbers of bone marrow . cells. It is expected that this effect will be further increased in reconstitution with optimal, rather than limiting numbers of bone marrow cells.
Synthetic peptides derived from casein enhance leukocyte reconstitution following syngeneic bone marrow transplantation in mice:
When sub-lethally irradiated (600 cGy), minimally bone marrow-reconstituted,
BALB/c mice (n = 5 per synthetic peptide, n = 10 in the control group) received
I mg per mouse synthetic peptides (13-26 amino acids in length, sec Table 3) derived from casein via an intraperitoneal injection one day after bone marrow transplantation, a clear enhancement of leukocyte reconstitution was observed.
Significant increases in peripheral white blood cell counts over a 10 to 14 day period were noted with peptides having 15 (day 10: 1.72 x 10° cells per ml: day 12: 6.54 x 10° cells per ml) and 17 (day 10: 2.74 cells x 10° per ml: day 12: 5.20 x 10° cells per ml) amino acids (sec Table 3), compared to controls receiving human serum albumin (day 10: 1.67 x 10° cells per ml; day 12: 4.64 x 10° cells per ml). Thus, synthetic peptides derived from casein enhance leukocyte reconstitution following transplantation with limiting numbers of bone marrow cells.
Synthetic peptides derived from casein enhance platelet reconstitution following syngeneic bone marrow transplantation in mice: In order to 2s confirm the observed ability of synthetic peptides derived from casein to enhance megakaryocyte proliferation in hematopoietic stem cell cultures (see .
Figures 6 and 7), the peptides’ effects on platelet reconstitution in vivo was investigated. When lethally irradiated (800 cGy), minimally bone marrow-reconstituted, mice (n = 5 per group) received 100 pg per mouse 50 synthetic peptides 4P and 3a (6 and 12 amino acids in length, respectively - see ’
Table 3) in 4 daily intraperitoneal injections (4-7 days post-transplantation), a clear enhancement of platelet reconstitution over untreated controls was . observed. Significant increases in platelet counts at 10 and 12 days post transplantation were noted for both peptides. Treatment with peptide 4P increased counts by 29 % (872 X 10°/m1 compared with 676 X 10%ml in the control group) at 12 days post transplantation while treatment with peptide 3a increased counts by up to 35.5 % (229 X 10°/ml compared with 169 X 10*/ml in the control group) at 10 days, and up to 13.5 % (622 X 10%/ml compared with 461 X 10° /ml in the control group) at 12 days post transplantation. Thus, the same synthetic peptides derived from casein enhance megakaryocyte proliferation in vitro and platelet reconstitution following bone marrow transplantation in vive.
Peptides derived from natural casein inhibit in vitro infection of lymphocytic T cell lines by HIV-1 virus
Penetration of peptides derived from natural casein into lymphocytic
Tcells: In order to investigate the mechanisms of immune stimulatory and anti-viral effects of peptides derived from natural casein, susceptible Sup-T1 and CEM cultured human T-cells were treated with peptides derived from natural casein prior to in vitro infection with HIV-1 virus. Fluorescent " microscopy revealed that FITC-conjugated peptides derived from natural casein (100 pg per ml) penetrated the Sup-T1 cells when incubated therewith as described above (Figures 10a-f). A small amount of label was observed in the cytoplasm of the cells after 15 minutes (Figures 10a-b). At 30 minutes (Figures 10c-d) more label was observed in the cytoplasm, with limited nuclear uptake.
From 1-hour incubation and on (Figures 10e-f), FITC-labeled peptides derived 3 from natural casein were observed in the cytoplasm, but mostly they were : concentrated in the cell nucleus. Analysis of the Sup-T1 cells by flow cytometry confirmed increasing uptake of the labeled peptides derived from natural casein from 5 minutes post incubation.
Peptides derived from natural casein enhance human lymphocyte proliferation: The presence of peptides derived from natural casein in the culture medium resulted in increased Sup-T1 cell counts over a period of 14 days. The greatest increases in cell number at 7 days was observed for 30 pe ) per ml peptides derived from natural casein (42 %), for 1000 pg at 10 days (30 : %) and for 600 pg (32 %) at 14 days incubation (data not shown).
Measurement of ["H]-thymidine incorporation by the cultured cells, providing a proliferation index, reflected the increase in cell number, with the most significant effect noted for 600 ug per ml peptides derived from natural casein on day 10 and 50 pg per ml on day 14 (Figure 11). The reduced proliferation indices at 14 days probably reflect cell overgrowth and nutrient depletion.
Synthetic peptides derived from casein enhance human lymphocyte proliferation: The presence of synthetic peptides derived from casein (all peptides listed in Table 3) in the culture medium resulted in increased Sup-T! cell counts over a period of 10 days. The increase was similar for all synthetic peptides. The greatest increases in lymphocyte cell number in infected cells were observed for 250 pg and 500 ug per ml of peptide representing the first 9 amino acids (80 % and 33 %, respectively) (data not shown).
Peptides derived from natural casein inhibit HIV-1 infection in human lymphocyte cells: Susceptible CEM lymphocyte cells pretreated with peptides derived from natural casein (50-1000 pg per ml) 24 or 48 hours prior to incubation with HIV-1, or exposed to HIV-1 pretreated 3 hours with peptides from natural casein, exhibited enhanced cell proliferation and reduced levels of viral infection compared to untreated controls. Cell counts and HIV-1 p- antigen assay at 15 days post infection revealed 100 % inhibition of viral infection after 3 hours incubation of viruses with 600-1000 ug per ml peptides ) derived from natural casein and 98 % and 99 % inhibition after 24 hours p incubation of cells with 50 and 600 pg per ml peptides, respectively (comparing cell numbers with uninfected controls UIF). Longer incubation times were not found to be more effective (Figure 12). Although increasing concentrations of peptides derived from natural casein enhanced cell proliferation at 3 and 24 hours post infection, viral infection is most significantly inhibited in these ! fastest growing cultures. An even more dramatic enhancement of cell v s proliferation and inhibition of HIV-1 infection was observed in Sup-T1 cells pretreated with peptides derived from natural casein before HIV-1 infection (average inhibition of viral infection of 96.7 %, 88.7 % and 95.7 % for 3 hours pretreatment of virus, and 24 hours and 48 hours pretreatment of cells, respectively) (not shown). Thus, peptides derived from natural casein penetrate human cultured lymphocyte cells and their nuclei, enhance cell growth, and significantly reduce the susceptibility of CD4 cells to HIV-1 infection. As such, peptides derived from natural casein are expected to be useful both at preventing HIV infection and for post infection treatment of HIV infected and
AIDS patients.
Synthetic peptides derived from casein inhibit HIV-1 infection in human lymphocyte cells: The ability of Synthetic peptides derived from casein to inhibit HIV-1 infection in human lymphocyte cells was demonstrated using
CEM-lymphocyte cells under the same conditions outlined above. Three hours pretreatment of CEM lymphocytes with the synthetic peptide derived from casein representing the first 3 amino acids of aSlcasein conferred a significant degree of resistance to infection following incubation with HIV-1. Lymphocyte cell numbers were 1.29 x 10° (100ug per mi) and 2.01 x 10° (500ug per ml) in the treated cells as compared to the infected HIV-1 control of 1.06 x 10° (Figure 13). HIV-1 infection levels in the same cells, measured by the HIV-P* antigen assay at 7 days post infection, was significantly reduced in the peptide treated cells (0.17 and 0.14ng P** Antigen/ml with100pg/ml and 500ug/ml respectively), as compared to the untreated controls (0.52 ng P* Ag/ ml).
Likewise, significant inhibition of HIV-1 infection was observed in the
CEM cells exposed to viruses pre-treated (3 hours) with the synthetic casein derived peptide representing the first 5 amino acids of aSlcasein.
Cell counts in the cultures incubated with 10 and 25ug peptide 3P per ml s were 1.17 x 10° and 1.26 x 10° respectively, as compared to the infected HIV-1 ; control of 1.06 x 10°,
HIV- P* antigen assay at 7 days post infection, revealed significant reduction in HIV-1 infection levels in treated cultures (0.26 and 0.18ng P' Ag per ml for 10 and 25ug per ml respectively, as compared to the control of 0.52 ng P* Ag per mi).
Likewise, 3 hours preincubation of the virus with the synthetic peptide derived from casein 4P, representing the first 6 amino acids of aSlcasein had a significant effect on the susceptibility of CEM lymphocyte cells to infection with HIV-1.
Is Cell numbers were most aftected at concentrations of 25 and 230ng per mi (1.26 x 10°, and 1.59 x 10° respectively, as compared to the infected control value of 1.06 Xx 10%).
Assay of HIV-P?* antigen at 7 days post infection, revealed a dose dependent reduction in viral particles as compared to the untreated, infected control cultures (Figure 13). Thus, the protection from HIV-1 infection afforded
Ivmphocyte cells by the peptides derived from natural casein is rctained in
Synthetic peptides derived from casein representing as few as the first five
N-terminal amino acids of aS-1 casein.
Peptides derived from natural casein prevent development of glucosuria in Non-Obese Diabetic (NOD) mice: Non-Obese Diabetic (NOD) mice spontaneously develop Juvenile (Type I, IDDM) Diabetes, an autoimmune condition causing inflammation of the pancreatic f§ cells and ending in disease and death. Female NOD mice are extremely susceptible, demonstrating evidence of macrophage invasion of the pancreatic islet interstitial matrix as early as 5 weeks old. A once or twice weekly injection of 100 pg peptides derived from natural casein for 5 weeks (5 or 10 injections total) were \ completely effective in preventing the glucosuria associated with the onset and course of the disease. By 200 days 100 % of the untreated control mice (n = 35) ‘ 5s had become diabetic, and subsequently died, while the treated mice (n = 3) remained 100 % euglycemic, all still surviving at 365 days (Figure 14). Thus, peptides derived from natural casein effectively protected genetically susceptible mice against the onset of this autoimmune inflammatory condition.
Synthetic peptides derived from casein prevent development of glucosuria in Non-Obese Diabetic (NOD) mice:
The preventative effect of Synthetic peptides derived from casein on the development of glucosuria in NOD mice was demonstrated under the same conditions outlined above, except that the mice were injected only once weekly for five (5) weeks with 100 pg of Synthetic peptides derived from casein. The results of these experiments are presented in Table 4 below:
Table 4
The effect of synthetic peptides on IDDM in NOD mice
IPGT TEST
Peptide
Derivative 0 min] code Healthy/Total" reload) bo min. post load
CA CR Ec RC
Cp fee pm
I EN LC ON LN
IE NO Lc A A
CN CR a J
I NS LL A AS
EN EO LL SN A hegatve i pd
I EE ewatve pot RM ee me
EE A LL LI A
2 LL I LA
I I Co CLI A
A —
I a — —
Ts fee [mw eee eee je jee
I I Lc a Gc fen pm foes
I A i A
EE EO 0 cH A po
Blood was drawn from the paraorbital plexus at 0 min and 60 min after the intraperitoneal injection of glucose 1 g/kg body weight. Plasma glucose levels were determined with a
Glucose Analyzer 2 (Beckman Instruments, Fullerton, CA) and expressed as mmol/L. * Healthy and well = Sugar not detected in urine.
Glucosuria = > 1000 mg/dL.
IPGTT performed with 6 healthy female control mice: 0 min- 110 mmol/L; 60 min- 106 mmol/L blood glucose.
The synthetic peptides derived from casein representing the first 9 (X), 11 (2a) and 12 (3a) amino acids and higher chain length of aS casein, were highly effective in preventing the glucosuria associated with the onset and . course of the disease.
Effect of treatment with synthetic peptides derived from casein was ) 1s evaluated after 25 weeks. At that time, all 5 mice in the untreated control group
(n = 5) had become diabetic, as indicated by the presence of frank (>1000 mg/dl) glucosuria (Table 4).
No glucosuria was detected in three of the five (3/5) NOD mice treated » with the synthetic peptide representing the first nine (9) amino acids from the N ¢ 5 terminal of aS] casein. Of the group injected with the synthetic peptide of eleven (11) amino acids from the N terminal of aS] casein, no glucosuria was detected in four out of five (4/5) of the NOD mice
In the groups of peptide treated mice in which glucosuria was detected, the onset was generally significantly delayed (by 3- 5 weeks) relative to the onset of glucosuria in untreated controls (data not shown), indicating a clearly protective effect of the peptides even when incomplete.
The protective effects of shorter Synthetic peptides derived from casein have also been studied in NOD mice. In an additional series of experiments similar to the abovementioned, administration of peptides representing the first 3 (IP) and 4 (2P) N-terminal amino acids of aS1 casein effectively prevented the onset of glucosuria in the treated mice (assayed at week 16), while the untreated controls had all become diabetic (100 % glucosuria) (data not shown).
The glucose tolerance (IPGT) test performed after 25 weeks with the healthy and well NOD mice, of the group injected with the synthetic casein derived peptide of the first 9 amino acids, showed no evidence of abnormal glucose metabolism (normal glycemic values pre- and 60 minutes post- glucose loading).
In the group treated with the synthetic peptide derived from casein representing the first 11 amino acids of the N-terminal of aS1 casein (2a), resting plasma glucose levels were somewhat elevated in two of the five mice - (215 and 159 mmol/L), and remained mildly elevated at (183 and 204 mmol/L) 60 minutes post load, indicating mild diabetic tendencies. The other two mice remained within normal glycemic range throughout the test (Table 4). In general, the normal results of the IPGTT reflected the absence of glucosuria in the healthy. surviving peptide-treated mice (Table 4). Thus. synthetic peptides representing only a few amino acids from the N-terminal of aS1 casein. as well as peptides derived from native casein dramatically reduce the susceptibility of genetically predisposed NOD mice to onsct of autoimmune diabetic disease.
Synthetic casein- derived peptides significantly reduce Total Cholestrol blood levels (TC), Low Density Lipoprotein (LDL) and High Density
Lipoprotein (HDL): Intraperitoneal administration of Synthetic peptides derived from casein caused a significant reduction in the blood lipid (HDL.
LDL and TC) values in experimentally hypercholesterolemic mice. After one week of the atherogenic Thomas Hartroft diet, the blood cholesterol levels of the mice had risen to the levels of 318mg/dl.
One week post treatment with 1 mg synthetic peptides derived from casein per mouse, the group treated with the Synthetic peptides derived from casein representing the first 5 (3P) and 11 (2a) amino acids of aS1 casein. had ts significantly reduced TC, HDL and LDL values, compared to those of the control group [TC: 308 and 279mg/dl respectively: HDL: 42.5 mg/dl and 41mg/dl respectively and LDL: 247mg/dl and 221mg/dl respectively as compared to 393mg/dl (TC), 54.5 mg/dl (HDL) and 326 mg/dl (LDL) in the diet-induced hypercholesterol-/hyperlipidemic control group] (Figure 15).
Thus. synthetic peptides representing the first few N-terminal amino acids of aS] casein effectively reduced experimentally induced hyperlipidemia and hypercholesterolemia within one weck after a single, intraperitoneal administration.
Clinical trials with peptides derived from natural casein: 23 Patients received intramuscular injections of 50 mg peptides derived from natural casein each, divided into three depots, as indicated.
Peptides derived from natural casein stimulates hematopoiesis in cancer patients: The hematology profiles of six cancer patients who had received or were receiving chemotherapy were examined before and following administration of peptides derived from natural casein, as indicated. Special attention was paid to changes in the Platelet (PLT), Leukocyte (WBC),
Erythrocyte (RBC) and Hemoglobin (HGB) values, representing thrombocytopoiesis, leukocytopoiesis, and erythrocytopoiesis, respectively. $ G.T., (Female patient, Patient 1): Patient had ovarian cancer, . s undergone a hysterectomy followed by chemotherapy. She received two intramuscular injections of peptides derived from natural casein at two and then two and one half months post operation. No chemotherapy was administered between the first and second administrations of peptides derived from natural casein. Blood tests from 6 days post first injection, 7, and 13 days post second injection reflect a considerable increase in platelet and WBC components, as well as increased RBC (Figure 16).
E.C., (Female patient, Patient 2): Patient underwent a radica! mastectomy for lobular carcinoma in 1983, and six years later suffered from gastric metastases. Three days prior to commencement of chemotherapy, she received one intramuscular injection (in three depots) of peptides derived from natural casein by injection, and a second 10 days after the chemotherapy.
Although the blood counts from 10 and 16 days post chemotherapy indicated an attenuation of the depressed hematological profile usually encountered following chemotherapy, the most significant effects of peptides derived from natural casein were noted 3 days after the first injection, prior to the chemotherapy (Figure 16).
E.S., (Female patient, Patient 3): Patient was suffering from widespread metastatic dissemination of a breast carcinoma first discovered in 1987. Two years later, she received a first intramuscular injection of peptides 2s derived from natural casein, and a second 23 days later. No additional therapy - was administered during this period. Blood tests indicate a strong enhancement of PLT seven days after the first treatment and a significant increase in RBC ) and WBC seven days after the second treatment (Figure 16).
J.R., (Female patient, Patient 4): Patient’s diagnosis Is breast cancer with bone metastases. She received one intramuscular injection of peptides derived from natural casein 8 days before commencing chemotherapy. and another, 14 days later. The most significant effect 1s clearly seen in the rapid return of WBC levels following chemotherapy-induced depression (Figure 16).
D.M., (Female patient, Patient 5): Patient suffering from hepatic : s cancer with widespread metastatic dissemination. She received three . intramuscular injections of peptides derived from natural casein at 10. 8 and 6 days before receiving chemotherapy. A second series of injections was initiated 10, 12 and 14 days following the chemotherapy treatment. Although a significant effect on the hematological profile is noted following the first series io of injections and prior to the chemotherapy, the most dramatic improvements are seen in the rapid return of depressed post-chemotherapy values to normalized cell counts following the second series of peptides derived from natural casein injections (Figure 16).
Thus, administration of peptides derived from natural casein to cancer is patients results in improved hematological profiles, specifically enhanced ervthropoiesis. leukocytopoiesis and thrombocytopoiesis. and is capable of moderating and shortening the duration of chemotherapy-induced depression of blood components.
Peptides derived from natural casein stimulates thrombocytopoiesis in transplant recipients with resistant thrombocytopenia: Prolonged transfusion-resistant thrombocytopenia with episodes of severe bleeding. may be a life threatening complication of bone marrow transplantation, especially where traditional therapies are ineffective. Two patients with severe resistant thrombocytopenia were treated with peptides derived from natural casein.
M-1 (Female patient): 32 year old patient suffering from Acute
Myeloid Leukemia in complete remission, following autologous stem cell . transplantation. She had experienced two life-threatening bleeding episodes, involving pulmonary hemorrhage and a large obstructive hematoma in the soft palate. At more than 114 days post transplantation, platelet counts were 50 refractive to rhIL-3, rhiL-6, intravenous gamma globulin, and recombinant erythropoietin. Following two intra muscular treatments of 50 mg peptides derived from natural casein (each treatment divided into three depots), her condition improved immediately. Along with the rapid return of normal : platelet counts (Figure 17), her distal limb bleeding with exertion and patechyae
J s subsided, she was able to resume walking, and returned to her home overseas with no complications or side effects.
M-2 (Male patient): 30 year old patient suffering from Acute Myeloid
Leukemia in a second complete remission following autologous stem cell transplantation, exhibiting totally resistant platelet counts and massive gastrointestinal bleeding episodes. He required daily transfusions of packed cells, had developed hypoalbuminia, and failed to respond to extensive therapy with rhlL-3, rhiL-6 and gamma globulin. Following two intramuscular treatments, each of 50 mg peptides derived from natural casein in three depots 86 days post transplantation, rapid platelet reconstitution (Figure 18) and gradual discontinuation of the bleeding was observed. No further treatment was required, and the patient is presently completely asymptomatic with normal platelet count.
Thus, one course of two intramuscular injections of peptides derived from natural casein at 0.7- 1.0 mg per kg body weight, each divided into three depots, was effective in rapidly reconstituting platelet counts and diminishing associated clinical symptoms in patients suffering from prolonged, transfusion resistant thrombocytopenia with life-threatening bleeding episodes.
Peptides derived from natural casein decreases triglycerides and T otal
Cholesterol in familial hyperlipidemia:
M.S. (Female patient): Patient is a 38 year old female with family . history of hyperlipidemia. Before treatment with peptides derived from natural casein, blood chemistry profile revealed elevated total cholesterol (321 mg per dl), triglycerides (213 mg per di; normal range 45 — 185 mg per dl) and elevated
LDL-cholesterol (236.4 mg per dl; normal range 75 — 174 mg per dl). One 350 month after a single administration of 50 mg peptides derived from natural
WO (13/018606 PCT/ILO2/00720 casein (in three intra muscular depots) the hyperlipidemia was stabilized: total cholesterol was reduced to 270 mg per dl. triglycerides were 165 mg per dl and
LDL-cholesterol was 201 mg per dl, still higher than normal range but significantly reduced from the pretreatment value. No additional treatment was s administered. Thus, treatment with peptides derived from natural casein 1s effective in rapidly bringing about a significant reduction in otherwise untreated hyperlipidemia in humans.
Peptides derived from natural casein stimulate normoglobinemia in a case of occult bleeding:
D. G. (Male patient): Patient is a 75 year old male suffering from anemia and hypoglobinemia (depressed RBC, HGB, HCT, MCH and MCHC) associated with extensive occult bleeding. One month after receiving one intramuscular injection of 50 mg peptides derived from natural casein (in three depots). a significant reduction of the anemia was observed. After two months. is RBC approached normal values (4.32 instead of 3.44 M per pl), HGB increased (11.3 instead of 8.9 g per dl) and HCT, MCH and MCHC all improved to nearly normal values, despite the persistence of occult bleeding. Thus. one injection of peptides derived from natural casein seemed capable of stimulating erythropoiesis and reducing anemia associated with blood Joss in humans.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single 25s embodiment, may also be provided separately or in any suitable subcombination.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and 50 variations will be apparent to those skilled in the art. Accordingly. 1t 1s
Q
: 1s intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent applications and sequences identified by an accession number, ’ mentioned in this specification are herein incorporated in their entirety by v s refercnce into the specification, to the same extent as if each individual publication, patent, patent application or sequence was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any refercnce in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims (1)

  1. WHAT IS CLAIMED IS:
    1. Use of a peptide derived from an N terminus portion of aS1 casein in the manufacture of a medicament for preventing or treating an autoimmune disease.
    2. The use of claim 1, wherein said peptide is a fragment derived by fragmentation of aSI casein.
    3. The use of claim 1, wherein said peptide is a synthetic peptide.
    4. The use of claim I, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    5. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for preventing or treating a viral infection.
    6. The use of claim 5, wherein said peptide is a fragment derived by fragmentation of aS! casein.
    7. The use of claim 5, wherein said peptide is a synthetic peptide.
    8. The use of claim 5, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25. AMENDED SHEET
    9. Use of a peptide derived from an N terminus portion of aS1 casein in the manufacture of a medicament for preventing viral infection.
    10. The use of claim 9, wherein said peptide is a fragment derived by fragmentation of aSl casein.
    11. The use of claim 9, wher:zin said peptide is a synthetic peptide.
    12. The use of claim 9, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    13. Use of a peptide derived from an N terminus portion of aS] casein in the manufacture of a medicament for inducing hematopoiesis.
    14. The use of claim 13, wherein said peptide is a fragment derived by fragmentation of aS] casein.
    15. The use of claim 13, wherein said peptide is a synthetic peptide.
    16. The use of claim 13, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    17. Use of a peptide derived from an N terminus portion of aS1 casein in the manufacture of a medicament for inducing hematopoietic stem cells proliferation. AMLNDED SHeEeT
    18. The use of claim 17, wherein said peptide is a fragment derived by fragmentation of aS] casein.
    19. The use of claim 17, wherein said peptide is a synthetic peptide.
    20. The use of claim 17, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    21. Use of a peptide derived from an N terminus portion of aS1 casein in the manufacture of a medicament for inducing hematopoietic stem cells proliferation and differentiation.
    22. The use of claim 21, wherein said peptide is a fragment derived by fragmentation of aSl casein.
    23. The use of claim 21, wherein said peptide is a synthetic peptide.
    24. The use of claim 21, wherein said peptide has a sequence as set forth in one of SEQ ID NOs:I-25.
    25. Use of a peptide derived from an N terminus portion of aS! casein in the manufacture of a medicament for inducing megakaryocytopoiesis. AmenoeD SHEET
    26. The use of claim 25, wherein said peptide is a fragment derived by fragmentation of aSI casein.
    27. The use of claim 25, wherein said peptide is a synthetic peptide.
    28. The use of claim 25, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    29. Use of a peptide derived from an N terminus portion of aS1 casein in the manufacture of a medicament for inducing erythropoiesis.
    30. The use of claim 29, wherein said peptide is a fragment derived by fragmentation of aSI casein.
    31. The use of claim 29, wherein said peptide is a synthetic peptide.
    32. The use of claim 29, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    33. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for inducing leukocytopoiesis.
    34. The use of claim 33, wherein said peptide is a fragment derived by fragmentation of aS] casein. FinchoD SHEET
    35. The use of claim 33, wherein said peptide is a synthetic peptide.
    36. The use of claim 33, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    37. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for inducing thrombocytopoiesis.
    38. The use of claim 37, wherein said peptide is a fragment derived by fragmentation of aS! casein.
    39. The use of claim 37, wherein said peptide is a synthetic peptide.
    40. The use of claim 37, wherein said peptide has a sequence as set forth in one of SEQ ID NOs:1-25.
    41. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for inducing plasma cell proliferation.
    42. The use of claim 41, wherein said peptide is a fragment derived by fragmentation of aSl casein.
    43. The use of claim 41, wherein said peptide is a synthetic peptide. PevieNLL Sai]
    44. The use of claim 41, wherein said peptide has a sequence as set forth in one of SEQ 1D NOs: 1-25.
    45. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for inducing dendritic cell proliferation.
    46. The use of claim 45, wherein said peptide is a fragment derived by fragmentation of aSl casein.
    47. The use of claim 45, wherein said peptide is a synthetic peptide.
    48. The use of claim 45, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    49. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for inducing macrophage proliferation.
    50. The use of claim 49, wherein said peptide is a fragment derived by fragmentation of aSl casein.
    51. The use of claim 49, wherein said peptide is a synthetic peptide.
    52. The use of claim 49, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25. AMENDED SHEET
    53. Use of a peptide derived from an N terminus portion of aSI casein in the manufacture of a medicament for preventing or treating thrombocytopenia.
    54. The use of claim 53, wherein said peptide is a fragment derived by fragmentation of aSl casein.
    55. The use of claim 53, wherein said peptide is a synthetic peptide.
    56. The use of claim 53, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    57. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for preventing or treating pancytopenia.
    58. The use of claim 57, wherein said peptide is a fragment derived by fragmentation of aSI casein.
    59. The use of claim 57, wherein said peptide is a synthetic peptide.
    60. The use of claim 57, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    61. Use of a peptide derived from an N terminus portion of aS! casein in the manufacture of a medicament for preventing or treating granulocytopenia.
    62. The use of claim 61, wherein said peptide is a fragment derived by fragmentation of aS! casein.
    63. The use of claim 61, wherein said peptide is a synthetic peptide. AMENDED SHEET
    64. The use of claim 61, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    65. Use of a peptide derived from an N terminus portion of aS casein in the manufacture of a medicament for preventing or treating hyperlipidemia.
    66. The use of claim 65, wherein said peptide is a fragment derived by fragmentation of aSlI casein.
    67. The use of claim 65, wherein said peptide is a synthetic peptide.
    68. The use of claim 65, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    69. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for preventing or treating cholesteremia.
    70. The use of claim 69, wherein said peptide is a fragment derived by fragmentation of aS] casein. AMENDED srikEL
    71. The use of claim 69, wherein said peptide is a synthetic peptide.
    72. The use of claim 69, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    73. Use of a peptide derived from an N terminus portion of aSlI casein in the manufacture of a medicament for preventing or treating glucosuria.
    74. The use of claim 73, wherein said peptide is a fragment derived by fragmentation of oS! casein.
    75. The use of claim 73, wherein said peptide is a synthetic peptide.
    76. The use of claim 73, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    77. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for preventing or treating diabetes.
    78. The use of claim 77, wherein said peptide is a fragment derived by fragmentation of aSl casein.
    79. The use of claim 77, wherein said peptide is a synthetic peptide. AMENDED SHEET
    80. The use of claim 77, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    81. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for preventing or treating AIDS.
    82. The use of claim 81, wherein said peptide is a fragment derived by fragmentation of aSl casein.
    83. The use of claim 8! wherein said peptide is a synthetic peptide.
    84. The use of claim 81, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    85. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for preventing or treating infection by HIV.
    86. The use of claim 85, wherein said peptide is a fragment derived by fragmentation of aSl casein.
    87. The use of claim 85, wherein said peptide is a synthetic peptide.
    88. The use of claim 85, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25. AMENDED SHEET
    89. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for preventing or treating conditions associated with myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT).
    90. The use of claim 89, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    91. The use of claim 89, wherein said peptide is a synthetic peptide.
    92. The use of claim 89, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    93. Use of a peptide derived from an N terminus portion of aS] casein in the manufacture of a medicament for treating an erythropoietin treatable condition.
    94. The use of claim 93, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    95. The use of claim 93, wherein said peptide is a synthetic peptide.
    96. The use of claim 93, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25. AMENDED 8hicel
    97. Use of a peptide derived from an N terminus portion of aSI casein in the manufacture of a medicament for augmenting the effect of erythropoietin.
    98. The use of claim 97, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    99. The use of claim 97, wherein said peptide is a synthetic peptide.
    100. The use of claim 97, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    101. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for treating a thrombopoietin treatable condition.
    102. The use of claim 101, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    103. The use of claim 101, wherein said peptide is a synthetic peptide.
    104. The use of claim 101, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    105. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for augmenting the effect of thrombopoietin.
    106. The use of claim 105, wherein said peptide is a fragment derived by fragmentation of aS1 casein. AMENDED Stil
    107. The use of claim 105, wherein said peptide is a synthetic peptide.
    108. The use of claim 105, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    109. Use of a peptide derived from an N terminus portion of aSl casein in the manufacture of a medicament for enhancing peripheral stem cell mobilization.
    110. The use of claim 109, wherein said peptide is a fragment derived by fragmentation cf aS! caseir.
    111. The use of claim 109, wherein said peptide is a synthetic peptide.
    112. The use of claim 109, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    113. A pharmaceutical composition for preventing or treating an autoimmune disease, the pharmaceutical composition comprising, as an active AMENDED Bilin ingredient, a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
    114. The pharmaceutical composition of claim 113, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    115. The pharmaceutical composition of claim 113, wherein said peptide is a synthetic peptide.
    11h. The pharmazeutical corposition of claim 115, wwherzin z2id peptide has a sequence as set forth in one of SEQ 1D NOs:1-25.
    117. A pharmaceutical composition for preventing or treating a viral disease, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
    118. The pharmaceutical composition of claim 117, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    119. The pharmaceutical composition of claim 117, wherein said peptide is a synthetic peptide.
    120. The pharmaceutical composition of claim 117, wherein said peptide has a sequence as set forth in onc of SEQ ID NOs: 1-25.
    121. A pharmaceutical composition for preventing viral infection, the pharmaceutical composition comprising, as an active Ingredient, a2 peptide ALZNOED SHEET derived from an N terminus portion of aS! casein and a pharmaceutically acceptable cammier.
    122. The pharmaceutical composition of claim 121, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    123. The pharmaceutical composition of claim 121, wherein said peptide is a synthetic peptide. 174, The phermacentical composition of cluim 121, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    125. A pharmaceutical composition for inducing hematopoiesis, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    126. The pharmaceutical composition of claim 117, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    127. The pharmaceutical composition of claim 117, wherein said peptide is a synthetic peptide.
    128. The pharmaceutical composition of claim 117. wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    129. A pharmaceutical composition for inducing hematopoictic stem cells proliferation, the pharmaceutical composition comprising, as an active AMENDED Shick ingredient, a peptide derived from an N terminus portion of aS1 casein and 2 pharmaceutically acceptable carrier.
    130. The pharmaceutical composition of claim 12}, wherein said peptide is a fragment derived by fragmentation of aS! casein.
    131. The pharmaceutical composition of claim 121. wherein said peptide is a synthetic peptide.
    132. The pharmaceutical composition of claim 121, whercin said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    133. A pharmaceutical composition for inducing hematopoietic stem cells proliferation and differentiation, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaccutically acceptable carrier.
    134. The pharmaceutical composition of claim 133, wherein said peptide is a fragment derived by fragmentation of aS] cascin.
    135. The pharmaceutical composition of claim 133, wherein said peptide is a synthetic peptide.
    136. The pharmaceutical composition of claim 133, wherein said peptide has a sequence as set forth in onc of SEQ ID NOs: 1-25.
    137. A pharmaceutical composition for inducing megakaryocytopoiesis, the pharmaceutical composition comprising, as an active Fudsieons SHEET ingredient, a peptide derived from an N terminus portion of aS] casein and 2a pharmaceutically acceptable carrier.
    138. The pharmaceutical composition of claim 137, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    139. The pharmaceutical composition of claim 137, wherein said peptide is a synthetic peptide.
    140. The pharmaceunsal compesiton of claim (37, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    141. A pharmaceutical composition for inducing erythropoiesis, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carner.
    142. The pharmaceutical composition of claim 141, wherein said peptide is a fragment derived by fragmentation of «S1 casein.
    143. The pharmaceutical composition of claim 141, wherein said peptide is a synthetic peptide.
    144. The pharmaceutical composition of claim 141, wherein said peptide has a sequence as set forth in one of SEQ 1D NOs: 1-25.
    145. A pharmaccutical composition for inducing leukocytopoiesis, the pharmaceutical composition comprising, as an active ingredient, a peptide AMENDS Blin derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    146. The pharmaceutical composition of claim 145, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    147. The pharmaceutical composition of claim 145, wherein said peptide is a synthetic peptide. 148, The pharmaceutical composition of claim 145, whercin said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    149. A pharmaceutical composition for inducing thrombocytopoiesis, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
    150. The pharmaceutical composition of clam 149, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    151. The pharmaceutical composition of claim 149, wherein said peptide is a synthetic peptide.
    152. The pharmaceutical composition of claim 149, wherein said peptide has a sequence as set forth in one of SEQ ID NOs:1-25.
    153. A pharmaceutical composition for inducing plasma cell proliferation, the pharmaceutical composition comprising, as an active machin az ingredient a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable camer.
    154. The pharmaceutical composition of claim 153, wherein said peptide is a fragment derived by fragmentation of aS1 casein. .
    155. The pharmaceutical composition of claim 153, wherein said peptide is a synthetic peptide. 156, The pharmaceutical composition of claim 1. wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    157. A pharmaceutical composition for inducing dendritic cell proliferation, the pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aS] casein and a pharmaceutically acceptable carrier.
    158. The pharmaceutical composition of claim 137, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    159. The pharmaceutical composition of claim 157, wherein said peptide is a synthetic peptide.
    160. The pharmaceutical composition of claim 157, wherein said peptide has a sequence as set forth in one of SEQ 1D NOs: 1-25.
    161. A pharmaceutical composition for inducing macrophage proliferation, the pharmaceutical composition comprising a peptide derived franisissls Silt from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    162. The pharmaceutical composition of claim 161, wherein sad peptide is a fragment derived by fragmentation of aS1 casein.
    163. The pharmaceutical composition of claim 161, wherein said peptide is a synthetic peptide.
    64. The pharmaceutical composition of claim 161, wherein 20d peptide has a sequence as set forth in one of SEQ 1D NOs: 1-25.
    165. A pharmaceutical composition for preventing or treating thrombocytopenia, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    166. The pharmaceutical composition of claim 165, wherein said peptide is a fragment derived by fragmentation of aS] casein.
    167. The pharmaceutical composition of claim 165, wherein said peptide is a synthetic peptide.
    168. The pharmaceutical composition of claim 1635. wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    169. A pharmaceutical composition for preventing or treating pancytopenia, the pharmaceutical composition comprising, as an aclive Fisiorsts eis GLEET ingredicnt, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    170. The pharmaceutical composition of claim 169, wherein said peptide is a fragment derived by fragmentation of-aS1 casein.
    171. The pharmaceutical composition of claim 169, wherein said peptide is a synthetic peptide.
    172. The pharmaceutical composition of lam 169, whersin said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    173. A pharmaceutical composition for preventing or treating oranulocytopenia, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    174. The pharmaceutical composition of claim 173, wherein said peptide is a fragment derived by fragmentation of aS} casein.
    175. The pharmaceutical composition of claim 173, wherein said peptide is a synthetic peptide.
    176. The pharmaceutical composition of claim 173, wherein suid peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    177. A pharmaceutical composition for preventing or treating hyperlipidemia, the pharmaceutical composition comprising, as an active Lon TE ingredient, a peptide derived from an N terminus portion of «$1 casein and a pharmaceutically acceplable carrier.
    178. The pharmaceutical composition of claim 177, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    179. The pharmaceutical composition of claim 177, wherein said peptide is a synthetic peptide. 180, The pharmaceutical composition of claim 172, wherein aid peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    181. A pharmaceutical composition for preventing or treating cholesteremia, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    182. The pharmaceutical composition of claim 181, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    183. The pharmaceutical composition of claim 181, wherein said peptide is a synthetic peptide.
    184. The pharmaceutical composition of claim 181, wherein said peptide has a sequence as sect forth in onc of SEQ ID NOs: 1-25.
    185. A pharmaceutical composition for preventing or treating glucosuria, the pharmaceutical composition comprising, as an active ingredient, fio hed a peptide derived from an N terminus portion of oS! casein and a pharmaceutically acceptable carrier.
    186. The pharmaceutical composition of claim 185, wherein said peptide is a fragment derived by fragmentation of «S1 casein.
    187. The pharmaceutical composition of claim 185, wherein said peptide is a synthetic peptide.
    188. The vsharmaczutical comonsition of claim 183, wherain said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    189. A pharmaceutical composition for preventing or treating diabetes, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of «Sl casein and a pharmaceutically acceptable carrier.
    190. The pharmaceutical composition of claim 189, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    191. The pharmaceutical composition of claim 189, wherein said peptide is a synthetic peptide.
    192. The pharmaceutical composition of claim 189, wherein said peptide has a sequence as set forth in one of SEQ ID NOs:1-25.
    193. A pharmaceutical composition for preventing or treating AIDS, the pharmaceutical composition comprising, as an active ingredient, a peptide films Solll¥ derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
    194. The pharmaceutical composition of claim 193, wherein said peptide is a fragment derived by fragmentation of aS! casein.
    195. The pharmaceutical composition of claim 193, wherein said peptide is a synthetic peptide.
    126. The pharmaceutical composition of claim 197, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    197. A pharmaceutical composition for preventing or treating infection by HIV, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable camer.
    198. The pharmaceutical composition of claim 197, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    199. The pharmaceutical composition of claim 197, wherein said peptide is a synthetic peptide.
    200. The pharmaceutical composition of claim 197, wherein said peptide has a sequence as sect forth in one of SEQ ID NOs: 1-25.
    201. A pharmaceutical composition for preventing or treating conditions associated with mycloablative doses ol chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell Fite i wind NY ined transplantation (ASCT) or allogencic bone marrow transplantation (BMT), the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    . 202. The pharmaceutical composition of claim 201, wherein said peptide is a fragment derived by fragmentation of aS] cascin.
    203. The pharmaceutical composition of claim 201, wherein said peptide is a synthetic peptide.
    204. The pharmaceutical composition of claim 201, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    205. A pharmaceutical composition for treating a thrombopoietin treatable condition, the pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
    206. The pharmaceutical composition of claim 205, wherein said peptide 1s a fragment derived by fragmentation of aS1 casein.
    207. The pharmaceutical composition of claim 205, wherein said peptide is a synthetic peptide.
    208. The pharmaceutical composition of claim 205, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25. J TT HE
    209. A pharmaceutical composition for augmenting the effect of thrombepoietin, the pharmaceutical composition comprising, as an active ingredient a peptide derived from an N terminus portion of aSl1 casein and a pharmaceutically acceptable carricr.
    210. The pharmaceutical composition of claim 209, wherein said peptide is a fragment derived by fragmentation of aS1 cascin.
    211. The pharmaceutical composition of claim 209, wherein said peptide is a synthetic peptide.
    212. The pharmaceutical composition of claim 209, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    213. A pharmaceutical composition for enhancing peripheral stem cell mobilization, the pharmaceutical composition comprising, as active ingredients thrombopoictin and a peptide derived from an N terminus portion of a SI casein and a pharmaceutically acceptable carrier.
    214. The pharmaceutical composition of claim 213, wherein said peptide is a fragment derived by fragmentation of aS] casein.
    215. The pharmaceutical composition of claim 213, wherein said peptide is a synthetic peptide.
    216. The pharmaceutical composition of claim 213, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25. Eifion LiRT
    217. A pharmaceutical composition for inducing hematopoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    218. The pharmaceutical composition of claim 217, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    219. The pharmaceutical composition of claim 217, wherein said peptide is a synthetic peptide.
    220. The pharmaceutical composition of claim 217, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    221. A pharmaceutical composition for inducing hematopoietic stem cells proliferation, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS] cascin and a pharmaceutically acceptable carrier.
    222. The pharmaceutical composition of claim 221, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    223. The pharmaceutical composition of claim 221, wherein said peptide is a synthetic peptide.
    224. The pharmaceutical composition of claim 221, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    225. A pharmaceutical composition for inducing hematopoietic stem cells proliferation and differentiation, the pharmaceutical composition Aincisoe os SLI comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of «S1 cascin and a pharmaceutically acceptable carrier.
    226. The pharmaceutical composition of claim 225, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    227. The pharmaceutical composition of claim 225, wherein said peptide 1s a synthetic peptide. 228% The pharmaceutical composition of claim 22%, wherein seid peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    229. A pharmaceutical composition for inducing megakaryocytopoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    230. The pharmaceutical composition of claim 229, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    231. The pharmaceutical composition of claim 229, wherein said peptide is a synthetic peptide.
    232. The pharmaceutical composition of claim 229, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    233. A pharmaceutical composition for inducing erythropoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoictin [rail tines Stina and a peptide derived from an N terminus portion of aSl casein and a pharmaceutically acceptable carrier.
    234. The pharmaceutical composition of claim 233, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    235. The pharmaceutical composition of claim 233, wherein said peptide is a synthetic peptide.
    224. The pharmaceutical composition of claim 233, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    237. A pharmaceutical composition for inducing feukocytopoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein and a pharmaceutically acceptable carrier.
    238. The pharmaceutical composition of claim 237, wherein said peptide is a fragment derived by fragmentation of aS} casein.
    239. The pharmaceutical composition of claim 237, wherein said peptide is a synthetic peptide.
    240. The pharmaceutical composition of claim 237, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    241. A pharmaceutical composition for inducing thrombocytopoiesis, the pharmaceutical composition comprising, as active ingredients, Fira irom ovum}
    thrombopoietin and a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
    242. The pharmaceutical composition of claim 241, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    243. The pharmaceutical composition of claim 241, wherein said peptide is a synthetic peptide. 244, The pharmaceutical composition of claim 241, wherein said peptide has a sequence as set forth in one of SEQ ID NOs:1-25.
    245. A pharmaceutical composition for preventing or treating thrombocytopenia, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
    246. The pharmaceutical composition of claim 245, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    247. The pharmaceutical composition of claim 245, wherein said peptide is a synthetic peptide.
    248. The pharmaceutical composition of claim 245, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    249. A pharmaceutical composition for preventing or ucating pancytopenia, the pharmaceutical composition comprising, as aclive RET ingredicnts, thrombopoietin and a peptide derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    250. The pharmaceutical composition of claim 249, wherein said peptide is a fragment derived by fragmentation of «Si casein.
    251. The pharmaceutical composition of claim 249, wherein said peptide is a synthetic peptide.
    252. The pharmaceutical compesition of Claim 246, wherzin said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    253. A pharmaceutical composition for preventing or treating granulocytopenia, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS! casein and a pharmaceutically acceptable carrier.
    254. The pharmaceutical composition of claim 253, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    255. The pharmaceutical composition of claim 233, wherein said peptide is a synthetic peptide.
    256. The pharmaceutical composition of claim 233, where said peptide has a sequence as set forth in one of SEQ ID NOs: 1-23.
    257. A pharmaceutical composition for treating or preventing an indication selected from the group consisting of autoimmune disease or condition, viral disease, viral infection, hematological disease, hematological LLIENTED Sitciy
    ® deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, glucosuria, hyperglycemia, diabetes, AIDS, HIV-1, helper T-cell disorders, dendrite cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, human immune system disorders resulting from treatment of diseases of immune deficiency and bacterial infections, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus porticn of &S! casein and a pharmuceutically acceptable carrier.
    258. The pharmaceutical composition of claim 257, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    259. The pharmaceutical composition of claim 257, wherein said peptide is a synthetic peptide.
    260. The pharmaceutical composition of claim 257, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    261. A pharmaceutical composition for treating or preventing an indication selected from the group consisting of hematological disease, hematological deficiencies, thrombocytopenia, pancytopenia. granulocytopenia, dendrite cell deficiencies, macrophage deficiencics, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, leukemic conditions, myelodysplastic syndrome, aplastic anemia and bone marrow insufficiency, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide Funin O20 Sh derived from an N terminus portion of aS1 casein and a pharmaceutically acceptable carrier.
    262. The pharmaceutical composition of claim 261, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    263. The pharmaceutical composition of claim 261, wherein said peptide is a synthetic peptide.
    264. The pharmaceutical composition of claim 261, wherein said peptide has a sequence as set forth in one of SEQ iD NOs: 1-25.
    265. A purified peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-25.
    266. A pharmaceutical composition comprising a purified peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-25 and a pharmaceutically acceptable carrier.
    267. A pharmaceutical composition comprising thrombopoietin and a purified peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-25 and a pharmaceutically acceptable carrier.
    268. Use of a peptide derived from an N terminus portion of aSl casein in the preparation of a medicament for enhancing colonization of donated blood stem cells in a myeloablated recipient.
    269. The use of claim 268, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    270. The use of claim 268, wherein said peptide is a synthetic peptide.
    271. The use of claim 268, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    272. The use of a peptide derived from an N terminus portion of aS casein for the preparation of a medicament for enhancing colonization of blood stem cells in a myeloablated recipient.
    273. The use of claim 272, wherein said peptide is a fragment derived by fragmentation of aS! casein.
    274. The use of claim 272, wherein said peptide is a synthetic peptide.
    275. The use of claim 272, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    276. A method of preparing donated blood stem cells for transplantation in a myeloablated recipient, the method comprising treating said donated blood stem cells with a peptide derived from an N terminus portion of aS1 casein prior to implanting the donated blood stem cells in the recipient.
    277. The method of claim 276, wherein said peptide is a fragment derived by fragmentation of aS1 casein. LLU SIZSICIR
    278. The method of claim 276, wherein said peptide is a synthetic peptide.
    279. The method of claim 276, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    280. A method of preparing blood stem cells for transplantation in a myeloablated recipient, the method comprising treating said blood stem cells with a peptide derived from an N terminus portion of aS1 casein prior to implanting the blood stem cells in the recipient.
    281. The method of claim 280, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    282. The method of claim 280, wherein said peptide is a synthetic peptide.
    283. The method of claim 280, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    284. Use of a peptide derived from an N terminus portion of aS! casein and thrombopoietin for the preparation of a medicament for enhancing colonization of donated blood stem cells in a myeloablated recipient.
    285. The use of claim 284, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    286. The use of claim 284, wherein said peptide is a synthetic peptide.
    287. The use of claim 284, wherein said peptide has a sequence as set Fonl SID GUTH forth in one of SEQ ID NOs: 1-25.
    288. A method of preparing donated blood stem cells for transplantation in a myeloablated recipient, the method comprising treating said donated blood stem cells with a peptide derived from an N terminus portion of aS1 casein and thrombopoietin prior to implanting the donated blood stem cells in the recipient.
    289. The method of claim 288, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    290. The method of claim 288, wherein said peptide is a synthetic peptide.
    291. The method of claim 288, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    292. A method of preparing donated blood stem cells for transplantation in a myeloablated recipient, the method comprising treating said donated blood stem cells with a peptide derived from an N terminus portion of aS1 casein and thrombopoietin prior to implanting the blood stem cells in the recipient.
    293. The method of claim 292, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    294. The method of claim 292, wherein said peptide is a synthetic peptide.
    295. The method of claim 292, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25. [ATR gt §
    296. A method of preparing blood stem cells for transplantation in a myeloablated recipient, the method comprising treating said blood stem cells with a peptide derived from an N terminus portion of aS1 casein and thrombopoietin prior to implanting the blood stem cells in the recipient.
    297. The method of claim 296, wherein said peptide is a fragment derived by fragmentation of aS1 casein.
    298. The method of claim 296, wherein said peptide is a synthetic peptide.
    299. The method of claim 296, wherein said peptide has a sequence as set forth in one of SEQ ID NOs: 1-25.
    300. The use of a peptide derived from an N terminus portion of aS1 casein for preventing or treating an autoimmune disease.
    301. The use of a peptide derived from an N terminus portion of aS! casein for the preparation of a medicament for preventing or treating a viral disease.
    302. The use of a peptide derived from an N terminus portion of aSI casein for preventing or treating a viral infection.
    303. The use of a peptide derived from an N terminus portion of aSl casein -for preventing or treating a viral disease.
    304. The use of a peptide derived from an N terminus portion of aSI casein for preventing viral infection. FTL
    305. The use of a peptide derived from an N terminus portion of «SI casein for inducing hematopoiesis.
    306. The use of a peptide derived from an N terminus portion of aSI casein for inducing hematopoietic stem cells proliferation.
    307. The use of a peptide derived from an N terminus portion of aSl casein for inducing hematopoietic stem cells proliferation and differentiation.
    308. The use of a peptide derived from an N terminus portion of aSl casein for inducing megakaryocytopoiesis.
    309. The use of a peptide derived from an N terminus portion of aS] casein for inducing erythropoiesis.
    310. The use of a peptide derived from an N terminus portion of aS] casein for inducing leukocytopoiesis.
    311. The use of a peptide derived from an N terminus portion of aS] casein for inducing thrombocytopoiesis.
    312. The use of a peptide derived from an N terminus portion of aSl casein for inducing plasma cell proliferation. aSI casein =
    313. The use of a peptide derived from an N terminus portion of «Sl casein for inducing dendritic cell proliferation.
    314. The use of a peptide derived from an N terminus portion of aSl casein for inducing macrophage proliferation. FRR NA
    315. The use of a peptide derived from an N terminus portion of aS] casein for preventing or treating thrombocytopenia.
    316. The use of a peptide derived from an N terminus portion of aSl casein for preventing or treating pancytopenia.
    317. The use of a peptide derived from an N terminus portion of aSl casein for preventing or treating granulocytopenia.
    318. The use of a peptide derived from an N terminus portion of aS] casein for preventing or treating hyperiipidemia.
    319. The use of a peptide derived from an N terminus portion of aSl casein for preventing or treating cholesteremia.
    320. The use of a peptide derived from an N terminus portion of aSl casein for preventing or treating glucosuria.
    321. The use of a peptide derived from an N terminus portion of aSl casein for preventing or treating diabetes.
    322. The use of a peptide derived from an N terminus portion of aSl casein for preventing or treating AIDS.
    323. The use of a peptide derived from an N terminus portion of aS] casein for preventing or treating infection by HIV.
    324. The use of a peptide derived from an N terminus portion of aSl casein for preventing or treating conditions associated with myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood SRE stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT).
    325. The use of a peptide derived from an N terminus portion of aSl casein for preventing or treating a thrombopoietin treatable condition.
    326. The use of a peptide derived from an N terminus portion of aSl casein for augmenting the effect of thrombopoietin.
    327. The use of a peptide derived from an N terminus portion of aS! casein for enhancing peripheral stem cell mobilization.
    328. The use of a peptide derived from an N terminus portion of aS! casein for the preparation of a medicament for enhancing colonization of blood stem cells in a myeloablated recipient.
    329. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for preventing or treating an autoimmune disease.
    330. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for preventing or treating a viral disease.
    331. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for preventing or treating a viral infection. Fil Lull DLL
    -—
    332. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSI casein, and a pharmaceutically acceptable carrier for inducing hematopoiesis.
    333. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSI casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cell proliferation.
    334. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSI casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cell proliferation and differentiation.
    335. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSI casein, and a pharmaceutically acceptable carrier for inducing megakaryocytopoiesis.
    336. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for inducing crythropoiesis.
    337. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for inducing leukocytopoiesis.
    338. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for inducing thrombocytopoiesis. [LTNRID TD
    339. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for inducing plasma cell proliferation.
    340. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for inducing dendritic cell proliferation.
    341. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for inducing macrophage proliferation.
    342. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating thrombocytopenia.
    343. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating pancytopenia.
    344. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia.
    345. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for preventing or treating hyperlipidemia.
    346. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSI casein, and a pharmaceutically acceptable carrier for preventing or treating cholesteremia. ire ES bs wb fmf
    347. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating glucosuria.
    348. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for preventing or treating diabetes.
    349. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating AIDS.
    350. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating infection by HIV.
    351. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating conditions associated with myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT).
    352. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for treating a thrombopoietin treatable condition.
    353. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSI casein, and a pharmaceutically acceptable carrier for augmenting the effect of thrombopoietin. ER a
    )
    354. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier peptide derived from an N terminus portion of aSl casein for enhancing colonization of donated blood stem cells in a myeloablated recipient.
    355. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier enhancing colonization of blood stem cells in a myeloablated recipient.
    356. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a pepteide derived from an N terminus portion of aS1 casein, and a pharmaceutically acceptable carrier for enhancing peripheral stem cell mobilization.
    357. he use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for inducing hematopoiesis.
    358. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS! casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cells proliferation.
    359. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cells proliferation and differentiation. Cede D OLICET
    360. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for inducing megakaryocytopoiesis.
    361. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for inducing erythropoiesis.
    362. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for inducing leukocytopoiesis.
    363. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aS] casein, and a pharmaceutically acceptable carrier for inducing thrombocytopoiesis.
    364. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSI casein, and a pharmaceutically acceptable carrier for preventing or treating thrombocytopenia.
    365. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating pancytopenia. Fiivicn vi mr Se fred
    366. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSlI casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia.
    367. The use of a pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating an indication selected from the group consisting of autoimmune disease or condition, viral disease, viral infection, haematological disease, haematological deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, glucosuria, hyperglycemia, diabetes, AIDS, HIV-1, helper T-cell disorders, dendrite cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, human immune system disorders resulting from treatment of diseases of immune deficiency and bacterial infections.
    368. The use of a pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of aSl casein, and a pharmaceutically acceptable carrier for preventing or treating an indication selected from the group consisting of haematological disease, haematological deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, dendrite cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, leukemic conditions, myelodysplastic syndrome, aplastic anemia and bone marrow insufficiency.
    369. The use of a pharmaceutical composition comprising, as active ingredients, a peptide derived from an N terminus portion of aSl casein and rial ies SUES thrombopoietin, and a pharmaceutically acceptable carrier for enhancing colonization of donated blood stem cells in a myeloablated recipient.
    370. The use of a pharmaceutical composition comprising, as active ingredients, a peptide derived from an N terminus portion of aS casein and thrombopoietin, and a pharmaceutically acceptable carrier for enhancing colonization of blood stem cells in a myeloablated recipient.
    371. The use of any of claims 300-370, wherein said peptide is a fragment derived by fragmentation of aS! casein.
    372. The use of any of claims 300-370, wherein said peptide is a purified peptide having a sequence as set forth in one of SEQ ID NO’s:1-25.
    373. The use of any of claims 300-370, wherein said peptide is a synthetic peptide.
    374. The use of any of claims 300-370, wherein said peptide has a sequence as set forth in one of SEQ ID NO’s: 1-25.
    375. The use of any one of claims 1, 5,9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 268, 272, 284 or 300-370, substantially as herein described and exemplified and/or described with reference to the accompanying figures.
    376. The pharmaceutical composition of any one of claims 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 266 or 267 substantially as herein described and exemplified and/or described with reference to the accompanying figures. Frown ivrimm Ov iiied
    377. The method of any one of claims 276, 280, 288, 292 or 296, substantially as herein described and exemplified and/or described with reference to the accompanying figures.
    378. The purified peptide of claim 265, substantially as herein described and exemplified and/or described with reference to the accompanying figures. borer iris dra
ZA200401574A 2001-08-30 2004-02-26 Casein derived peptides and uses thereof in therapy ZA200401574B (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US09/942,121 US20020147144A1 (en) 2000-03-01 2001-08-30 Casein derived peptides and uses thereof in therapy

Publications (1)

Publication Number Publication Date
ZA200401574B true ZA200401574B (en) 2007-02-28

Family

ID=25477603

Family Applications (1)

Application Number Title Priority Date Filing Date
ZA200401574A ZA200401574B (en) 2001-08-30 2004-02-26 Casein derived peptides and uses thereof in therapy

Country Status (15)

Country Link
EP (1) EP1556074A4 (en)
JP (1) JP2005511499A (en)
KR (1) KR20040078639A (en)
CN (1) CN1694719A (en)
AU (1) AU2002324323A2 (en)
BR (1) BR0212625A (en)
CA (1) CA2458924A1 (en)
CZ (1) CZ2004335A3 (en)
HU (1) HUP0500995A3 (en)
IL (1) IL160548A0 (en)
MX (1) MXPA04001890A (en)
NO (1) NO20040880L (en)
PL (1) PL375113A1 (en)
WO (1) WO2003018606A2 (en)
ZA (1) ZA200401574B (en)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0313892D0 (en) * 2003-06-16 2003-07-23 Hannah Res Inst Control of lactation
CA2554775A1 (en) * 2004-01-30 2005-08-18 Chemgenex Pharmaceuticals, Inc. Naphthalimide dosing by n-acetyl transferase genotyping
CA2558155A1 (en) * 2004-03-01 2005-09-09 Zvi Sidelman Casein derived peptides and therapeutic uses thereof
GB0423352D0 (en) * 2004-10-21 2004-11-24 Hannah Res Inst "Control of mammary cell number"
ES2319475B1 (en) * 2005-06-08 2010-02-16 Consejo Superior Investig. Cientificas BIOACTIVE PEPTIDES IDENTIFIED IN ENZYMATIC HYDROLYZES OF LACTEE CASEINS AND PROCEDURE OF OBTAINING.
CN101410131B (en) * 2006-06-09 2012-09-12 森永乳业株式会社 Lipid-metabolism-improving agent
JP2011026213A (en) * 2009-07-22 2011-02-10 Snow Brand Milk Prod Co Ltd Type 1 diabetes-inhibiting peptide
JP2015520129A (en) * 2012-04-16 2015-07-16 ザ クリーブランド クリニック ファウンデーション Multivalent breast cancer vaccine
EP3590519A4 (en) * 2017-03-03 2020-12-16 Morinaga Milk Industry Co., Ltd. Glp-1 secretagogue and composition
CN107814835B (en) * 2017-12-01 2020-04-28 熊猫乳品集团股份有限公司 Bioactive polypeptide AVPITPTLNREQ, and preparation method and application thereof
CN108017709B (en) * 2017-12-12 2020-04-10 浙江辉肽生命健康科技有限公司 Bioactive polypeptide KEPMIGVNQELA, and preparation method and application thereof
CN113952446B (en) * 2021-09-03 2023-12-05 河南省医药科学研究院 Application of bioactive peptide in inhibiting bone marrow toxicity

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3778426A (en) * 1970-12-16 1973-12-11 Research Corp Therapeutically useful polypeptides
CA2287082A1 (en) * 1997-03-21 1998-10-01 Snow Brand Milk Products Co., Ltd. Iron hydrolyzates of casein complexes and processes for preparation thereof
IL134830A0 (en) * 2000-03-01 2001-05-20 Chay 13 Medical Res Group N V Peptides and immunostimulatory and anti-bacterial pharmaceutical compositions containing them

Also Published As

Publication number Publication date
WO2003018606A3 (en) 2005-05-06
EP1556074A4 (en) 2008-05-07
IL160548A0 (en) 2004-07-25
CA2458924A1 (en) 2003-03-06
JP2005511499A (en) 2005-04-28
EP1556074A2 (en) 2005-07-27
CN1694719A (en) 2005-11-09
MXPA04001890A (en) 2004-06-18
PL375113A1 (en) 2005-11-28
KR20040078639A (en) 2004-09-10
NO20040880L (en) 2004-04-20
HUP0500995A3 (en) 2008-09-29
CZ2004335A3 (en) 2004-07-14
WO2003018606A2 (en) 2003-03-06
WO2003018606A9 (en) 2004-04-08
BR0212625A (en) 2007-06-19
HUP0500995A2 (en) 2007-05-02
AU2002324323A2 (en) 2003-03-10

Similar Documents

Publication Publication Date Title
AU782662B2 (en) Casein derived peptides and uses thereof in therapy
MXPA06010014A (en) Casein derived peptides and therapeutic uses thereof.
US7666996B2 (en) Casein derived peptides and uses thereof
ES2246566T3 (en) APPLICATION OF HSP70 PROTEINS.
ZA200401574B (en) Casein derived peptides and uses thereof in therapy
JP2004508412A (en) Thionine as an antineoplastic and immunostimulant
TWI658832B (en) Composition for inhibiting myeloid-derived suppressor cells
WO2004035081A1 (en) Activity of osteogenic growth peptide (ogp) for promoting proliferation of hemopoietic progenitor cell in erythron and its use