HK1083851A - Casein derived peptides and uses thereof in therapy - Google Patents

Description

Casein derived peptides and therapeutic uses thereof

no marking

Technical Field

The present invention relates to bioactive peptides derived from or similar to the partial N-terminal sequence of milk casein alpha S1. These peptides are capable of stimulating and enhancing immune responses, protecting against viral infections, normalizing serum cholesterol levels, and stimulating hematopoiesis. The casein derived peptides are non-toxic and can be used for the treatment and prevention of immunological disorders, hypercholesterolemia, hematological disorders and viral-related disorders.

Background

Bioactive molecules in nutrients:

in addition to the nutritional value of many foods, certain parts and products produced during digestion have a role in influencing physiological processes. Some of these "extra-nutrient" components are present in their active form in whole nutrition products, such as immunoglobulins in breast milk and colostrum, phytoestrogens in soy products, polyphenol antioxidants in fruits and vitamins. Other components are contained in nutritional molecules and are released in active form during digestion or food breakdown, such as antihypertensive peptides from lactoglobulin [ Kitts, D.D. (1999), can.j. physiol. pharmacol.72: 4; 423-434].

Biological activity in milk proteins:

casein, the major component of milk proteins, is conventionally defined as containing three parts, α, β and γ according to its electrophoretic mobility [ n.j.hipp, et al (1952), Dairy sci., 35: 272]. Casein is now defined according to the amino acid sequence of each subgroup, including α S1, α S2, β and κ [ w.n.engel et al (1984), j.dairy sci.67: 1599].

During digestion, casein is subjected to proteolytic cleavage mainly by acidic proteases such as chymosin, trypsin and pepsin, resulting in shorter peptides and, by the resulting protein fragments, in curdling and calcium sequestration. Several studies on emulsion compounds have shown casein-related bactericidal activity. Us patent 3,764,670 discloses that proteolytic digests of casein have antibiotic properties against microorganisms. Israel patent 42863 describes a casein-derived peptide consisting of the N-terminal 23 amino acids of casein, which peptide has antibacterial activity. In addition, casein or its derivatives have other physiological activities, such as opioid and growth factor-like activities [ Kitts, d.d. (1999), ibid ].

Immunomodulatory activity is also observed in casein peptides. An increase in lymphocyte proliferation capacity was observed after treatment of rats with a peptide derived from the C-terminus of beta-casein (1992, Immun. Lett.33: 41-46). However, none of these studies identified specific sequences in these casein peptides that were responsible for the "nutrient supplementation" properties.

Hematopoiesis in cancer treatment:

patients are at high risk for pancytopenia following high-dose chemotherapy, particularly high-dose myeloablative radiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT). Granulocytopenia can lead to the development of serious and sometimes even fatal complications of infections that can be common bacterial, viral, fungal and parasitic. Similarly, thrombocytopenia often results in bleeding tendencies and sometimes even requires long-term platelet dependence. Once platelet resistance occurs, bleeding events can be life threatening, and bleeding complications are often fatal. Since the risk of granulocytopenia can be partially alleviated by supportive therapy, the most effective approach is to administer recombinant human cytokines capable of enhancing granulocyte reconstitution, particularly granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF). These drugs are very expensive (about 200 and 400 mellitis per patient per day), and can cause rare adverse events due to allergic reactions, fever, and bone pain, with occasional development of vascular leak syndromes, including pericarditis and pleuritis. Some side effects may also be due to other cytokines that these hematopoietic growth factors may inherently release. Moreover, these hematopoietic growth factors may also prohibit their use in patients with certain tumor cells carrying G-CSF or GM-CSF receptors, such as acute and chronic myelogenous leukemia and myelodysplastic syndrome. Despite the great progress made in the treatment of patients at risk of pancytopenia by the use of hematopoietic cytokines, there has been no progress in the treatment of thrombocytopenia. After high dose chemotherapy, particularly ASCT, patients may be at risk for thrombocytopenia for months, even up to 3 years, and some thrombocytopenic patients may fail to recover for life. Many patients develop platelet resistance from previous treatments with multiple blood products, and thus even frequent intensive infusions with platelets from a single donor may render thrombocytopenia overwhelming or even temporary remission impossible. The long duration of platelet resistance and thrombocytopenia is a common cause of death in the ASCT center 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 have shown that although rhIL3 and rhIL6 can enhance platelet remodeling, these effects are not significant and take a long time.

It is clear that long-term persistence of thrombocytopenia is a major problem in the current clinical bone marrow transplant centers, for which no satisfactory solution has been available to date.

Therefore, there is a well-recognized need for a safe, inexpensive, therapeutically effective, and defined hematopoietic cell stimulating factor, particularly megakaryocyte production stimulating factor, that avoids the above limitations.

Thrombopoietin (TPO) that modulates hematopoietic and platelet function:

although the increase in kidney and liver-derived growth factors in platelet defects is not due to adaptation of TPO biosynthesis in these organs, TOP appears to be a major regulator of platelet production in vivo. There appears to be a "feedback loop" in which the number of circulating platelets determines how many cycles of TPO are available to the bone marrow for platelet production. Furthermore, TPO has been demonstrated to be an early acting cytokine with important multi-cell lineage effects: TPO alone or in combination with other early acting cytokines can (I) promote progenitor cell viability and inhibit progenitor apoptosis; (ii) regulating hematopoietic stem cell production and function; (iii) triggering cell division of resting pluripotent cells; (iv) (iv) inducing differentiation of the multi-cell line and (v) enhancing the formation of multi-cell line colonies comprising granulocytes, erythrocytes, macrophages and megakaryocytes (MK, CFU-GEMM). Moreover, TPO stimulates the production of more limited progenitors of granulocytes/monocytes, megakaryocytes and erythroid colonies, and stimulates the adhesion of primitive human bone marrow and megakaryocytes to fibronectin and fibrinogen. Thus, TPO is an important cytokine for clinical hematologists/transplanters: stem cells and committed precursor cells for autologous and allogeneic transplantation [ von demborn, a.e.g.kr., et al., (1998) Thrombopoietin: it's roll in sheet discs and as a new drug in a clinical media. InBailliers client. Hematol. June: 11(2),427-45].

In addition to TPO affecting hematopoiesis, this potent growth factor is a diverse agonist that stimulates platelets and modulates platelet-extracellular matrix interactions. While not causing platelet aggregation by itself, TPO upregulates ADP-induced aggregation, particularly the second round of aggregation, upregulated particle (ADP, ATP, 5-hydroxytryptamine, etc.) release and thromboxane B2 is produced, increases platelet adhesion to collagen, and potentiates shear-induced platelet aggregation. TPO also stimulates PMN activation, induces IL-8 release and stimulates oxygen metabolite production, likely enhancing antimicrobial defense.

Clinical studies suggest the value of TPO in understanding and treating a variety of hematopoietic conditions. Elevated TPO levels persist in patients with idiopathic Aplastic Anemia (AA), even in remission following immunosuppressive therapy, suggesting a hematopoietic deficiency. TPO is also elevated in other forms of aplastic thrombocytopenia, but not in conditions of increased platelet destruction. It is clear that the reactive increase in TPO production is sufficient in devastating thrombocytopenia. Thus, TPO is not only a treatment option for aplastic thrombocytopenia, but also for destructive thrombocytopenia.

Thrombopoietic agents are of widespread clinical interest for the prevention and/or treatment of pathology or treatment-induced thrombocytopenia, and as a replacement for platelet transfusions. Of the cytokines evaluated, all but critically effective IL-11 were considered not to be clinically useful. TPO is widely recognized as a cytokine of choice for the treatment of thrombocytopenia. Recently, recombinant human tpo (genentech) has become available, enabling accurate pharmacokinetic assays and clinical trials. Thus, potential uses for TPO include the fields of supportive care (chemotherapy/radiotherapy, post bone marrow and stem cell transplantation), hematologic disorders (AA, myelodysplasia, congenital and acquired thrombocytopenia), liver disease, blood transfusion (expanding, harvesting, mobilizing and storing platelets), and surgery (including liver transplantation). Of particular interest are the potential uses of TPO/EPO/G-CSF mixtures for myelodysplasia, the combination of G-CSF and TPO for peripheral stem cell mobilization, and TPO in harvested CD 34+ cells for good platelet reconstitution, as well as ex vivo expansion of megakaryocytes. However, like other hematopoietic agents that are being considered for clinical use, TPO is very expensive and may be antigenic at therapeutically effective levels. Therefore, it would be advantageous to develop safe, inexpensive and readily available platelet production stimulators capable of increasing TPO activity.

α S1 portion of casein:

the α S1 portion of casein can be obtained from milk proteins by a variety of methods [ d.g.schmidth and t.a.j.paynes (1963), biochim, biopys.acta, 78: 492; m.p.thompson and c.a.kiddy (1964), j.direct sci., 47: 626; mercier, et al, (1968), ball, soc, chim, biol.50: 521], the complete amino acid sequence of the α S1 part of casein was also determined j.c. mercier, etal (1968) (eur.j.biochem.23: 41). The genome and coding sequence of the bovine casein α S1 portion was also cloned and sequenced by recombinant DNA techniques [ d.koczan, et al (1991), nucleic.acids res.19 (20): 5591; McKnight, r.a., et al (1989), j.dairy sci.72: 2464-73]. The literature describes the proteolysis and identification of the N-terminal fragment of the α S1 part of casein [ j.c. mercier, et al (1970) eur.j.biochem.16: 439; p.l.h.mcsweeney et al, (1993), j.diaryres, 60: 401], after ingestion of intact milk proteins, which are absorbed via the small intestine, the fragment appears in the plasma of mammals [ Fiat, a.m., et al (1998) biochemie, 80 (2): 2155-65]. Meisel, H and Bockelmann, W. [ (1999), Antonie van leuwenhoek, 76: 207-15 the amino acid sequences of the immunopeptides, casokinin and casomorphins were examined in peptides released by the lactic acid bacteria digestion of the alpha and beta fractions of casein. Of particular interest, the C-terminal part of the alpha and kappa casein fractions showed anti-aggregation and hemolytic activity [ chapance, b.et al (1997), biochem. mol.biol. int.42(1) 77-84; caen j.et al (1993), j.dairy sci.76 (1): 301-310].

Previous studies have documented potential bioactive peptides present in the N-terminal α S1 amino acid sequence, but no mention has been made of the 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 for preventing or treating an autoimmune disease by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein to a subject in need thereof.

According to the present invention, there is also provided a method for preventing or treating a viral disease by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein to a subject in need thereof.

According to the present invention, there is also provided a method of inducing hematopoiesis by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing hematopoietic stem cell proliferation by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for inducing proliferation and differentiation of hematopoietic stem cells by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein to a subject in need thereof.

According to the present invention, there is also provided a method of inducing megakaryocytopoiesis by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing erythropoiesis by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing leukopoiesis by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing thrombopoiesis by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing plasma cell proliferation by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing dendritic cell proliferation by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing macrophage proliferation by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for preventing or treating thrombocytopenia by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for preventing or treating pancytopenia by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for preventing or treating granulocytopenia by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for preventing or treating hyperlipidemia by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for preventing or treating hypercholesterolemia by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for preventing or treating glucosuria (glucosuria) by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for preventing or treating diabetes by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for preventing or treating aids by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein to a subject in need thereof.

According to the present invention, there is also provided a method for preventing or treating HIV infection by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein to a subject in need thereof.

According to the present invention, there is also provided a method for preventing or treating a condition associated with myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT) by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.

According to the present invention, there is also provided a method of treating an erythropoietin-treatable condition by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for increasing the effect of erythropoietin by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method of treating a thrombopoietin-treatable condition by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for increasing the effect of thrombopoietin by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from the N terminus portion of α s1 casein.

According to the present invention, there is also provided a method of enhancing peripheral stem cell mobilization by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating an autoimmune disease, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating viral diseases, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing viral infection, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing hematopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing hematopoietic stem cell proliferation, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing proliferation and differentiation of hematopoietic stem cells, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing megakaryocytopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing erythropoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing leukopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing thrombopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing proliferation of plasma cells, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing proliferation of dendritic cells, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing macrophage proliferation, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating thrombocytopenia, which comprises, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also 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 α S1 casein and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating granulocytopenia, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating hyperlipidemia, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating hypercholesterolemia, which comprises, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating glucosuria, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating diabetes, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating aids, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating HIV infection, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating a condition associated with myeloablative doses of radiotherapy and chemotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT), comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for the treatment of an erythropoietin-treatable condition, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for increasing the effect of erythropoietin, which comprises, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for treating a thrombopoietin-treatable condition, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for increasing the effect of thrombopoietin, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for enhancing peripheral stem cell mobilization, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing hematopoiesis, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing the proliferation of hematopoietic stem cells, which comprises, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing proliferation and differentiation of hematopoietic stem cells, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing megakaryocytopoiesis, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing erythropoiesis, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing leukopoiesis, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing thrombopoiesis, which comprises, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating thrombocytopenia, which comprises, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also 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 α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating granulocytopenia, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention there is also provided a pharmaceutical composition for the treatment or prevention of an indication selected from the group consisting of: an autoimmune disease or condition, a viral disease, a viral infection, a hematologic disease, a hematologic defect, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, glucosuria, hyperglycemia, diabetes, aids, HIV-1, helper T cell disorders, dendritic cell defects, macrophage defects, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, human immune system disorders resulting from disease treatment for immunodeficiency and bacterial infection, comprising, as an active ingredient, a peptide derived from the N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention there is also provided a pharmaceutical composition for the treatment or prevention of an indication selected from the group consisting of: a hematologic disease, a defect in the blood system, thrombocytopenia, pancytopenia, granulocytopenia, dendritic cell deficiency, macrophage deficiency, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, myelodysplastic syndrome, aplastic anemia, and bone marrow deficiency, comprising, as active ingredients, a peptide derived from the N-terminal portion of thrombopoietin and α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention there is also provided a method of enhancing the colonization of donated blood stem cells in a myeloablative recipient by treating a donor of the donated blood stem cells with a peptide derived from an N terminus portion of α S1 casein prior to donation and implantation of the donated blood stem cells in the recipient.

According to the present invention there is also provided a method of enhancing the colonization of the provided blood stem cells in a myeloablative recipient by treating the provided blood stem cells with a peptide derived from an N terminus portion of α S1 casein prior to implanting the provided blood stem cells in the recipient.

According to the present invention there is also provided a method of enhancing the colonization of blood stem cells in a myeloablative recipient by treating the blood stem cells with a peptide derived from an N terminus portion of α S1 casein prior to implantation of the blood stem cells in the recipient.

According to the present invention there is also provided a method of enhancing the colonization of donated blood stem cells in a myeloablative recipient by treating a donor of the donated blood stem cells with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to donation and implantation of the donated blood stem cells in the recipient.

According to the present invention there is also provided a method of enhancing the colonization of donated blood stem cells in a myeloablative recipient by treating the donated blood stem cells with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to implantation of the donated blood stem cells in the recipient.

According to the present invention there is also provided a method of enhancing the colonization of blood stem cells in a myeloablative recipient by treating the blood stem cells with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to implantation of the blood stem cells in the recipient.

According to the invention, the application of the peptide derived from the N terminal part of the alpha S1 casein in preparing the medicament for preventing or treating the autoimmune disease is also disclosed.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of a viral disease is also disclosed.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention of viral infection is also disclosed.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing hematopoiesis is also disclosed.

According to the invention, the application of the peptide derived from the N terminal part of the alpha S1 casein in preparing the medicament for inducing the proliferation of the hematopoietic stem cells is also disclosed.

According to the invention, the application of the peptide derived from the N terminal part of the alpha S1 casein in preparing the medicament for inducing the proliferation and differentiation of hematopoietic stem cells is also disclosed.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing megakaryocytopoiesis.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing erythropoiesis is also disclosed.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing leukopoiesis.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing thrombopoiesis is also disclosed.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing proliferation of plasma cells is also disclosed.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing dendritic cell proliferation is also disclosed.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing macrophage proliferation is also disclosed.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of thrombocytopenia is also disclosed.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of pancytopenia is also disclosed.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of granulocytopenia is also disclosed.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein in the preparation of a medicament for the prevention or treatment of hyperlipidemia.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of hypercholesterolemia.

According to the invention, the application of the peptide derived from the N terminal part of the alpha S1 casein in preparing the medicament for preventing or treating the grape diabetes is also disclosed.

According to the invention, the application of the peptide derived from the N terminal part of the alpha S1 casein in preparing the medicament for preventing or treating diabetes is also disclosed.

According to the invention, the application of the peptide derived from the N terminal part of the alpha S1 casein in preparing the medicament for preventing or treating AIDS is also disclosed.

According to the invention, the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of HIV infection is also disclosed.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of a condition associated with myeloablative doses of radiotherapy supported with autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT).

According to the invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the treatment of a thrombopoietin-treatable condition.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for increasing the effect of thrombopoietin.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for enhancing peripheral stem cell mobilization.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for enhancing the colonization of blood stem cells provided in vivo by a myeloablative receptor.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for enhancing the colonization of blood stem cells in the myeloablative receptor.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating an autoimmune disease.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating viral diseases.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating viral infection.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing hematopoiesis.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cell proliferation.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing proliferation and differentiation of hematopoietic stem cells.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing megakaryocytopoiesis.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing erythropoiesis.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing leukopoiesis.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing thrombopoiesis.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing proliferation of plasma cells.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing dendritic cell proliferation.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing macrophage proliferation.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating thrombocytopenia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating pancytopenia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating hyperlipidemia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating hypercholesterolemia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating glucosuria.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating diabetes.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating aids.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating HIV infection.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating a condition associated with myeloablative doses of radiotherapy supported with autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT).

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for the treatment of a thrombopoietin-treatable condition.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for increasing the effect of thrombopoietin.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing the colonization of provided blood stem cells in a myeloablative recipient.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing blood stem cell colonization in a myeloablative receptor.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing peripheral stem cell mobilization.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing hematopoiesis.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing hematopoietic stem cell proliferation.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing the proliferation and differentiation of hematopoietic stem cells.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing megakaryocytopoiesis.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing erythropoiesis.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing leukopoiesis.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing thrombopoiesis.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for the prevention or treatment of thrombocytopenia.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for the prevention or treatment of pancytopenia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating an indication selected from the group consisting of: autoimmune diseases or conditions, viral diseases, viral infections, hematologic diseases, hematologic defects, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, diabetes mellitus, hyperglycemia, diabetes mellitus, aids, HIV-1, helper T cell disorders, dendritic cell defects, macrophage defects, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, human immune system disorders resulting from disease treatment of immune deficiencies and bacterial infections.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating an indication selected from the group consisting of: hematologic disorders, hematologic deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, dendritic cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, myelodysplastic syndromes, aplastic anemia, and bone marrow deficiencies.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for enhancing the colonization of provided blood stem cells in a myeloablative recipient.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for enhancing the colonization of blood stem cells in a myeloablative recipient.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for enhancing peripheral stem cell mobilization.

According to still further features in preferred embodiments of the invention described below, the peptide is a fragment derived by fragmentation of α S1 casein.

According to still further features in the described preferred embodiments the peptide is a synthetic peptide.

According to still further features in preferred embodiments of the invention the peptide has a sequence as shown in one of sequences 1-25.

According to the present invention there is also provided a purified peptide having an amino acid sequence selected from the group consisting of sequences 1-25.

According to the present invention there is also provided a pharmaceutical composition comprising a purified peptide having an amino acid sequence selected from the group consisting of sequences 1-25 and a pharmaceutically acceptable carrier.

According to the present invention there is also provided a pharmaceutical composition comprising thrombopoietin and a purified peptide having an amino acid sequence selected from the group consisting of sequences 1-25 and a pharmaceutically acceptable carrier.

The present invention successfully overcomes the deficiencies of the currently known treatments by providing peptides for the treatment of human diseases, which peptides are derived from the N-terminal part of α S1 casein, have no detectable toxicity and have a high therapeutic effect.

Brief Description of Drawings

The invention is described by way of example only with reference to the accompanying drawings. With respect to the details in the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most effective 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 figure:

FIG. 1 depicts the use of native casein-derived peptides to stimulate Natural Killer (NK) cell activity in cultured murine bone marrow cells. Pairs of cultured murine bone marrow cells in the presence or absence of 100. mu.g/ml native casein-derived peptide³⁵Lysis of S-labeled YAC target cells with the proportion of total radioactivity released (% released) by YAC cells into the culture supernatant³⁵S). The NK activities represented in FIG. 1 are effector cell to target cell ratios of 25: 1 and 50: 1.

FIGS. 2a and 2b depict the use of native casein-derived peptides to stimulate Natural Killer (NK) cell activity in cultured human Peripheral Blood Stem Cells (PBSCs). Derived from granulocyte colony stimulating factor (G-CSF) treated cultures incubated in the absence (0. mu.g) or increasing concentrations (5-500. mu.g/ml) of native casein-derived peptidesPairs of cultured human PBSCs in vivo³⁵Lysis of S-labeled K562 target cells, the proportion of total radioactivity released (% released) into the culture supernatant by K562 cells³⁵S). FIG. 2a shows NK activity of two blood samples from the same patient incubated with different effector to target cell ratios (1: 25 and 1: 50). FIG. 2b shows the expression of a gene from the same effector cell: target cell ratio of NK activity of blood samples from normal and affected donors incubated. The squares represent an effector to target ratio of 100: 1, and the diamonds represent an effector to target ratio of 50: 1.

FIGS. 3a-3c depict native casein-derived peptides stimulating the proliferation of Natural Killer (NK) cells and T-lymphocytes (T) cells from cultured human Peripheral Blood Stem Cells (PBSCs). NK and T cell proliferation in cultured PBSCs from granulocyte colony stimulating factor-treated donors incubated with or without native casein-derived peptides as binding to anti-CD₃FITC fluorescent anti-T cell antibody UCHT1 or anti-CD₅₆Percentage (%) of cells of the RPE fluorescent anti-NK cell antibody MOC-1(DAKO A/SDenmark). Controls were FITC and RPE conjugated anti-mouse IgG antibodies. FIG. 3a shows binding of fluorescent antibody CD after 10 days incubation with (peptide) or without (control) 100. mu.g/ml native casein derived peptide₅₆Percentage of cultured human PBSC cells (5 independent samples). FIG. 3b shows binding of fluorescent anti-CD after 14 days incubation with (peptide) or without (control) 100. mu.g/ml native casein derived peptide₃Percentage of cultured human PBSC cells of (T cell) antibody. FIG. 3c shows binding of fluorescent anti-CD after 28 days incubation with (peptide) or without (control) 100. mu.g/ml native casein derived peptide₃Cultured human PBSC cells and bound CD of (T cell) antibodies₃And CD₅₆Cell percentage of both (T and NK-like cells) antibodies.

FIG. 4 depicts the use of casein-derived synthetic peptides to stimulate Natural Killer (NK) cell activity in cultured human Peripheral Blood Stem Cells (PBSCs). Pairs of cultured human PBSCs (from a breast cancer patient)³⁵Lysis of S-labeled K562 target cells and release to culture supernatant Using K562 cellsThe proportion of total radioactivity (% release) in the fluid is expressed, where cultured human PBSCs (from breast cancer patients) were incubated with increasing concentrations of casein-derived synthetic peptide (10-500 μ g/ml) or without (0 μ g) casein-derived synthetic peptide. The peptides represent 1-10(1a, diamonds), 1-11(2a, squares) and 1-12(3a, triangles) of the starting amino acids of the N-terminal part of α S1 casein (synthetic peptide sequences are shown in Table 3 below).

FIGS. 5a-c depict the use of native casein-derived peptides to stimulate proliferation of cultured human cells of various origins. After the cultured human cells are incubated with increasing concentrations of the native casein-derived peptide for 14 to 21 days, the proliferation thereof is determined by the amount of the peptide incorporated in each sample³H]The amount of thymidine is indicated. FIG. 5a represents the incorporation of label into two samples (PBSC1, incubated for 15 days; PBSC2, incubated for 20 days) of human peripheral blood stem cells incubated with or without (control) 50-600. mu.g/ml native casein-derived peptide. FIG. 5b is a photograph representing the result of 21 days after incubating human bone marrow cells cultured with or without (control) 50 to 600. mu.g/ml of a native casein-derived peptide ³H]Incorporation of thymidine. Bone marrow was from remission cancer patients (BMAuto, solid squares, BM1, triangles and BM2, open triangles) or healthy volunteers (normal BM, diamonds). FIG. 5c represents the time period of 14 days after incubation of human cord blood cells cultured with or without (control) 50-1000. mu.g/ml native casein-derived peptide³H]Incorporation of thymidine. Cord blood cells were supplied by two independent donors (c.b.1, triangle, c.b.2, square).

FIG. 6 shows a table depicting the proliferation of blood progenitor cells from human bone marrow and cord blood in response to incubation with native casein-derived peptides. Relative cell number reflecting proliferation of cultured cells X10⁴The/ml, is determined by the counting cell method described in the examples section below. Bone marrow (bone marrow) from healthy volunteers and cord blood (cord blood) from normal labor were incubated in the presence of growth factors and AB serum for 13 (cord blood) or 14 (bone marrow) days, respectively, with or without the addition of increasing concentrations of native casein-derived peptides (25-500. mu.g/ml) during the incubation period.

FIG. 7 shows a table depicting the effect of in vitro incubation with casein-derived synthetic peptides on the relative distribution of megakaryocytes, erythrocytes, plasma cells and dendritic cells in CFU-GEMM colonies from murine bone marrow progenitors (differential counts). Cells were scored in macroscopic colonies grown from murine bone marrow cells prepared in a similar manner as previously described for CFU-GEMM colonies. Cells were incubated with hematopoietic factors and 25 μ g or more casein-derived synthetic peptides for 14 days. Differential counts are expressed as a percentage of total cells of each cell type. FIG. 8 shows stimulation of peripheral blood leukocyte reconstitution in myelocleared, bone marrow transplanted mice in response to native casein-derived peptide treatment. Cell count indicates the number of leukocytes (. times.10) ⁴Ml, as counted in a hemocytometer). Mice (n-6/group) received sublethal radiation and syngeneic bone marrow transplantation the next day (10)⁶Individual cells/mouse), 1mg of native casein-derived peptide/receptor (peptide: squares) or 1mg human serum albumin/receptor (control: diamond shaped).

Figure 9 depicts stimulation of platelet reconstitution in myeloablative, bone marrow transplanted mice in response to native casein-derived peptide treatment. Platelet (PLT) counts represent the number of platelets (× 10)⁶Ml, as counted in a hemocytometer). Mice (60 per group) received lethal radiation and were syngeneic bone marrow transplanted on day 1 (10)⁶Individual cells/mouse) and 1mg native casein derived peptide/receptor (peptide, diamonds) or 1mg human serum albumin/receptor (control, squares) administered intravenously.

FIGS. 10a-10f depict the penetration and nuclear uptake of FITC-conjugated native casein-derived peptides by cultured human T lymphocytes as recorded using a fluorescence microscope. F1 and F2 are identical parts of FITC-conjugated native casein-derived peptides. Sup-T was incubated with 100. mu.g/ml FITC-conjugated native casein-derived peptides as described in the examples section below₁A cell. During incubation, cells were washed, freed of labeled material, fixed with formalin, ready for observation using a laser scanning confocal microscope, And (6) recording. FIGS. 10a-10f are images of selected cells during sequential incubations showing penetration of FITC-conjugated native casein-derived peptides through Sup-T₁Cell membrane (FIGS. 10a, 10b) and concentrated in the nucleus (FIGS. 10c-10 f).

FIG. 11 shows a table depicting Sup-T incubated in response to native casein-derived peptides₁Stimulation of lymphocyte proliferation. Incubation of Sup-T with increasing concentrations (50-1000. mu.g/ml) of native Casein-derived peptides₁Cells (5000 per well), counted in the well thereof at a specified time after the culture, and used³H]Thymidine was pulsed for 18 hours. The proliferation index is in a cell cultured with a peptide derived from natural casein³H]The ratio of the average incorporation of thymidine (triplicate samples) to the incorporation of cells not cultured with native casein-derived peptide (control).

FIG. 12 shows a table depicting the inhibition of HIV-1 infected CEM lymphocytes by native casein-derived peptides. As described in the examples section below, CEM cells were either contacted with HIV-1 virus pre-incubated with native casein-derived peptides for 3 hours (3 hours) or pre-incubated with increasing concentrations (50-1000. mu.g/ml) of native casein-derived peptides on their own for the indicated number of hours (24 and 48 hours) prior to contact with HIV-1 virus. At day 15 post-infection, cell numbers were counted and counted by P, as described in the examples section below ²⁴Antigen assays analyze the severity of HIV-1 infection. Control cultures were CEM cells from IF exposed to HIV-1 virus that were not pretreated with native casein-derived peptides, and CEM cells from UIF cultured under the same conditions without native casein-derived peptides and without exposure to HIV-1.

FIG. 13 shows a table depicting the inhibition of HIV-1 infected CEM lymphocytes by casein derived synthetic peptides. As described in the examples section below, CEM cells were contacted (3 hours) with HIV-1 virus pre-incubated for 3 hours with various concentrations (10-500. mu.g/ml) of natural casein-derived synthetic peptides (1P, 3P and 4P). At day 7 post-infection, cell numbers were counted and counted by P, as described in the examples section below²⁴Antigen assayThe severity of HIV-1 infection was analyzed. Control cultures (IF) were CEM cells exposed to HIV-1 virus that had not been pretreated with native casein-derived peptides.

Figure 14 depicts the prevention of type I (IDDM) diabetes in female non-obese diabetic (NOD) mice by native casein derived peptides. In female NOD mice receiving one (triangle and square) or two 100 μ g injections of native casein-derived peptide for 5 weeks (5 or 10 total injections) a week and untreated controls, glucose diabetes was monitored over a period of 365 days post-treatment. All controls developed diabetes and subsequent death of grapes.

Figure 15 depicts that casein-derived synthetic peptides reduced diet-induced high cholesterol/hyperlipidemia in female C57B 1/6 mice. Total Cholesterol (TC), High Density (HDL) and Low Density Lipoprotein (LDL) were determined in pooled blood samples (2 mice per sample) that received (IP) casein derived peptides B, C, 2a or 3P, or untreated (control) mice. The "normal" samples represent control mice that were not fed the atherogenic diet.

FIG. 16 shows a table depicting the stimulation of hematopoiesis in cancer patients in response to injection of native casein-derived peptides. Peripheral blood of 5 female cancer patients undergoing chemotherapy or having undergone chemotherapy, as described above, before (n) and after (n) intramuscular injection of the native casein-derived peptide⁺…), counting the total number of leukocytes (WBC, × 10) respectively³) Total number of platelets (PLT,. times.10)³) Total number of red blood cells (RBC,. times.10)³) And hemoglobin amount (g/dl). Patient 1 is g.t.; patient 2 is e.c.; patient 3 is e.s.; patient 4 is j.r.; patient 5 is d.m.

FIG. 17 depicts native casein-derived peptides stimulating thrombopoiesis in platelet resistant patients of acute myeloid leukemia (M-1). Peripheral platelets (PLT,. times.10) for platelet reconstitution ⁶Per ml) and platelet counts were performed as described above at specified time intervals after intramuscular injection (as described in the examples section below) of 100 μ g native casein-derived peptide.

FIG. 18 depicts native casein-derived peptides stimulating thrombopoiesis in platelet resistant patients of acute myeloid leukemia (M-2). Peripheral platelets (PLT,. times.10) for platelet reconstitution⁶Per ml) and platelet counts were performed as described above at specified time intervals after intramuscular injection (as described in the examples section below) of 100 μ g native casein-derived peptide.

Description of the preferred embodiments

The present invention relates to bioactive peptides derived from or similar to the N-terminus of the α S1 portion of milk casein, compositions containing the bioactive peptides, and methods of using the bioactive peptides, such as stimulating and enhancing immune responses, protecting against viral infections, normalizing serum cholesterol levels, and stimulating hematopoiesis. The casein derived peptides are non-toxic and can be used for the treatment and prevention of, for example, immunological disorders, hypercholesterolemia, hematological disorders and viral-related disorders.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptive text.

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 illustrated in the examples. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology or terminology employed herein is for the purpose of description and should not be regarded as limiting.

The term "treatment" as used herein includes substantial inhibition, delay or reversal of disease progression, substantial amelioration of clinical symptoms of the disease.

The term "prevention" as used herein includes substantially preventing the appearance of clinical symptoms of a disease.

The term "peptide" as used herein includes natural peptides (or degradation products, synthetic peptides or recombinant peptides)Peptides) and peptidomimetics (typically, synthetic peptides), such as peptoids and semipeptoids, which are peptide analogs, may have modifications that, for example, make the peptides more stable in the body. Such modifications include, but are not limited to, cyclization, N-terminal modifications, C-terminal modifications, peptide bond modifications, including, but not limited to CH₂-NH，CH₂-S，CH₂-S＝O，O＝C-NH，CH₂-O，CH₂-CH₂S ═ C-NH, CH ═ CH or CF ═ CH, backbone modifications and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are described in detail in, for example, Quanttitive Drug Design, C.A. Ramsen Gd., Chapter 17.2, F.Choplin Pergamon Press (1992), which is incorporated herein by reference. More details on this are provided below.

Thus, according to the invention, the peptide may be a cyclized peptide. Cyclization can be achieved, for example, by formation of an amino bond, e.g., by incorporation of glutamic acid, aspartic acid, lysine, ornithine, diaminobutyric acid (Dab), diaminopropionic acid (Dap) at various sites in the chain (-CO-NH or-NH-CO bonds). Backbone-to-backbone cyclization can be achieved by incorporating modified amino acids of the formula H-N ((CH)₂)_n-COOH) -C * H-COOH or H-N ((CH)₂)_n-COOH)-C*H-NH₂Where n-1-4, R may be any natural or unnatural side chain of an amino acid.

Cyclization can also be achieved by incorporating two cysteine residues to form an S-S bond. Other side chains may be cyclized with the side chain by reaction of the formula- (-CH)₂-)_n-S-CH₂-C-, wherein n is 1 or 2, can be obtained by, for example, incorporating cysteine or homocysteine, and reacting the free thiol thereof with, for example, bromoacetyl lysine, Orn, Dab or Dap.

Peptide bonds (-CO-NH-) within peptides may be replaced by, for example, N-methyl bonds (-N (CH)₃) -CO), ester linkage (-C * H-C-O-O-C * -N-), ketomethylene linkage (-CO-CH)₂-), α -aza linkage (-NH-N * -CO-), whereinR is any alkyl group, e.g. methyl, carba linkage (-CH) ₂-NH), hydroxyethyl linkage (-CH (OH) -CH₂-), thioamide bond (- -CS- -NH- -), olefinic double bond (- -CH- -), inverse amide bond (- -NH- -CO- -), peptide derivative (N * - -CH- -), and their use in the preparation of pharmaceutical compositions₂-CO), wherein R is a "normal" side chain naturally occurring on a carbon atom.

These modifications may occur at any bond on the peptide chain, and may even occur at several (2-3) positions simultaneously.

Synthetic unnatural acids such as TIC, napthylelasanine (Nol), cyclomethyl derivatives of phenylalanine, phenylalanine or halogenated derivatives of o-methyl tyrosine can be used in place of the natural aromatic amino acids, tryptophan, tyrosine and phenylalanine.

Tables 1-2 below list all natural amino acids (Table 1) and non-traditional or modified amino acids (Table 2).

TABLE 1

Amino acids	Three letter abbreviations	Single letter symbols
			Alanine	Ala	A
Arginine	Arg	R
			Asparagine	Asn	N
Aspartic acid	Asp	D
			Cysteine	Cys	C
Glutamine	Gln	Q
			Glutamic acid	Glu	E
Glycine	Gly	G
			Histidine	His	H
Isoleucine	Ile	I
			Leucine	Leu	L
Lysine	Lys	K
			Methionine	Met	M
Phenylalanine	Phe	F
			Proline	Pro	P
Serine	Ser	S
			Threonine	Thr	T
Tryptophan	Trp	W

Tyrosine	Tyr	Y
			Valine	Val	V
Any of the above amino acids	Xaa	X

TABLE 2

Non-traditional amino acids	Encoding	Non-traditional amino acids	Encoding
				Alpha-aminobutyric acid	Abu	L-N-methylalanine	Nmala
Alpha-amino-alpha-methylbutyric acid	Mgabu	L-N-methyl arginine	Nmarg
				Aminocyclopropane-	Cpro	L-N-methyl asparagine	Nmasn
Carboxylic acid esters		L-N-methyl aspartic acid	Nmasp
				Aminoisobutyric acid	Aib	L-N-methyl cysteine	Nmcys
Amino n-borneol base-	Norb	L-N-methylglutamine	Nmgin
				Carboxylic acid esters		L-N-methyl glutamic acid	Nmglu
Cyclohexylalanine	Chexa	L-N-methylhistidine	Nmhis
				Cyclopentaalanine	Cpen	L-N-methylisoleucine	Nmile
D-alanine	Dal	L-N-methylleucine	Nmleu
				D-arginine	Darg	L-N-methyl lysine	Nmlys
D-aspartic acid	Dasp	L-N-Methylmethionine	Nmmet
				D-cysteine	Dcys	L-N-Methylnorleucine	Nmnle
D-glutamine	Dgln	L-N-methylnorvaline	Nmnva
				D-glutamic acid	Dglu	L-N-methylornithine	Nmorn
D-histidine	Dhis	L-N-Methylphenylalanine	Nmphe
				D-isoleucine	Dile	L-N-methylproline	Nmpro
D-leucine	Dleu	L-N-methylserine	Nmser
				D-lysine	Dlys	L-N-methyl threonine	Nmthr
D-methionine	Dmet	L-N-methyltryptophan	Nmtrp
				D-ornithine	Dorn	L-N-methyl tyrosine	Nmtyr
D-phenylalanine	Dphe	L-N-methylvaline	Nmval
				D-proline	Dpro	L-N-methylethylglycine	Nmetg
D-serine	Dser	L-N-methyl-tert-butylglycine	Nmtbug
				D-threonine	Dthr	L-norleucine	Nle

D-tryptophan	Dtrp	L-norvaline	Nva
				D-tyrosine	Dtyr	Alpha-methyl-aminoisobutyric acid	Maib
D-valine	Dval	Alpha-methyl-gamma-aminobutyric acid	Mgabu
				D-alpha-methylalanine	Dmala	Alpha-methylcyclohexylalanine	Mchexa
D-alpha-methyl arginine	Dmarg	Alpha-methylcyclopentylalanine	Mcpen
				D-alpha-methyl asparagine	Dmasn	Alpha-methyl-alpha-naphthylalanine	Manap
D-alpha-methyl aspartic acid	Dmasp	Alpha-methyl penicillamine	Mpen
				D-alpha-methyl cysteine	Dmcys	N- (4-Aminobutyl) glycine	Nglu
D-alpha-methylglutamine	Dmgln	N- (2-aminoethyl) glycine	Naeg
				D-a-methylhistidine	Dmhis	N- (3-aminopropyl) glycine	Norn
D-alpha-methylisoleucine	Dmile	N-amino-alpha-methylbutyric acid	Nmaabu
				D-alpha-methylleucine	Dmleu	Alpha-naphthylalanine	Anap
D-alpha-methyl lysine	Dmlys	N-Phenylmethylglycine	Nphe
				D-a-methyl methionine	Dmmet	N- (2-carbamoylethyl) glycine	Ngln
D-alpha-methyl ornithine	Dmorn	N- (carbamoyl) glycine	Nasn
				D-alpha-methyl phenylalanine	Dmphe	N- (2-carboxyethyl) glycine	Nglu
D-alpha-methylproline	Dmpro	N- (carboxymethyl) glycine	Nasp
				D-alpha-methylserine	Dmser	N-Cyclobutylglycine	Ncbut
D-alpha-methyl threonine	Dmthr	N-cycloheptylglycine	Nchep
				D-alpha-methyltryptophan	Dmtrp	N-cyclohexyl glycine	Nchex
D-alpha-methyl tyrosine	Dmty	N-cyclodecylglycine	Ncdec
				D-alpha-methylvaline	Dmval	N-cyclododecyl glycine	Ncdod
D-alpha-methylalanine	Dnmala	N-Cyclooctylglycine	Ncoct
				D-alpha-methyl arginine	Dnmarg	N-Cyclopropylglycine	Ncpro
D-alpha-methyl asparagine	Dnmasn	N-cycloundecylglycine	Ncund
				D-alpha-methyl aspartic acid	Dnmasp	N- (2, 2-diphenylethyl) glycine	Nbhm
D-alpha-methyl cysteine	Dnmcys	N- (3, 3-Diphenylpropyl) glycine	Nbhe
				D-N-methylleucine	Dnmleu	N- (3-indolylethyl) glycine	Nhtrp
D-N-methyl lysine	Dnmlys	N-methyl-gamma-aminobutyric acid	Nmgabu
				N-methylcyclohexylalanine	Dmchexa	D-N-Methylmethionine	Dnmmet
D-N-methylornithine	Dnmorn	N-methylcyclopentylalanine	Nmcpen

N-methylglycine	Nala	D-N-Methylphenylalanine	Dnmphe
				N-methylaminoisobutyric acid	Nmaib	D-N-methylproline	Dnmpro
N- (1-methylpropyl) glycine	Nile	D-N-methylserine	Dnmser
				N- (2-methylpropyl) glycine	Nile	D-N-methylserine	Dnmser
N- (2-methylpropyl) glycine	Nleu	D-N-methyl threonine	Dnmthr
				D-N-methyltryptophan	Dnmtrp	N- (1-methylethyl) glycine	Nva
D-N-methyl tyrosine	Dnmtyr	N-methyl-naphthylalanine	Nmanap
				D-N-methylvaline	Dnmval	N-methyl penicillamine	Nmpen
Gamma-aminobutyric acid	Gabu	N- (p-carboxyphenyl) glycine	Nhtyr
				L-tert-butylglycine	Tbug	N- (thiomethyl) glycine	Ncys
L-ethylglycine	Etg	Penicillin amines	Pen
				L-homophenylalanine	Hphe	L-alpha-methylalanine	Mala
L-alpha-methyl arginine	Marg	L-alpha-methyl asparagine	Masn
				L-alpha-methyl aspartic acid	Masp	L-alpha-methyl-tert-butylglycine	Mtbug
L-alpha-methyl cysteine	Mcys	L-methyl ethyl glycine	Metg
				L-alpha-methylglutamine	Mgln	L-alpha-methyl glutamic acid	Mglu
L-alpha-methylhistidine	Mhis	L-alpha-methylhomophenylalanine	Mhphe
				L-alpha-methylisoleucine	Mile	N- (2-methylthioethyl) glycine	Nmet
D-N-methylglutamine	Dnmgln	N- (3-guanidinopropyl) glycine	Narg
				D-N-methyl glutamic acid	Dnmglu	N- (1-carboxyethyl) glycine	Nthr
D-N-methylhistidine	Dnmhis	N- (carboxyethyl) glycine	Nser
				D-N-methylisoleucine	Dnmile	N- (Imidazolyl) glycine	Nhis
D-N-methylleucine	Dnmleu	N- (3-indolylethyl) glycine	Nhtrp
				D-N-methyl lysine	Dnmlys	N-methyl-gamma-aminobutyric acid	Nmgabu
N-methylcyclohexylalanine	Dmchexa	D-N-Methylmethionine	Dnmmet
				D-N-methylornithine	Dnmorn	N-methylcyclopentylalanine	Dnmmet
N-methylglycine	Nala	D-N-Methylphenylalanine	Nmcpen
				N-methylaminoisobutyric acid	Nmaib	D-N-methylproline	Dnmpro
N- (1-methylpropyl) glycine	Nile	D-N-methylserine	Dnmser
				N- (2-methylpropyl) glycine	Nleu	D-N-methyl threonine	Dnmthr
D-N-methyltryptophan	Dnmtrp	N- (1-methylethyl) glycine	Nval

D-N-methyl tyrosine	Dnmtyr	N-methyl-naphthylalanine	Nmanap
				D-N-methylvaline	Dnmval	N-methyl penicillamine	Nmpen
Gamma-aminobutyric acid	Gabu	N- (p-carboxyphenyl) glycine	Nhtyr
				L-tert-butylglycine	Tbug	N- (thiomethyl) glycine	Ncys
L-ethylglycine	Etg	Penicillin amines	Pen
				L-homophenylalanine	Hphe	L-alpha-methylalanine	Mala
L-alpha-methyl arginine	Marg	L-alpha-methyl asparagine	Masn
				L-alpha-methyl aspartic acid	Masp	L-alpha-methyl-tert-butylglycine	Mtbug
L-alpha-methyl cysteine	Mcys	L-methyl ethyl glycine	Metg
				L-alpha-methylglutamine	Mgln	L-alpha-methyl glutamic acid	Mglu
L-alpha-methylhistidine	Mhis	L-alpha-methylhomophenylalanine	Mhphe
				L-alpha-methylisoleucine	Mile	N- (2-methylthioethyl) glycine	Nmet
L-alpha-methylleucine	Mleu	L-alpha-methyl lysine	Mlys
				L-alpha-methyl methionine	Mmet	L-alpha-methyl norleucine	Mnle
L-alpha-methylnorvaline	Mnva	L-alpha-methyl ornithine	Morn
				L-alpha-methyl phenylalanine	Mphe	L-alpha-methylproline	Mpro
L-alpha-methylserine	Mser	L-alpha-methyl threonine	Mthr
				L-alpha-methylvaline	Mtrp	L-alpha-methyl tyrosine	Mtyr
L-alpha-methylleucine	Mval	L-N-Methylphenylalanine	Nmhphe
				N- (N- (2, 2-diphenylethyl) carbamoylmethylglycine	Nnbhm	N- (N- (3, 3-Diphenylpropyl) carbamoylmethyl) (1) glycine	Nnbhe
1-carboxy-1- (2, 2-diphenylethylamino) cyclopropane	Nmbc

The peptides of the invention may be used in individual form or may be part of components, such as proteins, and display components, such as display bacteria and phages. The peptides of the invention may also be chemically modified in a polypeptide chain or covalently cross-linked side chains to become active dimers or multimers.

Furthermore, the peptides of the invention may comprise at least 2, optionally at least 3, optionally at least 4, optionally at least 5, optionally at least 6, optionally at least 7, optionally at least 8, optionally at least 9, optionally at least 10, optionally at least 11, optionally at least 12, optionally at least 13, optionally at least 14, optionally at least 15, optionally at least 16, optionally at least 17, optionally at least 18, optionally at least 19, optionally at least 20, optionally at least 21, optionally at least 22, optionally at least 23, optionally at least 24, optionally at least 25, optionally at least 26, optionally at least 27 and 60, or more amino acid residues (also referred to herein as amino acids).

Thus, it is to be understood that the term "amino acid", as used herein, includes the 20 natural amino acids; these amino acids are typically post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other unusual amino acids, including but not limited to 2-amino fatty acids, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. Furthermore, the term "amino acid" also includes both D-and L-form amino acids.

The term "derived from an N-terminal part of α S1 casein" as used herein refers to peptides as defined by the term herein, e.g., cleavage products of α S1 casein (herein referred to as native casein-derived peptides), synthetic peptides chemically synthesized to correspond to the amino acid sequence of the N-terminal part of α S1 casein (herein referred to as casein-derived synthetic peptides), peptides similar (homologous) to the N-terminal part of α S1 casein and functional homologues thereof, e.g., peptides characterized by substitution of one or more amino acids, such as, but not limited to, permissible substitutions, provided that at least 70%, preferably at least 80%, more preferably at least 90% similarity is maintained. The terms "homologue" and "functional homologue" as used herein refer to a peptide containing insertions, deletions and substitutions which do not affect the biological activity of the peptide.

The term "α S1 casein" as used herein refers to α S1 casein of mammals, including, but not limited to, livestock mammals (e.g., cows, sheep, goats, mares, camels, deer and buffalo), humans and marine mammals. A series of α S1 caseins of known amino acid sequence are provided below, identified by their genbank (ncbi) accession and source: CAA26982 (ovine aries), CAA51022(Capra hircus (goat)), CAA42516(Bos taurus (cow)), CAA55185 (human (Homo sapiens)), CAA38717 (suscrofa (pig)), P09115 (rabbit) and 097943(camelus dromeduus (camel)).

The term "N-terminal portion" as used herein refers to the M amino acids of α S1 casein, derived from the first 60 amino acids of α S1 casein, wherein M may be any integer from 5 to 60 (including integers 5 and 60). Preferably, the term refers to the first M amino acids of α S1 casein.

The peptides of the invention can be obtained by extraction from milk as described above, or by solid phase peptide synthesis, the latter being a standard method known to the person skilled in the art. The peptides of the invention may be subjected to purification procedures, such as High Performance Liquid Chromatography (HPLC), by standard techniques known to those skilled in the art. The cleavage of milk casein to obtain the peptide of the present invention can be carried out using various enzymatic and/or chemical methods.

As described in further detail below and exemplified in the examples section, the peptides of the invention have a variety of therapeutic effects. In the examples section, various assays are provided by which one of ordinary skill in the art, following the teachings of the present invention, can determine whether a particular designed peptide has a particular therapeutic effect. Any of the peptides described herein can be used alone or can be prepared into pharmaceutical compositions for the treatment or prevention of disease. Such compositions include, for example, any of the active ingredients of the peptides described herein, and a pharmaceutically acceptable carrier.

As used herein, "pharmaceutical composition" refers to a formulation that includes one or more of the peptides described herein, as well as other chemical components, such as suitable pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to enable easier application of the compound to an organism.

Hereinafter, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent which does not cause significant irritation to the organism nor affect the biological activity and properties of the compound used. Examples of carriers are, but not limited to: propylene glycol, saline, emulsions and mixtures of organic solvents and water. The term "excipient" as used herein refers to an inert substance added to a pharmaceutical composition to facilitate administration of the compound. Examples of excipients include, but are not limited to: calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for preparing and administering drugs are available in the literature, "Remington's pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

Suitable routes of administration may include, for example, oral, rectal, transmucosal, transdermal, intestinal or parenteral administration, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injections.

The pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Thus, for use in the present invention, the pharmaceutical compositions may be formulated in conventional manner using one or more pharmaceutically acceptable carriers, including excipients and adjuvants, to facilitate incorporation of the active peptide into the pharmaceutical formulation. Suitable formulations depend on the chosen route of administration.

For injection, the peptides of the invention may be formulated in aqueous solution, preferably in physiologically soluble 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 will be used in the formulation. Such penetrants are well known in the art.

For oral administration, the active peptides may be conveniently formulated by combining them with pharmaceutical carriers well known in the art. These carriers can formulate the peptides of the invention into tablets, pills, dragees, capsules, liquids, colloids, syrups, slurries, suspensions and the like, for oral administration to a patient. Pharmaceutical preparations for oral use can be prepared using solid excipients, optionally grinding the mixture obtained, if desired after adding suitable adjuvants, and granulating the mixture 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 corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate, may also be added.

The cores of the dragees are treated with a suitable coating. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, co-solvents and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings to identify different active ingredient dosage compositions or to impart different characteristics to different active ingredient dosage compositions.

Pharmaceutical compositions which can be administered orally include push-fit capsules prepared using gelatin, as well as soft, sealed capsules prepared using gelatin and a plasticizer such as glycerol or sorbitol. Capsules suitable for push-fit can contain the active ingredient in admixture with fillers such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. For 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 also be added. The dosage of all formulations for oral administration should be appropriate for the chosen route of administration.

For buccal administration, the composition may be in the form of tablets or lozenges formulated in a conventional manner.

For administration by inhalation, the peptides of the invention may conveniently be delivered in the form of an aerosol spray presentation from a pressurised pack or nebuliser, using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. For pressurized aerosols, the dosage unit can be determined by delivering the amount of the formulation using a valve. Capsules and pellets of, for example, 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 also be formulated for parenteral administration, such as by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an optional preservative. The compositions may be in the form of 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 agents in water-soluble form. In addition, suspensions of the active peptides can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may contain suitable stabilizers or agents that increase the solubility of the peptide, and highly concentrated solutions may be prepared.

In addition, the active ingredient may be presented as a powder for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The peptides of the invention may also be formulated into compositions for rectal use, for example as suppositories or retention enemas, for example, conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions described herein may also include suitable solid gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

Optimal dosages and methods of quantification of any of the peptides of the invention can be readily determined by one of ordinary skill in the art.

Any peptide used in accordance with the teachings of the present invention, in a therapeutically effective amount, also referred to as a therapeutically effective dose, can be initially estimated by methods of cell culture or in vivo animal testing. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC measured in cell culture₅₀Or IC₁₀₀. This information can be used to more accurately determine the dosage applied in humans. The starting dose can also be estimated from in vivo data. Based on these initial guidelines, one of ordinary skill in the art will be able to determine an effective dosage for 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 measuring LD₅₀And ED₅₀. The dose ratio of toxic to therapeutic effect is the therapeutic index, and LD can be used₅₀And ED₅₀The ratio therebetween. Peptides with high therapeutic indices are preferred. Using data from cell culture assays and animal studies, a non-toxic dose range for human use can be constructed. This is achieved byThe dosage of these peptides is preferably such that ED is included₅₀With little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration. The particular formulation, route of administration and dosage may be selected by a physician according to the condition of the patient (see, e.g., Fingl et al, 1975, In: the pharmacological Basis of Therapeutics, chapter 1, page 1).

The dosage and interval of administration should be adjusted to the individual condition so that the plasma levels of the active ingredient are sufficient to maintain the therapeutic effect. Typically, the dosage for oral administration to a patient will range from about 1 to about 1000 mg/kg/dose, usually from about 10 to about 500 mg/kg/dose, preferably from about 20 to about 300 mg/kg/dose, and most preferably from about 50 to about 200 mg/kg/dose. In some cases, therapeutically effective serum levels may be achieved by multiple doses per day. In the case of topical administration or selective uptake, the effective local concentration of the drug may not be related to the plasma concentration. One skilled in the art can optimize the local therapeutically effective dose without undue experimentation.

Depending on the severity of the condition to be treated and the response to treatment, administration may be a single administration of the sustained release composition, with the course of treatment lasting from several days to several weeks, or until cure or remission of the disease state is achieved.

The amount of the composition administered will, of course, depend on the subject being treated, the severity of the disease, the mode of administration, and the judgment of the prescribing physician, among other factors.

If desired, the compositions of the present invention may be presented in a packaging or dispensing device, such as an FDA approved kit, which may include one or more unit dosage forms containing the active ingredient. The packaging may comprise, for example, metal or plastic foil, such as blister packs. The packaging or dispensing device may be present with instructions for placement of the medication. The package or dispenser device may also be accompanied by a notice affixed to the container in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is in the form of approval by the agency of the composition or for human or livestock use. For example, the notification may be a label for a prescription drug approved by the U.S. food and drug administration or an approved product insert. The compositions may also be prepared to contain a peptide of the invention formulated in a pharmaceutically acceptable carrier, placed in a suitable container and indicated for use in the treatment or prevention of a certain indication or induction of a certain desired event. Suitable indications on the label may include treatment and/or prevention of autoimmune diseases or conditions, viral diseases, viral infections, hematological diseases, hematological defects, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, glucosuria, hyperglycemia, diabetes, aids, HIV-1, helper T cell disorders, dendritic cell defects, macrophage defects, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, immune system disorders resulting from chemotherapy or radiotherapy, human immune system disorders resulting from treatment of diseases with immunodeficiency and bacterial infections.

The pharmaceutical compositions of the present invention may be used to maintain and/or restore blood system architecture, balance blood cell counts, balance the levels of various metabolites in the blood, including sugars, cholesterol, calcium, uric acid, urea, and enzymes such as alkaline phosphatase. Furthermore, the pharmaceutical composition of the invention may also be used for inducing blood cell proliferation, regulating white blood cell and/or red blood cell count, in particular increasing white blood cell and/or red blood cell count, increasing blood hemoglobin level, and regulating platelet count.

The term "balance" as used herein in relation to certain physiological parameters means that the level of the parameter in question is changed so that it is closer to normal.

The term "normal value" as used herein in relation to certain physiological parameters refers to a value in the range of values for a healthy human or animal.

In a particularly preferred embodiment, the peptides of the invention can balance red blood cell, white blood cell, platelet and hemoglobin levels. The pharmaceutical composition of the present invention can be used for activating blood cell proliferation.

In addition, the pharmaceutical compositions may be used to treat and/or prevent hematopoietic stem cell diseases, including platelet, lymphocyte, plasma cell, and neutrophil diseases, as well as deficiencies and dysfunctions in pre-leukemic and leukocyte conditions, and thrombocytopenia.

Furthermore, the pharmaceutical composition may be used for the treatment and/or prevention of cell proliferative disorders. In this regard, it is noted that the pharmaceutical compositions of the present invention are advantageous in stimulating immune responses, reducing adverse effects, reducing emesis induced by chemotherapy and radiotherapy, and promoting rapid recovery during chemotherapy or radiotherapy.

Furthermore, the pharmaceutical compositions of the present invention may be used to stimulate an immune response in humans during the treatment of immunodeficiency associated diseases, such as HIV and autoimmune diseases.

The compositions of the invention may also be used in the veterinary field.

The pharmaceutical compositions of the invention may be used to treat and/or prevent, for example, diseases involving abnormal blood cell levels, diseases involving abnormal hematopoietic stem cell production and differentiation, treating platelet, lymphocyte and/or neutrophil disorders, treating pre-leukemic and leukemic conditions, and treating thrombocytopenia. The pharmaceutical compositions of the invention may also be used to treat cell proliferative disorders and diseases involving immunodeficiency, such as HIV, as well as autoimmune diseases. Furthermore, the pharmaceutical compositions of the present invention may be used to stimulate an immune response during chemotherapy or radiotherapy, for example, to reduce chemotherapy-related emesis.

When the peptides of the invention are used in practice, it was unexpectedly observed that the peptides of the invention exert a synergistic effect on human hematopoietic stem cell proliferation and differentiation upon the addition of other hematopoietic growth factors. Of particular interest is the stimulation and dose-dependent enhancement of erythropoietin-mediated erythropoiesis colony formation by the peptides of the invention to induce megakaryocyte proliferation by Thrombopoietin (TPO). Recombinant human (rh) EPO has been approved for the treatment of indications such as renal anemia, anemia of prematurity, cancer and AIDS-related anemia, as well as for pre-operative treatment at selective times (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 by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for increasing the effect of erythropoietin by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

Thrombopoietin is an early acting cytokine with important multi-cell lineage effects: TPO alone or in combination with other early acting cytokines can (I) promote progenitor cell viability and inhibit progenitor apoptosis; (ii) regulating hematopoietic stem cell production and function; (iii) triggering cell division of resting pluripotent cells; (iv) (iv) inducing differentiation of the multi-cell line and (v) enhancing the formation of multi-cell line colonies comprising granulocytes, erythrocytes, macrophages and megakaryocytes (MK, CFU-GEMM). Moreover, TPO stimulates the production of more limited progenitors of granulocytes/monocytes, megakaryocytes and erythroid colonies, and stimulates the adhesion of primitive human bone marrow and megakaryocytes to fibronectin and fibrinogen. Thus, TPO is an important cytokine for clinical hematologists/transplanters: for mobilization, expansion 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 plasmapheresis yields. However, clinical application of TPO therapy is complicated by other considerations, such as the relatively high price of the recombinant human cytokine rhTPO, and the potential antigenicity of repeated doses of TPO.

Combination therapy of TPO and a peptide of the invention, either administered together in a pharmaceutical composition comprising both, or administered separately, can provide an inexpensive, proven non-toxic enhancement of TPO to the proliferation and function of target cells. In the combination, the peptides of the present invention can be used for the treatment of myelodysplastic syndrome (MDS), aplastic anemia, and complications of liver failure, in addition to the above-mentioned conditions. Pretreatment of platelet donors with the peptides of the invention, alone or in combination with TPO, can even enhance plasmapheresis yields.

According to the present invention, there is also provided a method of treating a thrombopoietin-treatable condition by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for increasing the effect of thrombopoietin by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from the N terminus portion of α s1 casein.

According to the present invention, there is also provided a method of enhancing peripheral stem cell mobilization by administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of thrombopoietin and a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a pharmaceutical composition for treating a thrombopoietin-treatable condition, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for increasing the effect of thrombopoietin, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for enhancing peripheral stem cell mobilization, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing hematopoiesis, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing the proliferation of hematopoietic stem cells, which comprises, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing proliferation and differentiation of hematopoietic stem cells, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing megakaryocytopoiesis, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing erythropoiesis, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing leukopoiesis, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing thrombopoiesis, which comprises, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating thrombocytopenia, which comprises, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also 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 α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating granulocytopenia, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention there is also provided a pharmaceutical composition for the treatment or prevention of an indication selected from the group consisting of: a hematologic disease, a defect in the blood system, thrombocytopenia, pancytopenia, granulocytopenia, dendritic cell deficiency, macrophage deficiency, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, myelodysplastic syndrome, aplastic anemia, and bone marrow deficiency, comprising, as active ingredients, a peptide derived from the N-terminal portion of thrombopoietin and α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention there is also provided a pharmaceutical composition comprising thrombopoietin and a purified peptide having an amino acid sequence selected from the group consisting of sequences 1-25 and a pharmaceutically acceptable carrier.

According to the present invention there is also provided a method of enhancing the colonization of donated blood stem cells in a myeloablative recipient by treating a donor of the donated blood stem cells with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to donation and implantation of the donated blood stem cells in the recipient.

According to the present invention there is also provided a method of enhancing the colonization of donated blood stem cells in a myeloablative recipient by treating the donated blood stem cells with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to implantation of the donated blood stem cells in the recipient.

According to the present invention there is also provided a method of enhancing the colonization of blood stem cells in a myeloablative recipient by treating the blood stem cells with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to implantation of the blood stem cells in the recipient.

According to the present invention there is also provided a method of enhancing the colonization of donated blood stem cells in a myeloablative recipient by treating the donated blood stem cells with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to implantation of the donated blood stem cells in the recipient.

According to the present invention there is also provided a method of enhancing the colonization of blood stem cells in a myeloablative recipient by treating the blood stem cells with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to implantation of the blood stem cells in the recipient.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for the treatment of a thrombopoietin-treatable condition.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for increasing the effect of thrombopoietin.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing the colonization of provided blood stem cells in a myeloablative recipient.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing blood stem cell colonization in a myeloablative receptor.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing peripheral stem cell mobilization.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing hematopoiesis.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing hematopoietic stem cell proliferation.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing the proliferation and differentiation of hematopoietic stem cells.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing megakaryocytopoiesis.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing erythropoiesis.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing leukopoiesis.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing thrombopoiesis.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for the prevention or treatment of thrombocytopenia.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for the prevention or treatment of pancytopenia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating an indication selected from the group consisting of: autoimmune diseases or conditions, viral diseases, viral infections, hematologic diseases, hematologic defects, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, diabetes mellitus, hyperglycemia, diabetes mellitus, aids, HIV-1, helper T cell disorders, dendritic cell defects, macrophage defects, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, human immune system disorders resulting from disease treatment of immune deficiencies and bacterial infections.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating an indication selected from the group consisting of: hematologic disorders, hematologic deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, dendritic cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, myelodysplastic syndromes, aplastic anemia, and bone marrow deficiencies.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for enhancing the colonization of provided blood stem cells in a myeloablative recipient.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for enhancing the colonization of blood stem cells in a myeloablative recipient.

According to the present invention, there is also disclosed the use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for enhancing peripheral stem cell mobilization.

The invention further relates to antibacterial pharmaceutical compositions comprising as active ingredient at least one peptide of the invention, and to the use of the peptides of the invention as antibacterial agents.

As described in detail in the examples section below, the peptides of the present invention and pharmaceutical compositions comprising the peptides of the present invention as an active ingredient can be used for the treatment and prevention of blood cell diseases, cell proliferative diseases, diseases involving immunodeficiency, and autoimmune diseases.

According to the present invention, there is provided a method for preventing or treating an autoimmune disease by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein to a subject in need thereof.

According to the present invention, there is also provided a method for preventing or treating a viral disease by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein to a subject in need thereof.

According to the present invention, there is also provided a method of inducing hematopoiesis by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing hematopoietic stem cell proliferation by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for inducing proliferation and differentiation of hematopoietic stem cells by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein to a subject in need thereof.

According to the present invention, there is also provided a method of inducing megakaryocytopoiesis by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing erythropoiesis by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing leukopoiesis by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing thrombopoiesis by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing plasma cell proliferation by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing dendritic cell proliferation by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method of inducing macrophage proliferation by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for preventing or treating thrombocytopenia by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for preventing or treating pancytopenia by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for preventing or treating granulocytopenia by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for preventing or treating hyperlipidemia by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

According to the present invention, there is also provided a method for preventing or treating hypercholesterolemia by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for preventing or treating glucosuria by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein to a subject in need thereof.

According to the present invention, there is also provided a method for preventing or treating diabetes by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for preventing or treating aids by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein to a subject in need thereof.

According to the present invention, there is also provided a method for preventing or treating HIV infection by administering a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein to a subject in need thereof.

According to the present invention, there is also provided a method for preventing or treating a condition associated with myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT) by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of aS1 casein.

According to the present invention, there is also provided a method of treating an erythropoietin-treatable condition by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for increasing the effect of erythropoietin by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method of treating a thrombopoietin-treatable condition by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a method for increasing the effect of thrombopoietin by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from the N terminus portion of α s1 casein.

According to the present invention, there is also provided a method of enhancing peripheral stem cell mobilization by administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α s1 casein.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating an autoimmune disease, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating viral diseases, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing viral infection, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing hematopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing hematopoietic stem cell proliferation, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing proliferation and differentiation of hematopoietic stem cells, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing megakaryocytopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing erythropoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing leukopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing thrombopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing proliferation of plasma cells, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing proliferation of dendritic cells, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for inducing macrophage proliferation, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating thrombocytopenia, which comprises, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also 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 α S1 casein and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating granulocytopenia, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating hyperlipidemia, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating hypercholesterolemia, which comprises, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating glucosuria, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating diabetes, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating aids, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating HIV infection, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

According to the present invention, there is also provided a pharmaceutical composition for preventing or treating a condition associated with myeloablative doses of radiotherapy and chemotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT), comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein and a pharmaceutically acceptable carrier.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of autoimmune diseases.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of viral diseases.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention of viral infection.

According to the present invention, also disclosed is the use of a peptide derived from an N terminus portion of α S1 casein for inducing hematopoiesis.

According to the present invention, also disclosed is the use of a peptide derived from an N terminus portion of α S1 casein for inducing hematopoietic stem cell proliferation.

According to the present invention, also disclosed is the use of a peptide derived from an N terminus portion of α S1 casein for inducing hematopoietic stem cell proliferation and differentiation.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for inducing megakaryocytopoiesis.

According to the present invention, also disclosed is the use of a peptide derived from an N terminus portion of α S1 casein for inducing erythropoiesis.

According to the present invention, also disclosed is the use of a peptide derived from an N terminus portion of α S1 casein for inducing leukopoiesis.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for inducing thrombopoiesis.

According to the present invention, also disclosed is the use of a peptide derived from an N terminus portion of α S1 casein for inducing plasma cell proliferation.

According to the present invention, also disclosed is the use of a peptide derived from an N terminus portion of α S1 casein for inducing dendritic cell proliferation.

According to the present invention, also disclosed is the use of a peptide derived from an N terminus portion of α S1 casein for inducing macrophage proliferation.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of thrombocytopenia.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of pancytopenia.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of granulocytopenia.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of hyperlipidemia.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of hypercholesterolemia.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of glucosuria.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of diabetes.

According to the invention, the application of the peptide derived from the N terminal part of the alpha S1 casein in preventing or treating AIDS is also disclosed.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of HIV infection.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the prevention or treatment of conditions associated with myeloablative doses of radiotherapy and chemotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT).

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for the treatment of a thrombopoietin-treatable condition.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for increasing the effect of thrombopoietin.

According to the present invention, also disclosed is the use of a peptide derived from an N terminus portion of α S1 casein for enhancing peripheral stem cell mobilization.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for enhancing the colonization of provided blood stem cells in vivo in a myeloablative receptor.

According to the present invention, there is also disclosed the use of a peptide derived from an N terminus portion of α S1 casein for enhancing blood stem cell colonization in vivo in a myeloablative receptor.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating an autoimmune disease.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating viral diseases.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating viral infection.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing hematopoiesis.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cell proliferation.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing proliferation and differentiation of hematopoietic stem cells.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing megakaryocytopoiesis.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing erythropoiesis.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing leukopoiesis.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing thrombopoiesis.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing proliferation of plasma cells.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing dendritic cell proliferation.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing macrophage proliferation.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating thrombocytopenia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating pancytopenia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating hyperlipidemia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating hypercholesterolemia.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating glucosuria.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating diabetes.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating aids.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating HIV infection.

According to the present invention, there is also disclosed a use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating a condition associated with myeloablative doses of radiotherapy supported with autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT).

According to the present invention, there is also provided a purified peptide having an amino acid sequence selected from the group consisting of sequences 1-25.

According to the present invention there is also provided a pharmaceutical composition comprising a purified peptide having an amino acid sequence selected from the group consisting of sequences 1-25 and a pharmaceutically acceptable carrier.

The present invention successfully overcomes the deficiencies of the currently known treatments by providing peptides for the treatment of human diseases, which peptides are derived from the N-terminal part of α S1 casein, have no detectable toxicity and have a high therapeutic effect.

Other objects, advantages and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to limit the invention. Furthermore, experimental support may be found in the following examples, in connection with the various embodiments and aspects of the invention described above and in the claims that follow.

Examples

The invention is illustrated by way of non-limiting example with reference to the following examples, which together with the above description.

Materials and Experimental methods

Preparation of native casein-derived peptides: casein fractions were isolated from cow milk and subjected to complete proteolytic digestion at 30 ℃ using rennet (20ng/ml) as described by Hipp et al (1952), ibid. After the reaction is complete, the solution is heated to inactivate the enzyme, and then acidified with an organic acid, acetic acid or trichloroacetic acid to precipitate the digestive juice as paracaseinate. The paracaseinate salt is separated by centrifugation and the supernatant fraction containing the peptide fragment of interest is precipitated with a higher concentration of acid to precipitate the caseicidin. The obtained caseicidin was lyophilized after resuspension, dialysis and neutralization. The powder formulations obtained were tested for biological activity and separated by HPLC for peptide analysis as described below.

HPLC analysis of native casein-derived peptides: the native casein derived peptides obtained as described above were separated in two stages using HPLC. Initially, the lyophilized casein digest was separated using C18 reverse phase using a 0.1% aqueous trifluoroacetic acid (w/w) -acetonitrile gradient. Detection was based on UV absorption at 214 nm. Then, the sample was analyzed by an HPLC-Mass Spectrometry (MS) method equipped with an electron emission source. The mass calculation represents the mass of the ionized peptide sample over the retention time. After isolation, the amino acid composition of the peptides was determined using a gas phase microsequencer (applied biosystems 470A).

The following are representative data: 8 typical peptide peaks were observed, with 3 major peaks having Rt values of 17.79, 19.7, 23.02, and 5 minor peaks having Rt values of 12.68, 14.96, 16.50, 21.9 and 25.1, representing molecular weights of 2764, 1697, 1880, 2616, 3217, 2333, 1677 and 1669Da, respectively. A23 amino acid peptide main peak with an Rt value of 17.79 (corresponding to 2764Da) represents 1-23 amino acids of α S1 casein, with a sequence of RPKHPIKHQGLPQEVLNENLLRF (sequence 22, see McSweeny et al, 1993, ibid, for the complete sequence of α S1 casein). Other peptides were derived from the 208-224 sites of beta casein, 16-37 sites of alpha S1 casein and the 197-222 sites of alpha S2-like casein precursor. Other peptides are also present.

Synthesis of casein-derived peptides: peptides of increasing length, corresponding to the N-terminal 2-26 amino acids of α S1 casein, were synthesized by NoVetide ltd, Haifa, Israel, with a purity > 95% (HPLC). The quality control comprises the following steps: HPLC, mass spectrometry (EI), amino acid analysis and peptide content. The sequences of these peptides are provided in table 3 below.

TABLE 3

Number of amino acids in name sequence (N-terminal-C-terminal)

74 RP 2 1

1P RPK 3 2

2P RPKH 4 3

3P RPKHP 5 4

4P RPKHPI 6 5

5P RPKHPIK 7 6

Y RPKHPIKH 8 7

X RPKHPIKHQ 9 8

1a RPKHPIKHQG 10 9

2a RPKHPIKHQGL 11 10

3a RPKHPIKHQGLP 12 11

A RPKHPIKHQGLPQ 13 12

B RPKHPIKHQGLPQE 14 13

C RPKHPIKHQGLPQEV 15 14

D RPKHPIKHQGLPQEVL 16 15

E RPKHPIKHQGLPQEVLN 17 16

F RPKHPIKHQGLPQEVLNE 18 17

G RPKHPIKHQGLPQEVLNEN 19 18

H RPKHPIKHQGLPQEVLNENL 20 19

I RPKHPIKHQGLPQEVLNENLL 21 20

J RPKHPIKHQGLPQEVLNENLLR 22 21

K RPKHPIKHQGLPQEVLNENLLR 23 22

F

L RPKHPIKHQGLPQEVLNENLLR 24 23

FF

M RPKHPIKHQGLPQEVLNENLLR 25 24

FFV

N RPKHPIKHQGLPQEVLNENLLR 26 25

FFVA

Juvenile form (type I, IDDM) diabetes in non-obese diabetic (NOD) mice:

native casein-derived peptides: NOD mice are a common animal model for studying autoimmune diseases and human juvenile diabetes. Female NOD mice of 6 weeks of age were injected once or twice a week with 100 μ g native casein-derived peptide for a total of 5 or 10 treatments. Control mice received no treatment. Disease severity was determined based on grape diabetes using Combi paper [ Gross, d.j.et al (1994), diabetes, 37: 1195]. Results are expressed as the percentage of non-grape diabetic mice per sample group over the 365 day period.

Casein derived synthetic peptides: in another experiment, 6 week old female NOD mice were treated 5 times weekly with 100 μ g of casein-derived synthetic peptide. Control mice received no treatment. Results are expressed as the number of healthy mice in each treatment group.

Intraperitoneal glucose tolerance test (IPGTT): glucose tolerance tests are established methods for studying glucose metabolism and the tendency to diabetes in mammals. Responses to glucose load were assessed 25 weeks after receiving the casein-derived synthetic peptide using an intraperitoneal glucose tolerance assay. 1g/kg body weight of glucose was injected. The glucose values of the blood drawn before the test (0 min) and 60 min after the load were determined. Plasma glucose levels were determined with glucose analyzer 2(Beckman Instruments, Fullerton, Calif.) and expressed in mmol/L. Normal values do not exceed 140 mmol/L.

Stimulation of Natural Killer (NK) cell proliferation:

derived from human Peripheral Blood Stem Cells (PBSC). PBSC of an individual was treated with G-CSF by centrifugation using FICOLL gradient, and then the cells were washed twice with RPMI-1640 medium and seeded in 1.5ml wells with or without the indicated natural casein-derived peptide or casein-derived synthetic peptide (0-500. mu.g/ml). After 2 days of incubation, detection was carried out³⁵S-labeled K562 target cells (NEG-709A, 185.00MBq, 2.00mCi EASYTAGth Methionine, L-, [ 2 ]³⁵S]43.48 TBq/mmol, 1175.0Ci/mmol, 0.488ml, Boston USA) to determine the natural killer activity of the cells. In 96 well tissue culture plates with U-shaped bottom, each well is 5X 10 ³Target cells, with two concentrations of effector cells (2.5X 10 per well)⁵And 5 × 10⁵Cells) were incubated together (effector cells: target cells ratio 50: 1 and 100: 1). At 37 ℃ 5% CO₂Cells were incubated for 5 hours in 95% air and then centrifuged at 1000rpm for 5 minutes. Assay in 50. mu.l sample supernatant³⁵The amount of S released.

Mouse Bone Marrow (BM) cell origin: bone marrow was collected from untreated BALB/C and C57B1/6 mice. Bone marrow was harvested by injecting the mouse hind and hind limb long bones with medium using a 25 Gauge needle. The aspirated cells were washed with RPMI 1640, counted in a hemocytometer, stained as necessary (20. mu.l cells in 380. mu.l acetic acid/trypan blue), and then applied at 16402-5X 10 per ml RPMI⁶Concentration of individual cells were seeded in culture flasks, RPMI 1640 containing 10% fetal bovine serum, antibiotics and glutamine, with or without 100 μ g/ml native casein-derived peptides. At 37℃，5％ CO₂95% air for 12-15 days, then collecting the cells by centrifugation at 1500rpm for 10 minutes, counting, and inoculating in a medium containing⁵¹Cr (chromium-51, 740MBq, 2.00mCi activity) or³⁵S(NEG-709A，185.00MBq，2.00mCi EASYTAGth Methionine，L-[³⁵S]43.48TBq/mmol, 1175.0Ci/mmol, 0.488ml, Boston USA)) labelled murine lymphoma (YAC) in a U-bottom culture well at an effector to target ratio of 25: 1 or 50: 1. NK activity is expressed as a percentage of radioactivity in cell-free supernatant.

Proliferation of human cells in culture: peripheral Blood (PB) is collected from a healthy or diseased patient. Prior to plasmapheresis, the patient is not treated with any treatment other than G-CSF. Bone Marrow (BM) cells are aspirated from an informed consented healthy patient or a patient in remission following chemotherapy. Cord blood was collected during normal labor. Human cells of various origins were separated by centrifugation on FICOLL gradient, washed 2 times with RPMI-1640 medium, and seeded at the indicated concentration in 0.2ml flat-bottomed tissue culture wells with or without native casein-derived peptides or with or without casein-derived synthetic peptides. All treatments were repeated 3 times, including the control group. Cell proliferation by H³T incorporation assay: after incubation for a prescribed number of days, radioactive thymidine [ thymidine (methyl-, [ 2 ]) is added³H]Specific Activity 5Ci/ml 37MBq/ml, ICN Corp.)]To detect. The cells are then harvested after 16-20 hours incubation with the marker and washed with medium. Incorporated radioactivity was measured using a beta scintillation counter.

K562 leukemia and proliferation of colon cancer cell lines: colon and K562 are established tumor cell lines capable of growth in culture. Both cell lines were cultured in flasks at 37 ℃ with 5% CO ₂95% air at 4X 10 per well⁵Individual cell (K562) or 3X 10³Individual cells (colon) were harvested and washed 2 times with culture medium before being seeded into tissue culture wells. Native casein-derived peptides were added to the wells at the indicated concentrations and after 9 days (K562) or 3 days (colon) incubation, the standards as described above were addedThymidine is noted. Cells were harvested as described above and the amount of radioactivity uptake was determined.

NK and T cell proliferation was determined using fluorescent antibodies in human Peripheral Blood Stem Cells (PBSC):

peripheral Blood Stem Cells (PBSC) were collected from human subjects treated with G-CSF by plasmapheresis, the cells were separated by centrifugation using FICOLL gradient, washed 2 times using RPMI-1640 medium containing 10% fetal bovine serum, and then cultured in a flask at 37 ℃ and 5% CO₂Cells were incubated at 95% air conditions with or without the indicated concentration of native casein-derived peptides in the flasks. anti-CD application after 10, 14 or 28 days incubation with native casein-derived peptides₃Fluorescent antibodies (CD)₃/FITC clone UHCT1), anti-CD₅₆Fluorescent antibodies (CD)₅₆RPE clone MOC-1) (DAKO A/S, Denmark) and as controls mouse IgG1/RPE and IgG1/FITC antibodies, emerging T Cells (CD) were detected by direct immunofluorescence ₃Surface antigen) and NK Cells (CD)₅₆Surface antigens). Detection of fluorescently labeled cells was performed using Fluorescence Activated Cell Sorting (FACS).

Stimulation of hematopoiesis in cultured Bone Marrow (BM) cells:

proliferation of megakaryocytes in murine myelopluripotent colonies (CFU-GEMM): in serum-free methylcellulose IMDM medium, 5% CO at 37 ℃₂95% air culture of Primary bone marrow cells (1X 10) derived from 8-12 week old C3H/HeJ mice⁵/ml) for 8-9 days. The medium is very suitable for the growth of pluripotent colonies (CFU-GEMM) and contains 1% BSA (Sigma), 10^-4M thioglycerol (Sigma), 2.8X 10^-4M human transferrin (TF, Biological industries, Israel), 10% WEHI-CM as a source of IL-3, and 2 units/ml erythropoietin (rhEPO, R)&D Systems, Minneapolis). After 8-9 days, colonies were scored using an Olympus dark field microscope. Colonies were removed using a micropipette, centrifuged, stained with May-Grunwald-Giemsa, and counted by classification. At least 700 cells were counted at a time.

Proliferation of dendritic cells in CFU-GEMM: pluripotent (CFU-GEMM) colonies grown from primary bone marrow cells as described above for the megakaryocyte proliferation assay described above were collected, stained and dendritic cells were counted. At least 700 cells were counted for each preparation.

Proliferation of plasma cells in CFU-GEMM: pluripotent (CFU-GEMM) colonies grown from primary bone marrow cells as described above for the megakaryocyte proliferation assay described above were collected, stained and plasma cells were counted. At least 700 cells were counted for each preparation.

Proliferation of erythrocytes in CFU-GEMM: pluripotent (CFU-GEMM) colonies grown from primary bone marrow cells as described above for the megakaryocyte proliferation assay described above were collected, stained and red blood cells counted. At least 700 cells were counted for each preparation.

Proliferation of polymorphonuclear cells (PMN) in CFU-GEMM: pluripotent (CFU-GEMM) colonies grown from primary bone marrow cells as described above for the megakaryocyte proliferation assay described above were collected, stained and the polymorphonuclear cells were counted. At least 700 cells were counted for each preparation.

Proliferation of megakaryocytopoietic and erythrocytopoietic cells in human bone marrow and umbilical cord blood: histopaque-107(Sigma Diagnostics) density gradient separation was performed using healthy human bone marrow samples to obtain a purified Monocyte (MNC) population. Colony assays were performed in plate media containing methylcellulose at a final concentration of 0.92% (4000centripase powder, Sigma Diagnostics), rehydrated in Iscoves 'modified Dulbecco's medium containing 36mM sodium bicarbonate (Gibco), 30% Fetal Bovine Serum (FBS) (Hyclone), 0.292mg/ml glutamine, 100 units/ml penicillin and 0.01mg/ml streptomycin (Biological Industries, wait Haemek). Cord blood from normal labor was collected and processed as described above.

Will contain 10⁵MNC/ml colony assay medium was plated in 24 well tissue culture plates (Greiner), 0.33ml per well, in 3 replicates. At 37 ℃ 5% CO₂95% emptyCultures were incubated under air and 55% relative humidity with or without the indicated concentrations of native casein-derived peptides or casein-derived synthetic peptides per well. After 14 days, plates were scored for colonies containing more than 50 cells. Megakaryocytes are identified by an indirect immunofluorescence method by using a highly specific rabbit antibody for identifying human platelet glycoprotein and FITC-conjugated goat anti-rabbit IgG. The growth factors added included 15ng/ml leucomax (GM-CSF) (Sandoz Pharmma) and 5% v/v human phytohemagglutinin-m (Difco Lab) induced Conditioned Medium (CM), inducing the development of granulocytic, monocytic colonies (CFU-GM). Erythropoietin (EPO) was used at 2 units/ml to induce erythroid colony formation (burst colony forming unit-erythrocyte-BFU-E).

In addition, human bone marrow cells from informed volunteer donors or patients undergoing autologous bone marrow transplantation were pre-cultured in medium containing 10-1000. mu.g/ml native casein-derived peptide, grown in semi-solid agar, and scored for granulocyte-macrophage hematopoietic colonies (GM-CFU) after 7 or 14 days of treatment.

Megakaryocytopoiesis of normal bone marrow cells from an informed healthy donor can be determined by assessing the number of megakaryocytes in a sample in liquid medium (RPMI-1640 plus 10% human AB serum, glutamine and antibiotics) with or without 100. mu.g/ml native casein-derived peptide, and by assessing colony formation using a methylcellulose assay. 2 x 10 to⁵Individual bone marrow cells were seeded in a standard growth factor combination (medium) with or without native casein-derived peptides. In the methylcellulose assay, megakaryocytes are counted 12-14 days after seeding using an inverted microscope.

Clinical trials using native casein-derived peptides: in a series of experiments, a single dose of the drug containing 50mg of native casein-derived peptide was administered to human subjects intramuscularly over a 2 hour period, dividing into 3 injections. Clinical parameters were monitored at specified intervals. In other trials, patients at different stages of cancer and metastatic disease treatment and/or remission were treated once or twice with native casein-derived peptides and monitored for changes in peripheral blood counts.

Inhibition of HIV infection of human lymphocytes in vitro:

Peptide: peptides provided in lyophilized powder form (either native casein-derived peptides or casein-derived synthetic peptides (2-26 amino acids in length, see table 3) were resuspended in RPMI complete medium and added to the cell culture medium at a final concentration of 50-1000 μ g/ml.

Cell: several freshly isolated human cells (primary cells) and cell lines are known to be susceptible to HIV-1 infection in vitro, despite almost any CD₄Cells with very low molecular surface levels are considered potential targets for HIV-1 infection. Two commonly used human cell lines, CEM and Sup-T1, which are highly sensitive to HIV-1 infection, were selected.

CEM is a human T4 lymphoblastoid cell line originally derived from g.e.foley et al [ (1965), Cancer 18: 522] peripheral blood buffy coat of a 4 year old white girl with acute lymphoblastic leukemia. These cells are maintained in suspension continuously in culture and are widely used for analysis of infections, antiviral agents and neutralizing antibodies.

Sup-T1 is a human T lymphoblastoid cell line isolated from pleural fluid of an 8 year old boy with non-hodgkin's T cell lymphoma [ Smith, s.d.et al (1984) cancer research 44: 5657]. The cells express high levels of surface CD ₄It is useful in studying cell fusion, cytopathic effects and HIV-1 infection. Sup-T1 cells were grown in suspension in enrichment medium.

Culture medium: cells were grown in RPMI-1640 complete medium, which was also enriched with 10% fetal bovine serum, 2mM glutamine and 2mM penicillin-streptomycin (GIBCO).

Virus: the HIV strain used was HIV-1IIIB, initially designated HTLV-IIIB. Concentrated cultures of peripheral blood from several patients with AIDS or related diseases were used to establish persistent productive infections in H-9 cells. This subtype B virus is highly replication competent in human T cell lines. The virus titer of the stock solution was 5.38 ng/ml.

FITC-labeled peptide: FITC F-1300 (fluorescein isothiocyanato, isomer 1, Sigma (F25o-2) St.Louis, MI, USA) was used, with maximum excitation/emission at approximately 494/520nm, respectively. Amine reactive fluorescein derivatives are perhaps the most common fluorescent derivatizing reagents used to covalently label proteins. FITC-conjugated native casein-derived peptides were prepared by covalently binding FITC to the amino group of lysine.

HIV-1 P²⁴Antigen capture assay: HIV-1P adopted²⁴Antigen capture assay kits designed to quantify HIV-1P ²⁴A core antigen, which antigen is positively correlated with the extent of virus production in the cell. The kit is purchased from SAIC-NCI-Frederick tumor research institute, AIDS vaccine programs of P.O.BoxB, Frederick, M.D21702, USA, and comprises a vaccine coated with anti-HIV-1P²⁴Monoclonal antibody 96-well plate, primary antibody-rabbit anti-HIV P²⁴Serum, secondary antibody-goat anti-rabbit IgG (H + L) peroxidase conjugated antibody, TMB peroxidase substrate system and cleaved HIV-1P²⁴And (4) a standard substance. Analysis of HIV-1P at 450nm Using an Organon-Technica ELISA reader²⁴Antigen capture assay, reference wavelength 650 nm.

HIV-1 P²⁴Antigen capture ELISA: application in tissue culture medium for detecting HIV-1P²⁴Indirect enzyme immunoassay of core antigen determines HIV infection. Tissue culture supernatant and primary antibody rabbit anti-HIV-1P²⁴Antigen reaction, then using peroxidase coupled goat anti-rabbit IgG observation. By adding 4N H₂SO₄Terminating the reaction, wherein the shade of color produced is proportional to the amount of HIV-1 antigen present in the tissue culture supernatant.

Laboratory biohazard level (BL-3): all Virus production, isolation and infection, tissue culture of HIV-1 infected cells, harvesting of supernatant containing P24 antigen and P ²⁴Antigen capture ELISA, both performed at BL-3 laboratories of Hadasah medical college, Hebrew university, and which meet biosafety practice standards set by NIH and CDC (USA).

Flow cytometry: using FACStort cell sorter (Becton)&Dickinson, SanJose, Calif. (I) determination of CD before HIV-1 infection₄Percentage of positive CEM and sup-T1 cell batches to confirm that the extent of infection was the same in each experiment; and (ii) detecting T cells that have a presence of FITC-conjugated native casein-derived peptides in the cytoplasm and nucleus.

CO₂An incubator: during the experiment, for the purpose of culturing the produced cells with HIV-1 virus, the cells and virus pretreated with native casein-derived peptide, and the cells further incubated with HIV-1 were all in humidified CO₂And (5) storing in an incubator.

HIV infection of cultured human CD4 cells: for longer incubations, cells (CEM, Sup-T1)24 (synthetic and natural peptides) and 48 (natural peptide only) were pre-incubated with several increasing concentrations of either natural casein-derived peptides (50-1000. mu.g/ml) or casein-derived synthetic peptides (10-500. mu.g/ml) for hours, followed by addition of HIV-1IIIB (final concentration of 45pg/ml) per well. For a shorter incubation (3 hours), HIV-1IIIB3 hours were pre-incubated with the peptide and cells in tissue culture plates (5000 cells/well) were added. The control groups were IF (infected group, cells cultured with HIV-1 but no peptide), UIF (uninfected group, cells cultured without HIV-1 and no peptide) and UIF + Ch (uninfected + native casein-derived peptide group, { 50-1000. mu.g/ml } cultured cells in the presence of native casein-derived peptides), to examine the effect of native casein-derived peptides and casein-derived synthetic peptides on cell viability and growth. Cell viability and proliferation rate were counted at 7, 10 and 14 days post infection (day of harvest of P24 antigen culture supernatant). Cell and tissue culture supernatants (media) were harvested and immediately lysed in 1/10 volumes of 10% Triton X-100. These samples were incubated at 37 ℃ for an additional 1 hour and then stored at-80 ℃ until p was reached ²⁴And (4) detecting the antigen.

Confocal microscope: penetration of FITC-conjugated peptides into cells was detected using a Zeiss LSM 410 confocal laser scanning system using laser scanning confocal microscopy attached to a TW Zeiss Axiovert 135M inverted microscope. At 37 ℃ 5% CO₂After incubation in a 95% air incubator with FITC-conjugated native casein-derived peptides incubated with T cells, the cells were washed 3 times with Phosphate Buffered Saline (PBS) to remove unbound FITC peptides. Cells were fixed for 10 min using 3.8% formalin, washed 2 times with PBS, and resuspended in 50-100. mu.l PBS before microscopic examination. Images of cells selected from different incubation time points (15 min, 30 min, 1 h, 1.5 h and 3 h) showed the presence of varying amounts of FITC-native casein derived peptide in their cytoplasm and nucleus, these images were stored in a 3.5 "Zip drive (230MB) and image processed using Photoshop software.

[³H]Thymidine incorporation experiments: to examine the effect of native casein-derived peptides on T cell proliferation, different concentrations of native casein-derived peptides (10mg/ml in RPMI) were added to 96-well flat-bottomed microplates containing cultured Sup-T1 cells (5000 cells/well) as described in the HIV-1 infected Sup-T1 cell fraction. Cells were counted and cell activity was detected by trypan blue exclusion. Application at each time point (3, 7, 10 and 14 days) ³H]Cells were treated with thymidine pulses for 18 hours (overnight) and harvested using a glass fiber filter for radioactivity detection (incorporated in DNA of cells)³H]Thymidine levels are directly proportional to the degree of cell proliferation).

Toxicity of native casein-derived peptides in normal, myeloablative and transplant recipient mice and guinea pigs: natural casein derived peptides up to 5000mg/kg animal body weight are administered to normal animals intramuscularly or intravenously in a single dose or in 3 divided doses. Multiple mice were used, including BALB/C, C3H/HeJ and non-obese diabetic (NOD) mice. Mice were monitored for 10 months prior to sacrifice, necropsies (toxicity assay) after sacrifice, or observed for 200 days (survival). Each guinea pig received a single intramuscular injection of 20mg of native casein derived peptide. Guinea pigs were sacrificed after 15 days for pathological examination.

Reconstitution of leukocytes and platelets in bone marrow transplant recipient mice: BALB/c mice received sub-lethal radiation at 70cm distance from the skin at a dose of 50 cGy/min for a total dose of 600 cGy. As described above, mice receiving radiation were syngeneic bone marrow reconstituted and, following a double blind protocol, were injected intravenously with 1mg of native casein-derived peptide, casein-derived synthetic peptide (13-26 amino acids, see Table 3 above), or human serum albumin (control) per mouse after 24 hours. Reconstitution of leukocytes was determined from peripheral blood counts collected at specified time intervals 6-12 days after treatment. Platelet reconstitution was determined from the cell count of the retroorbital plexus collected at the indicated time interval 6-15 days after treatment, where the retroorbital plexus was drawn into heparinized capillaries.

In an additional series of experiments, CBA mice received lethal radiation (900cGy) and were then reconstituted and processed using BM cells and native casein-derived peptides or human serum albumin as described above. Platelet remodeling events were detected as described above.

In a third series of experiments, mice received radiation (800cGy) and were reconstituted and injected intraperitoneally 4, 5, 6 and 7 days post-transplantation with 100 μ g of casein-derived synthetic peptide (peptides 3a and 4P, representing the first 6 amino acids of the N-terminus of α S1 casein, respectively-see table 3 above). Platelet reconstitution was measured 10 and 12 days after transplantation.

Reconstitution of bone marrow transplant recipient mice: the C57/B1/6 mice received lethal radiation with a radiation source 70cm from the skin at a radiation dose of 50 cGy/min for a total radiation dose of 900 cCy. Irradiated mice were subjected to syngeneic bone marrow cell reconstitution following a double-blind protocol, derived from mice treated with 1 mg/mouse native casein-derived peptide or saline (control) 1 day prior to bone marrow collection. In one experiment, mice were monitored for 18 days of survival. In another experiment, mice were sacrificed after 8 days and spleen colonization was monitored.

Casein derived synthetic peptides significantly reduced cholesterol levels:

The ability of synthetic casein derived peptides to reduce cholesterol levels in 7-week-old female C57B1/6j mice was assessed after feeding an atherogenic diet. The mice were divided into 8 groups. One control group was fed a normal diet. The second control group was fed a modified Thomas Hartroft diet (# TD 88051: Teklad, Madison, Wis. [ Gerber, D.W.Et al., Journal of Lipid research.42, 2001] containing chocolate. The remaining groups were all fed the modified Thomas Hartroft diet. Serum cholesterol levels increased significantly after one week of feeding with the diet, 1 mg/mouse of casein-derived synthetic peptide was injected intraperitoneally, followed by a second injection of 0.1mg one week later.

Blood cholesterol levels were determined according to the Roche cholesterol assay based on the Roeschlou & Allin enzymatic method (Roche, inc., Germany).

Results of the experiment

Native casein-derived peptides: starting from the observation that curd sometimes does not support bacterial growth, a casein fragment with bactericidal properties was isolated from milk protein (katairkatcarasky, et al, U.S. patent No. 3,764,670). Crude peptides obtained from native casein cleavage products can be prepared by acid precipitation of the soluble fraction of the casein cleavage digest, dialysis and lyophilization. When the biological activity of the crude preparation is examined after long-term storage, it should be noted that the crude preparation retains the activity (in vitro and in vivo) for at least 12 months under the conditions of lyophilization and storage at 4 ℃.

As described above, in order to identify active peptides contained in native casein-derived peptides, High Performance Liquid Chromatography (HPLC) was used to fractionate the lyophilized crude preparation. All freeze-dried samples analyzed showed similar retention time characteristics, consistent with that described above.

Thus, one major component of a crude peptide preparation derived from native casein is the α S1 casein N-terminal fragment.

Native casein-derived peptides are non-toxic in rodents and humans: short and long term efficacy studies of large doses of native casein-derived peptides in mouse, rat, guinea pig and human volunteers confirmed that the formulation was free of toxic, teratogenic or side effects. In a series of experiments, a single dose of native casein-derived peptide representing 7000 times the estimated effective dose was injected intramuscularly to mice. Standard pathological necropsy of mice performed 14 days after treatment did not reveal toxic effects on internal organs and any abnormalities. Similar toxicity experiments in guinea pigs, 2 weeks after intramuscular injection of 20mg of native casein-derived peptide per dose, also did not find abnormalities. In another series of experiments, healthy mice were given large doses of native casein-derived peptides and after 2 weeks hematological parameters were examined and found to have no effect on these parameters, including White Blood Cells (WBC), Red Blood Cells (RBC), Hemoglobin (HGB), electrolytes, blood glucose, etc. The third series of experiments tested repeated application of large doses of 100mg/kg body weight for 2 weeks in mice and rats, with the result that no allergic, delayed skin or allergic reactions, nor pathological changes were found at necropsy. When testing the effect of native casein-derived peptides on the long-term survival of irradiated, bone marrow reconstituted BALB/C and C3H/HeJ mice, it was found that the survival of the treated mice (18 out of 27 BALB/C and C3H/HeJ mice, survival 66%) significantly exceeded the survival of the albumin-treated control group (4 out of 26 BALB/C and C3H/HeJ mice, survival 15%). Teratogenicity experiments in mice treated with native casein-derived peptides [ see, e.g., Drug Safety in Pregnancy, Folb and Dakes, p.336, Elsevier; amsterdam, New York, Oxford (1990) ] found that the peptide had no effect on any growth parameter.

As well as being tested in rodents to find no toxicity or side effects, native casein derived peptides are also safe for use in humans. Comparison of the blood and urine samples from 7 healthy volunteers 7 days before, during and after intramuscular injection of native casein-derived peptides revealed no change in any clinical index. No other adverse reactions were observed.

Thus, the use of native casein derived peptides for long-term treatment in rodents at high doses has not been found to be significantly toxic, pathological, allergic, teratogenic, serological or any other adverse reaction. Furthermore, the use of native casein derived peptides has a clear survival advantage over a period of 200- & 300 days for irradiated mice at risk for short and long term complications. These results, as well as the use of native casein-derived peptides by injection in healthy human volunteers, did not show any adverse reactions, clearly indicating that parenteral administration of the peptides is very safe.

Recipient mice for bone marrow transplant reconstitution: the C57/B1/6 mice received lethal radiation and were then reconstituted using syngeneic bone marrow derived from mice treated with 1 mg/mouse native casein-derived peptide or untreated mice 1 day prior to bone marrow aspiration, and as a result, the survival rate of the irradiated mice receiving bone marrow from the treated group was found to be much greater than that of the irradiated mice receiving untreated mouse bone marrow (after 10 days of radiation, 15 of 18 irradiated mice receiving treated mouse bone marrow survived; and only 4 of 17 irradiated mice receiving bone marrow cells from saline-treated control mice survived 10 days of radiation). The spleens of mice irradiated with bone marrow-treated group mouse bone marrow included approximately 2-3 times the number of colonies per spleen compared to spleen of mice irradiated with saline-treated control mouse bone marrow cells (1-5 colonies vs 0-3 colonies).

Native casein-derived peptides stimulate lymphocyte proliferation: natural Killer (NK) cells and cytotoxic T cells are essential for the immune system to protect the body from infectious pathogens and cancer cells, since both cells have active cytotoxic effects and are capable of secreting immunoregulatory lymphokines. Such as immune impairment following aids or chemotherapy, results in an abnormal reduction in T or NK cell activity. When normal murine bone marrow cells from BALB/C and C57B1/6 mice were cultured in the presence of 100. mu.g/ml native casein-derived peptide, the cells were cultured on two effector cells: a clear increase in NK activity was observed in all target cell proportion groups. Moreover, comparison between the two groups revealed a clear dose-response relationship. At an effector to target cell ratio of 1: 25, the mean NK activity increased from 13.93% to 30.77%, and at an effector to target cell ratio of 1: 50, the mean NK activity increased from 13.68% to 44.05% (FIG. 1). Similar experiments with human peripheral blood stem cells from donors treated with granulocyte colony stimulating factor demonstrated a more pronounced concentration-dependent stimulation of target cell lysis by native casein-derived peptides.

In a first set of experiments (fig. 2a), NK activity in blood samples from one patient was measured and incubated with increasing concentrations of native casein-derived peptides at two effector to target cell ratios. Only 4% was measured in control, untreated PBSC cultures³⁵And S is released. Almost the same percentage radioactivity (4%) was found at the lowest peptide concentration (5. mu.g/ml). However, at higher peptide concentrations, in the range of 10. mu.g/ml to 100. mu.g/ml, 10.8-14.9% was measured for an effector to target ratio of 100: 1³⁵S release, measured at an effector to target ratio of 50: 1, of 8.3-14.5%³⁵Release of S (fig. 2 a).

When PBS cells from normal (patient 1) and affected (patients 2-6) human donors were incubated with increasing concentrations of native casein-derived peptides, a significant increase in NK activity in the affected patients could be measured. Thus, although native casein-derived peptides have minimal effect on NK activity in normal patients (from 13-15%³⁵Increased S release, patient 1), PBS cells from breast cancer and non-Hodgkin lymphoma patients (e.g., patients 3 and 4) showed a significant, dose-dependent increase in NK activity (3.5-10.8% respectively) ³⁵S；12.2-19.1％ ³⁵S) (fig. 2 b).

Native casein-derived peptides stimulate CD56 surface antigen positive (NK) cell proliferation: in another series of experiments, Peripheral Blood Stem Cells (PBSC) from 5 human donors treated with GCSF were incubated with native casein-derived peptides for 10, 14, or 28 days and then assayed for the presence of CD56 antigen. A sometimes significant increase in CD56 antigen detection was observed in peptide-treated cells from all donors (except patient 1). A representative reaction is depicted in fig. 3 a: the presence of CD56 surface antigen positive (NK) cells was detected by direct immunofluorescence staining after 10 days of incubation in the presence or absence of native casein derived peptides. Overall, incubation with native casein-derived peptides increased the average percentage of cells staining positive for CD56 from 0.64% in the control group to 2.0% post-treatment (fig. 3 a).

Native casein-derived peptides stimulate CD3 surface antigen positive (T) cell proliferation: the effect of native casein-derived peptides on the proliferation of CD3 surface antigen positive (T) cells in PBS cells from 5 subjects was determined by direct immunofluorescence. In all donors (except patient 4), incubation with native casein-derived peptides for 14 days significantly increased T cell proliferation, in some cases by more than 5-fold at most. Overall, the average percentage of cells positively stained for CD3 was increased from 19.45% in the control group to 35.54% in the treatment group (fig. 3 b).

Native casein-derived peptides stimulate proliferation of CD56 and CD3(NK/T cells) positive cells: in another experiment, PBSCs from 7 patients were incubated with native casein-derived peptides for 28 days, and then the effect on NK/T cell proliferation (positive for CD56 and CD3 surface antigen) was detected by direct immunofluorescence. Incubation with native casein-derived peptides stimulated T cell proliferation in some cases (patient 6) more than 5-fold, while the average percentage of CD3 positive (T-) cells increased from 2.08% in the control group to 6.49% in the treatment group. Cells positive for both CD56 and CD3 surface antigen (NK/T) increased from 1.1% in the control group to 4.3% in the treatment group (fig. 3 c). Thus, native casein-derived peptides stimulate proliferation of T lymphocytes and natural killer cells from normal murine and human blood cell progenitors. Interestingly, the maximal immunostimulatory effect of native casein-derived peptides was noted in human donors with low initial T and NK cell levels (fig. 3 a-c).

Casein derived synthetic peptides stimulate human lymphocytes to proliferate in vitro: when a casein-derived synthetic peptide of the first 3-26 residues of α S1 casein was incubated with PBSCs of healthy and cancer patients (see below), a significant enhancement of NK cell activity was observed. When PBSCs from non-hodgkin lymphoma and breast cancer patients were incubated for 2 days with as little as 10 μ g/ml of peptide containing the first 9 or more residues of α S1 casein, the target cells were lysed most (3- >5 fold over control group) (fig. 4). Under the same conditions, none of the tested peptides had a significant effect on NK cell activity in healthy donor PBSC cultures. Thus, a peptide containing at least the first 10 residues of the N-terminal sequence of α S1 casein is capable of selectively stimulating the proliferation of lymphocytes in tumor patient cells in vitro, even at very low concentrations of the peptide.

Similar NK cell activity was observed when a casein-derived synthetic peptide representing the first 3 residues of α S1 casein was incubated with PBS cells from human donors with hematopoietic disease (see below). Incubation of PBS cells with peptide increased target cell lysis from 2-fold to more than 8-fold of untreated controls. Of the 5 patients tested, three (3) responded to a peptide concentration of 25. mu.g/ml, one (1) to a peptide concentration of 100. mu.g/ml and one (1) to a peptide concentration of 250. mu.g/ml. Three of five (5) patients responded at 25. mu.g/ml. No significant effect on NK activity in PBSC cultures from healthy donors treated with synthetic peptide representing the first 3 amino acids of α S1 casein was observed, demonstrating the selective nature of the human lymphocyte stimulating properties of the casein derived peptides.

Stimulation of human blood progenitor cells for hematopoiesis: blood progenitor cells can differentiate into a variety of blood cells: macrophages, monocytes, granulocytes, lymphocytes, erythrocytes and megakaryocytes. Progenitor cells are abundant in bone marrow, but also present in peripheral blood (PBSC cells) after treatment with granulocyte colony stimulating factor and fresh cord blood. Increasing concentrations (50-600. mu.g/ml) of the native casein-derived peptide are added to human bone marrow, PBSC and cord blood cultures and can be assayed ³H]Thymidine incorporation, an increase in cell proliferation was observed (FIGS. 5a-5 c). Proliferation of human PBSC was most pronounced after 15 days of culture at a concentration of 300. mu.g/ml (FIG. 5 a). Peptide derived from native casein (60)After incubation for 14 days (not after 7 days) with 0. mu.g/ml, a stronger effect on cord blood cells can be observed (,)³H]Thymidine incorporation increased 3-4 fold) (FIG. 5 c). Among 4 donors, cultured bone marrow cells from 3 of them also reacted strongly (3-5 fold increase in incorporation) to native casein-derived peptide (300. mu.g/ml) after 21 days of incubation (FIG. 5 b). Thus, native casein-derived peptides can stimulate the proliferation of human blood progenitor cells of bone marrow and other sources. Interestingly, cultured human K562 (chronic myelogenous leukemia) and colon (colon cancer) cell lines were incubated with high concentrations (up to 500. mu.g/ml) of native casein-derived peptide under similar conditions, and the results were for³H]Thymidine incorporation was not affected. Thus, native casein-derived peptides can stimulate the proliferation of human blood progenitor cells, but not the in vitro growth of tumor cells.

Casein-derived peptides stimulate megakaryocytopoiesis:

native casein-derived peptides stimulate megakaryocyte progenitor proliferation in cultured murine bone marrow cells: in bone marrow, megakaryocytes of multiple nuclei are derived from primitive stem cells and then mature into giant cells, each of which produces thousands of platelet cells. Platelets are essential for clot formation, while thrombocytopenia is a major concern in the myeloablative state (after chemotherapy or radiation therapy).

Primary bone marrow cell cultures can be induced to form CFU-GM (granulocytes and monocytes) and CFU-GEMM (granulocytes, erythrocytes, macrophages and megakaryocytes) colonies, the latter also including more blood cell types. Colony counts reflect the expansion of specific progenitor cells, cell numbers reflect the rate of proliferation, and differentiated cell counts reflect which specific cell line developed [ Patenkin, D.Et al. (1990), mol.Cel.biol.10, 6046-50 ]. In cultured bone marrow cells of mice incubated with erythropoietin and IL-3, and supplemented with 25. mu.g/ml native casein-derived peptide for 8 days, the number of CFU-GEMM was increased 2.5-fold compared to the control group, and the relative cell number per colony in CFU-GEMM was increased 3-fold. In a similar series of experiments, the addition of native casein-derived peptides to bone marrow cells incubated with erythropoietin and conditioned medium (see materials and experimental methods) stimulated a concentration-dependent increase in the percentage of early and late megakaryocytes (15% megakaryocytes without peptide; up to 50% megakaryocytes with 500. mu.g/ml native casein-derived peptide). Thus, treatment with native casein-derived peptides for 8 days can stimulate significantly increased megakaryocyte formation and development in murine primary bone marrow cultures.

Casein-derived synthetic peptides stimulate megakaryocyte progenitor proliferation in cultured murine bone marrow cells:

similar to the above experiment, under similar experimental conditions, the casein-derived peptide representing the first 5-24 amino acids of α S1 casein increased the percentage of early and late megakaryocytes from 15% without synthetic peptide to over 40% in the presence of 25 μ g/ml synthetic peptide (fig. 7). Thus, treatment with synthetic casein-derived peptides representing the first 5, 6, 11, 12, 17, 18, 19, 20, 21 and 24 amino acids for 8 days stimulated a significant increase in megakaryocyte formation and development of primary murine bone marrow cultures. Some mildness, but still perceptible irritation, was observed with other casein-derived synthetic peptides.

Native casein-derived peptides stimulate megakaryocytopoiesis in cultured human bone marrow cells: under similar conditions, 100. mu.g/ml native casein-derived peptide was added to bone marrow cell cultures of healthy donors, and CFU-GM colony formation increased with or without the addition of other stimulatory factors (GM-CSF, CM). Native casein-derived peptides may also stimulate red blood cells to form colonies in the presence of erythropoietin. Treatment of human bone marrow cells with Thrombopoietin (TPO) stimulates Megakaryocyte (MK) colony formation. Addition of 300. mu.g/ml native casein-derived peptide to TPO-treated cells increased MK colony proliferation by 2-fold or more (2X 10 cells without addition of peptide) ⁵The cells formed 16 colonies, and native casein-derived peptides were added, every 2X 10⁵Each cell formed 35 colonies).

Incubation with native casein-derived peptides for 14 days in the presence of other hematopoietic factors, such as erythropoietin, human IL-3, hSCF and AB serum, increased the number of CFU-GEMM colonies in human bone marrow cells by nearly 3-fold (158 colonies supplemented with 500. mu.g/ml native casein-derived peptide, 68 colonies with only hematopoietic factor), but had a lesser effect (1.5-fold) on the formation of CFU-GEMM colonies in cultured cord blood. The relative cell counts in cultured human bone marrow and cord blood colonies reflected the proliferation of megakaryocytes to which 25. mu.g/ml native casein-derived peptide was added (see Table shown in FIG. 6). Thus, incubation of cultured human primary bone marrow and cord blood cells with native casein-derived peptides can stimulate the development and proliferation of committed megakaryocyte and erythrocyte colonies. Notably, the observed synergy in stimulating megakaryocytopoiesis between TPO and native casein-derived peptides suggests a possible role for this potent hematopoietic growth factor in the mechanism of the stimulatory properties of native casein-derived peptides, and further suggests that native casein-derived peptides may similarly augment many TPO-mediated effects.

Natural casein-derived peptides and natural casein-derived synthetic peptides enhance the effect of Erythropoietin (EPO) in cultured human bone marrow cells: the effect of casein-derived natural and synthetic peptides on erythrocyte proliferation in cultured human bone marrow cells was evaluated under the same conditions as described above for megakaryocyte production. When added in the presence of EPO, 50-300. mu.g/ml of native casein-derived peptide, or 100. mu.g/ml of casein-derived synthetic peptide (F, Table 3, SEQ ID NO: 18), stimulated proliferation of erythrocyte precursors by 1.5 (synthetic peptide) -4 fold (appearance of BFU-E colonies) compared to treatment with EPO alone. Thus, the natural casein-derived peptides and synthetic derivatives thereof enhance the erythropoiesis stimulating effect of EPO, and thus can be used to enhance various clinically important EPO-mediated effects.

The casein-derived synthetic peptide stimulates dendritic cell proliferation in murine CFU-GEMM: the effect of casein-derived synthetic peptides on dendritic cell proliferation in murine primary bone marrow cells was evaluated under the same conditions described above for the stimulation of megakaryocytes. The first 2, 3, 5, 6, 7, 9, 11, 12, 16, 23, 24 and 26 amino acid casein-derived synthetic peptides representing α S1 casein stimulated dendritic cell proliferation from 2.2% to 23% of total cells, whereas the cell samples contained 0.1-0.2% dendritic cells when incubated in the absence of the casein-derived synthetic peptide (fig. 7).

The casein-derived synthetic peptide stimulates plasma cell proliferation in murine CFU-GEMM: the effect of casein-derived synthetic peptides on plasma cell proliferation in murine primary bone marrow cells was evaluated under the same conditions as described above for the stimulation of megakaryocytes. The first 2, 3, 5, 7, 11, 16, 17, 18, 19, 20, 21, 22, 23 and 24 and 26 amino acids of the casein-derived synthetic peptide representing α S1 casein stimulated plasma cell proliferation from 1.5% to 12.3% of the total cell count, compared to 0.3% in the absence of the casein-derived synthetic peptide (fig. 7).

The casein-derived synthetic peptide stimulates macrophage proliferation in CFU-GEMM: the effect of casein-derived synthetic peptides on macrophage proliferation in murine primary bone marrow cells was evaluated under the same conditions described above for the stimulation of megakaryocytes. Incubation with the casein-derived synthetic peptides representing the first 7, 9, 16 and 23 amino acids of α S1 casein significantly stimulated macrophage proliferation, increasing from approximately 17% of the total cell count of the control group to approximately 30% of the total cell number of the group incubated with the casein-derived synthetic peptides (fig. 7).

The casein-derived synthetic peptide stimulates erythrocyte proliferation in CFU-CEMM: the effect of casein-derived synthetic peptides on the proliferation of erythrocytes in murine primary bone marrow cells was evaluated under the same conditions as described above for the stimulation of megakaryocytes. Incubation with the casein-derived synthetic peptide representing the first 4 amino acids of α S1 casein significantly stimulated erythrocyte proliferation, increasing from 53% of the total cell count of the control group to 71% of the total cell count of the group incubated with the casein-derived synthetic peptide (fig. 7).

The casein-derived synthetic peptide stimulates Polymorphonuclear (PMN) cell proliferation in CFU-GEMM: the effect of casein-derived synthetic peptides on the proliferation of Polymorphonuclear (PMN) cells in murine primary bone marrow cells was demonstrated under the same conditions described above for the stimulation of megakaryocytes. Incubation with casein-derived synthetic peptides representing the first 3, 6, 7, 9, 16 and more amino acids of α S1 casein significantly stimulated PMNs proliferation, increasing from 1.6% of the total cell count of the unincubated control group to 2.9% -14.9% of the total cell number of the group incubated with the casein-derived synthetic peptides (fig. 7).

Native casein-derived peptides can stimulate hematopoiesis in vivo following radiation and bone marrow transplantation: myeloablative therapy can lead to a reduction in the number of life-threatening platelets and leukocytes, which can persist despite administration of blood cell and growth factor therapies. The following disclosure shows the effect of native casein-derived peptides following radiation and bone marrow transplantation.

Native casein-derived peptides can enhance leukocyte and platelet reconstitution following syngeneic bone marrow transplantation in mice: minimal bone marrow reconstituted BALB/c mice (n ═ 12) were injected intravenously with 1 mg/mouse native casein-derived peptide one day after bone marrow cell reconstitution following sub-lethal dose irradiation (600 cGy). At 4, 6 and 15 days post-treatment, a significant increase in peripheral blood leukocyte counts was observed compared to the control group receiving human serum albumin (figure 8). Peripheral platelet counts of bone marrow transplanted mice after irradiation were inhibited to an equal extent for up to 8 days after treatment, both in the treated and control groups. However, by day 13, mice treated with native casein-derived peptides showed a significant advantage, with a significant increase in advantage over the human serum albumin-treated control group (platelet count) by day 13, which was even more pronounced after 15 days (fig. 9). Thus, native casein-derived peptides may enhance the reconstitution of platelets and leukocytes after the application of a limited number of bone marrow cell transplants. It is expected that this effect may be further enhanced when an optimal number, rather than a limited number, of bone marrow cell reconstructions are employed.

After syngeneic bone marrow transplantation in mice, casein-derived synthetic peptides can enhance leukocyte reconstitution: BALB/c mice with minimal bone marrow reconstitution one day after bone marrow transplantation after sub-lethal dose irradiation (600cGy) (each combination)Peptide-forming group n-5 and control group n-10) were injected intraperitoneally with 1 mg/mouse casein-derived synthetic peptide (13-26 amino acids in length, see table 3). During the 10-14 days, the control group received human serum albumin (day 10: 1.67X 10)⁶Cells/ml; day 12: 4.64X 10⁶Cells/ml), using a protein with 15 amino acids (day 10: 1.72X 10⁶Cells/ml; day 12: 6.54X 10⁶Cells/ml) and 17 amino acids (day 10: 2.74X 10⁶Cells/ml; day 12: 5.20X 10⁶Cells/ml) a significant increase in peripheral blood leukocyte count was observed (see table 3). Thus, casein-derived synthetic peptides may enhance leukocyte reconstitution after transplantation using a limited number of bone marrow cells.

After syngeneic bone marrow transplantation in mice, casein-derived synthetic peptides can enhance platelet reconstitution: to confirm that the observed casein-derived synthetic peptides enhance megakaryocyte proliferation in hematopoietic stem cell cultures (see FIGS. 6 and 7), the effect of the peptides on in vitro platelet reconstitution was investigated. When minimally bone marrow reconstituted mice (n-5/group) received 100 μ g/mouse synthetic peptides 4P and 3a (6 and 12 amino acids in length, respectively-see table 3) given a total of 4 doses per day i.p. (4-7 days post-transplant) when receiving lethal radiation (800cGy), a significant enhancement in platelet reconstitution was observed compared to untreated controls. For both peptides, platelet counts increased significantly at 10 and 12 days post-transplantation. Treatment with peptide 4P increased the count by 29% (872X 10) at 12 days post-implantation ³Perml, and 676X 10 for the control group³/ml), whereas treatment with peptide 3a increased the count by as much as 35.5% on day 10 post-transplantation (229 × 10)³A/ml, and 169X 10 for the control group³Ml), increased by as much as 13.5% on day 12 post-transplant (622X 10)³Perml, and 461X 10 for the control group³In ml). Thus, the same casein-derived synthetic peptide enhances megakaryocyte proliferation in vitro and platelet reconstitution in vivo after bone marrow transplantation.

Natural casein derived peptides for inhibiting HIV-1 virus infection of T lymphocyte cell line in vitro

Penetration of native casein-derived peptides into T lymphocytes: to investigate the immunostimulatory mechanism and antiviral effect of native casein-derived peptides, susceptible Sup-T1 and cultured human T cells were treated with native casein-derived peptides prior to HIV-1 virus infection in vitro. Fluorescence microscopy revealed that FITC-conjugated native casein-derived peptides (100. mu.g/ml) penetrated Sup-T1 cells when incubated as described above (FIGS. 10 a-f). After 15 min, a small amount of labeling was observed in the cytoplasm (FIGS. 10 a-b). At 30 min (FIGS. 10c-d), more markers were observed in the cytoplasm with limited nuclear uptake. From 1 hour incubation (fig. 10e-f), FITC-labeled native casein-derived peptides were observed in the cytoplasm, but most of them were concentrated in the nucleus. Sup-T1 cell analysis by flow cytometry confirmed the increased uptake of labeled native casein-derived peptides starting 5 minutes after incubation.

Native casein-derived peptides can enhance human lymphocyte proliferation: the presence of native casein-derived peptides in the culture medium resulted in an increase in the count of Sup-T1 cells during 14 days of culture. The largest increase in cell number observed was 50. mu.g/ml native casein-derived peptide (42%) at 7 days of incubation, 1000. mu.g/ml (30%) at 10 days, and 600. mu.g/ml (32%) at 14 days. By measuring the cultured cell [ 2 ]³H]The thymidine incorporation of (a) provides a proliferation index, reflecting an increase in cell number, with the most significant effects observed being at day 10 and at day 14 with 600 μ g/ml native casein-derived peptide (figure 11). The decreased proliferation index at 14 days may reflect cell overgrowth and nutrient depletion.

Casein derived synthetic peptides can enhance human lymphocyte proliferation: the presence of casein derived synthetic peptides in the medium (all peptides listed in table 3) resulted in an increase in the count of Sup-T1 cells during 10 days of culture. This increase was similar in all synthetic peptides. The largest increase in lymphocyte numbers observed in infected cells was given 250 μ g and 500 μ g/ml of peptides representing the first 9 amino acids (80% and 33%, respectively) (data not shown).

Native casein-derived peptides suppress HIV-1 infection of human lymphocytes: pretreatment of susceptible CEM lymphocytes with native casein-derived peptides (50-1000 μ g/ml) for 24 or 48 hours prior to incubation with HIV-1, or exposure to HIV-1 pretreated with native casein-derived peptides for 3 hours, showed increased cell proliferation and decreased levels of viral infection compared to untreated controls. Cell counting and HIV P24 antigen experiments were performed 15 days after infection and found that after 3 hours of incubation with 600-1000. mu.g/ml native casein-derived peptide, the viral infection was 100% inhibited and after 24 hours of incubation with 50 and 600. mu.g/ml peptide, the infection inhibition was 98% and 99%, respectively (compare the cell number of untreated control UIF). No longer incubation times were found to be more effective (figure 12). Although increasing concentrations of native casein-derived peptides can enhance cell proliferation at 3 and 24 hours post-infection, inhibition of viral infection was also most pronounced in these most rapidly growing cultures. Prior to HIV-1 infection, Sup-T1 cells were pretreated with native casein-derived peptides, and more dramatic enhancement of cell proliferation and inhibition of HIV-1 infection were observed (mean inhibition of viral infection was 96.7%, 88.7% and 95.7% for 3 hours, 24 hours and 48 hours of virus pretreatment, respectively) (not shown). Thus, native casein-derived peptides penetrate cultured human lymphocytes and their nuclei, enhancing cell growth, significantly reducing susceptibility of CD4 cells to HIV-1 infection. Thus, native casein-derived peptides are expected to be useful in the prevention of HIV infection, as well as in post-infection treatment of HIV infection and AIDS patients.

Casein derived synthetic peptides inhibit HIV-1 infection of human lymphocytes: the ability of casein-derived synthetic peptides to inhibit HIV-1 infection in human lymphocytes was demonstrated using CEM-lymphocytes under the same conditions as described above. Pretreatment of CEM lymphocytes with a casein-derived synthetic peptide representing the first 3 amino acids of α S1 casein for 3 hours achieved significant resistance to infection following HIV-1 incubation. The number of lymphocytes in the treated cells was 1.29X 10⁶(100. mu.g/ml) and 2.01X 10⁶(500. mu.g/ml) compared to the infected HIV-1 control, which was 1.06X 10⁶(FIG. 13). Compared with untreated control group (0.52ng P)²⁴Ag/ml) in infectionsHIV-P7 days later²⁴Analysis of HIV-1 infection levels in the same cells measured significantly decreased in peptide-treated cells (0.17 and 0.14ng P at 100. mu.g/ml and 500. mu.g/ml, respectively)²⁴Antigen/ml).

Also, significant inhibition of HIV-1 infection was observed in CEM cells exposed to virus (3 hours) pretreated with synthetic casein-derived peptide representing the first 5 amino acids of α S1 casein.

1.06X 10 in comparison with infected HIV-1⁶In contrast, the cell counts in cultures incubated with 10 and 25. mu.g peptide 3P/ml were 1.17X 10, respectively⁶And 1.26X 10⁶。

HIV-P on day 7 post infection ²⁴Antigen analysis revealed a significant reduction in HIV-1 infection levels in the treated cultures (0.26 and 0.18ng P at 10 and 25. mu.g/ml, respectively)²⁴Ag/ml, while the control group was 0.52ng P²⁴ Agml)。

Also, pre-incubation of the virus with casein-derived synthetic peptide 4P, representing the first 6 amino acids of α S1 casein, for 3 hours had a significant effect on the susceptibility of CEM lymphocytes to infection with HIV-1.

Cell numbers were most significantly affected at concentrations of 25 and 250. mu.g/ml (1.26X 10, respectively)⁶And 1.59X 10⁶While the control value for infection was 1.06X 10⁶)。

HIV-P on day 7 post infection²⁴Antigen analysis found a dose-dependent reduction of viral particles compared to untreated infected control cultures (figure 13). Thus, the protective effect provided by the natural casein-derived peptides against HIV-1 infected lymphocytes is retained in the casein-derived synthetic peptides representing as little as the first 5N-terminal amino acids of α S-1 casein.

Native casein-derived peptides prevent non-obese diabetic (NOD) mice from developing glucosuria: non-obese diabetic (NOD) mice spontaneously develop type I (IDDM) diabetes, an autoimmune disease that can cause inflammation of the beta cells of the islets of langerhans, ultimately leading to illness and death. Female NOD mice are particularly susceptible, showing evidence of macrophage invasion of the islet stroma as early as 5 weeks of age. 100 ug of native casein-derived peptide was injected 1 or 2 times a week for 5 weeks (5 or 10 total injections), which was completely effective in preventing glucosuria associated with the onset and course of the disease. At 200 days, 100% of the untreated control mice (n-5) became diabetic and subsequently died, while 100% of the treated group of mice (n-5) remained normoglycemic and all survived at 365 days (fig. 14). Thus, native casein-derived peptides may be effective in protecting genetically susceptible mice from the development of this autoimmune inflammatory disease.

Casein derived synthetic peptides prevent non-obese diabetic (NOD) mice from developing glucosuria:

the preventive effect of casein-derived synthetic peptides on the onset of glucosuria in NOD mice was demonstrated under the same conditions as above, but the mice were injected with 100. mu.g of casein-derived synthetic peptides once a week for five (5) weeks. The results of these experiments are shown in table 4 below:

TABLE 4

Effect of casein-derived synthetic peptides on IDMM in NOD mice

Peptide derivative codes	Health/total-	Urine sugar	IPGT experiment
					0 minutes (before load)	60 minutes after loading
			Y	1/5			Negative of	121	138
X	3/5	Negative of			94	114
							Negative of	104	119
		Negative of			141	114
			1a	1/5			Negative of	88	106
2a	4/5	Negative of			215	183
							Negative of	112	119
		Negative of			95	107
							Negative of	159	204
3a	3/5	Negative of			135	137
							Negative of	205	197
		Negative of			201	211
			A	2/5			Negative of	134	164
		Negative of			105	107
			B	2/5			Negative of	130	117
		Negative of			130	97
			D	2/5			Negative of	99	108
		Negative of			130	136

I	2/5	Negative of	324	Not detected
							Negative of	124	138
J	3/5	Negative of	166	Not detected
							Negative of	193	Not detected
		Negative of	186	Not detected
					K	2/5	Negative of	116	143
		Negative of	443	Not detected
					Chay-13	2/5	Negative of	123	130
		Negative of	111	111
					Chay-13	2/5	Negative of	128	116
		Negative of	113	125
					Control	0/5

Blood was drawn from the paraorbital plexus at 0min and 60min after intraperitoneal injection of 1g/kg body weight of glucose. Plasma glucose levels were measured using a glucose analyzer 2(Beckman Instruments, Fullerton, Calif.) and expressed as mmol/L.

Healthy and good no detectable sugar in urine

Grape diabetes is > 1000mg/dL.

IPGTT with 6 healthy female control mice: 0min-110 mmol/L;

60min-106mmol/L blood sugar.

Synthetic peptides derived from casein, representing the first 9(X), 11(2a) and 12(3a) amino acids and longer chains of α S1 casein, are highly effective in preventing glucosuria associated with the onset and course of disease.

The effect of the casein derived synthetic peptides was assessed after 25 weeks. At this time, all 5 mice (n ═ 5) in the untreated control group developed diabetes, indicated by the presence of overt glucosuria (> 1000mg/dl) (table 4).

No glucosuria was detected in 3 of 5 NOD mice (3/5) treated with synthetic peptide representing the first nine (9) amino acids of the N-terminus of α S1 casein. In the group injected with the synthetic peptide of eleven (11) amino acids at the N-terminal of α S1 casein, glucosuria was not detected in 4 (4/5) of 5 NOD mice

In the peptide-treated groups of mice in which glucosuria was detected, the onset was generally significantly delayed (3-5 weeks delayed) relative to untreated controls (data not shown), indicating clear protection of even incomplete peptides.

The protective effect of shorter casein-derived synthetic peptides was also studied in NOD mice. In a further series of experiments similar to the above, administration of peptides representing the first 3 (1P) and 4 (2P) N-terminal amino acids of α S1 casein was effective in preventing the onset of glucosuria in untreated mice (measured at week 16), whereas the untreated controls developed diabetes (100% glucosuria) (data not shown).

In the group injected with synthetic casein-derived peptides representing the first 9 amino acids, glucose tolerance (IPGT) experiments were performed 25 weeks later with healthy and good NOD mice, showing no signs of abnormal glucose metabolism (normal blood glucose values before and 60 min post-glucose loading).

In the group treated with the synthetic peptide derived from casein representing the first 11 amino acids of the N-terminus of α S1 casein (2a), there was a certain increase in resting plasma glucose levels in two of 5 mice (215 and 159mmol/L) and a moderate increase was maintained 60 minutes after loading (183 and 04 mmol/L). Two other mice maintained a normal range of blood glucose throughout the experiment (table 4). In summary, the results of IPGTT reflect the absence of glucosuria in healthy, surviving peptide-treated mice (table 4). Thus, synthetic and natural casein-derived peptides representing only a few amino acids of the N-terminus of α S1 casein significantly reduced the susceptibility of NOD mice with genetic predisposition to the onset of autoimmune diabetes.

Synthetic casein-derived peptides significantly reduce Total Cholesterol (TC), Low Density Lipoprotein (LDL) and High Density Lipoprotein (HDL) levels in blood: intraperitoneal administration of casein-derived synthetic peptides resulted in a significant reduction in blood lipids (HDL, LDL and TC) in experimental high cholesterol mice. One week after the atherogenic Thomas Hartroft diet, the blood cholesterol level of the mice rose to a level of 318 mg/dl.

1 week after treatment with 1mg of casein-derived synthetic peptide per mouse, compared to control group [ TC: 308 and 279mg/dl, respectively; HDL: 42.5mg/dl and 41mg/dl, respectively, LDL: 247mg/dl and 221mg/dl, respectively, diet-induced high cholesterol/hyperlipidemia control groups were 393mg/dl (TC), 54.5mg/dl (HDL) and 326mg/dl (LDL) ], and TC, HDL and LDL values were significantly reduced in the groups treated with casein-derived synthetic peptides representing the first 5(3P) and 11(2a) amino acids of α S1 casein (fig. 15). Thus, synthetic peptides representing the first several N-terminal amino acids of α S1 casein reduced experimentally induced hyperlipidemia and hypercholesterolemia within 1 week after a single intraperitoneal administration.

Clinical trials of native casein-derived peptides:

the patient was given intramuscular injections of 50mg of native casein-derived peptide per injection, divided into 3 injections, as indicated.

Native casein-derived peptides stimulate hematopoiesis in tumor patients: as indicated, hematological markers were measured before and after administration of native casein-derived peptides to 6 tumor patients who had or were receiving chemotherapy. Of particular note are changes in Platelets (PLT), White Blood Cells (WBC), Red Blood Cells (RBC) and Hemoglobin (HGB), which are indicative of platelet, white blood cell and red blood cell production, respectively.

G.t., (female patient. patient 1): the patient suffered from ovarian cancer, underwent hysterectomy, and then was chemotherapy. The native casein-derived peptide was injected intramuscularly two times two months and two and a half months after surgery. During the first and second administrations of native casein-derived peptide, no chemotherapy was performed. Blood assays were performed 6 days after the first injection and 7 and 13 days after the second injection, and resulted in considerable increases in both platelet and WBC fractions, as well as RBC (fig. 16).

B.c. (female patient. patient 2): patients suffered from gastric metastatic cancer 6 years later due to lobular cancer with radical mastectomy in 1983. She received intramuscular injection of native casein derived peptide 3 days before the start of chemotherapy and a second (peptide injection) 10 days after chemotherapy. Although blood counts showed reduced inhibition of hematological indicators at 10 and 16 days post-chemotherapy, which is very common after chemotherapy, the most significant effect of native casein-derived peptides was observed 3 days after the first injection, i.e., before chemotherapy (fig. 16).

B.s., (female patient. patient 3): patients with widespread metastatic, disseminated breast cancer were first discovered in 1987. She received a first intramuscular injection of native casein-derived peptide 2 years later and a second 23 days later. During this period, no other treatment was given. Blood tests suggested a significant increase in PLT numbers 7 days after the first treatment and RBC and WBC numbers 7 days after the second treatment (fig. 16).

J.r., (female patient. patient 4): the diagnosis of the patient is breast cancer with bone metastasis. She received one dose of native casein-derived peptide intramuscularly 8 days before the start of chemotherapy and another 14 days later. The most significant effect seen clearly is the rapid return of WBC levels following chemotherapy-induced myelosuppression (fig. 16).

D.m. (female patient. patient 5): patients suffer from liver cancer with widespread metastatic spread. She received 3 intramuscular injections of native casein-derived peptide 10, 8 and 6 days prior to receiving chemotherapy. The second series of injections started 10, 12 and 14 days after chemotherapy. Although a significant effect of the preparation on hematological indices was observed after the first series of injections prior to chemotherapy, the most dramatic improvement was a rapid return of the inhibited indices to normal cell counts after the second series of native casein-derived peptide injections (fig. 16).

Thus, the use of native casein-derived peptides in patients with tumors may lead to an improvement of hematological indicators, in particular an enhancement of the production of erythrocytes, leukocytes and platelets, and a modulation and shortening of the chemotherapy-induced inhibition period of blood components.

In transplant recipients with resistant thrombocytopenia, native casein-derived peptides can stimulate thrombopoiesis: long-term transfusion-resistant thrombocytopenia combined with severe bleeding may be a life-threatening complication of bone marrow transplantation, especially when traditional therapies are ineffective. Two patients suffering from severe resistant thrombocytopenia were treated with native casein derived peptides.

M-1 (female patient): by age 32, with acute myeloid leukemia, there was complete remission after autologous stem cell transplantation. She had experienced two life-threatening bleeding events including pulmonary hemorrhage and massive obstructive hematoma of the soft palate. Platelet counts were completely unresponsive to rhIL-3, rhIL-6, intravenous gamma globulin and recombinant erythropoietin treatment for a period of more than 114 days post-infusion. Her condition improved immediately after intramuscular treatment with 50mg of native casein-derived peptide, which was injected in 3 injections, twice (each treatment was divided into 3 injections). At the same time as the rapid return of platelet counts to normal (fig. 17), her limb peripheral bleeding and patechyae were also relieved, she was able to return to walking and to foreign homes without any complications and adverse effects.

M-2 (male patient): by age 30, with acute myeloid leukocytes, there was a second complete remission after autologous stem cell transplantation, but there was complete platelet count resistance and major gastrointestinal bleeding. He required daily infusion of packed cells and developed hypoalbuminemia, which was not responsive to various treatments with rhIL-3, rhIL-6 and gamma globulin. A rapid reconstitution of platelets (fig. 18) and a gradual cessation of bleeding was observed 86 days post infusion following intramuscular treatment with two (50 mg each) native casein-derived peptides injected in 3 portions. No further treatment is required and the patient has normal platelet counts and no symptoms at all.

Therefore, it is effective in rapidly reconstituting platelet count and eliminating clinical symptoms associated with fatal bleeding episodes due to resistant thrombocytopenia caused by long-term infusion by administering a native casein-derived peptide twice intramuscularly in 3 divided doses at a dose of 0.7-1.0mg/kg body weight as one treatment course.

The natural casein derived peptides lower triglyceride and total cholesterol levels in patients with familial hyperlipidemia:

m.s. (female patients): the patient was a 38 year old female with a family history of hyperlipidemia. Prior to treatment with native casein-derived peptides, elevated levels of total cholesterol (321mg/dl), triglycerides (213 mg/dl; normal range 45-185mg/dl) and LDL-cholesterol (236.4 mg/dl; normal range 75-174mg/dl) were found by blood biochemical markers. Hyperlipidemia was stabilized by administering 50 μ g of native casein-derived peptide 3 times intramuscularly for 1 month; total cholesterol levels were reduced to 270mg/dl, triglyceride 165mg/dl, and LDL-cholesterol 201mg/dl, which, although still above the normal range, had a significant drop from pre-treatment levels. No other treatment was given. Thus, treatment with native casein-derived peptides can significantly reduce human hyperlipidemia compared to no treatment.

Native casein-derived peptides stimulate the formation of normoglobulinemia in one patient with occult bleeding:

d.c. (male patients): the patient was a 75 year old male with anemia and hypoglobulinemia due to long-term occult bleeding (RBC, HGB, HCT, MCH and MCHC were all decreased). A significant reduction in anemia was observed 1 month after receiving 50 μ g of native casein-derived peptide in two 3 intramuscular injections. After 2 months, RBC reached normal (4.32 rather than 3.44M/. mu.l), HGB increased (11.3 rather than 8.9g/dl), and HCT, MCH and MCHC all improved, approaching normal values, although occult bleeding was also present. Thus, multiple injections of native casein-derived peptides appear to stimulate erythropoiesis in humans, reducing anemia due to blood loss.

It is noted 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 embodiment, may also be provided separately or in any suitable subcombination.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alterations, modifications and variations that fall within the spirit and scope of the appended claims. All publications, patents, patent applications, and sequences identified by number mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, treatment application, or sequence was specifically and individually indicated to be incorporated herein by reference. In addition, citation or reference to any reference in this application is not intended as an admission that such reference is prior art to the present invention.

Sequence listing

Sequence listing

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Claims (365)

1. A method for preventing or treating an autoimmune disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

2. The method of claim 1, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

3. The method of claim 1, wherein the peptide is a synthetic peptide.

4. The method of claim 1, wherein the peptide has a sequence as set forth in one of sequences 1-25.

5. A method of preventing or treating a viral disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

6. The method of claim 5, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

7. The method of claim 5, wherein the peptide is a synthetic peptide.

8. The method of claim 5, wherein the peptide has a sequence as set forth in one of sequences 1-25.

9. A method of preventing a viral infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

10. The method of claim 9, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

11. The method of claim 9, wherein the peptide is a synthetic peptide.

12. The method of claim 9, wherein the peptide has a sequence as set forth in one of sequences 1-25.

13. A method of inducing hematopoiesis, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

14. The method of claim 13, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

15. The method of claim 13, wherein the peptide is a synthetic peptide.

16. The method of claim 13, wherein the peptide has a sequence as set forth in one of sequences 1-25.

17. A method of inducing hematopoietic stem cell proliferation, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

18. The method of claim 17, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

19. The method of claim 17, wherein the peptide is a synthetic peptide.

20. The method of claim 17, wherein the peptide has a sequence as set forth in one of sequences 1-25.

21. A method of inducing hematopoietic stem cell proliferation and differentiation, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

22. The method of claim 21, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

23. The method of claim 21, wherein the peptide is a synthetic peptide.

24. The method of claim 21, wherein the peptide has a sequence as set forth in one of sequences 1-25.

25. A method of inducing megakaryocytopoiesis, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

26. The method of claim 25, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

27. The method of claim 25, wherein the peptide is a synthetic peptide.

28. The method of claim 25, wherein the peptide has a sequence as set forth in one of sequences 1-25.

29. A method of inducing erythropoiesis, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

30. The method of claim 29, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

31. The method of claim 29, wherein said peptide is a synthetic peptide.

32. The method of claim 29, wherein the peptide has a sequence as set forth in one of sequences 1-25.

33. A method of inducing leukopoiesis, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

34. The method of claim 33, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

35. The method of claim 33, wherein said peptide is a synthetic peptide.

36. The method of claim 33, wherein the peptide has a sequence as set forth in one of sequences 1-25.

37. A method of inducing thrombopoiesis, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

38. The method of claim 37, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

39. The method of claim 37, wherein said peptide is a synthetic peptide.

40. The method of claim 37, wherein the peptide has a sequence as set forth in one of sequences 1-25.

41. A method of inducing proliferation of plasma cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

42. The method of claim 41, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

43. The method of claim 41, wherein said peptide is a synthetic peptide.

44. The method of claim 41, wherein the peptide has a sequence as set forth in one of sequences 1-25.

45. A method of inducing dendritic cell proliferation, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

46. The method of claim 45, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

47. The method of claim 45, wherein said peptide is a synthetic peptide.

48. The method of claim 45, wherein the peptide has a sequence as set forth in one of sequences 1-25.

49. A method of inducing macrophage proliferation, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

50. The method of claim 49, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

51. The method of claim 49, wherein said peptide is a synthetic peptide.

52. The method of claim 49, wherein the peptide has a sequence as set forth in one of sequences 1-25.

53. A method for preventing or treating thrombocytopenia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

54. The method of claim 53, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

55. The method of claim 53, wherein said peptide is a synthetic peptide.

56. The method of claim 53, wherein the peptide has a sequence as set forth in one of sequences 1-25.

57. A method for preventing or treating pancytopenia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

58. The method of claim 57, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

59. The method of claim 57, wherein said peptide is a synthetic peptide.

60. The method of claim 57, wherein the peptide has a sequence as set forth in one of sequences 1-25.

61. A method for preventing or treating granulocytopenia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

62. The method of claim 61, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

63. The method of claim 61, wherein said peptide is a synthetic peptide.

64. The method of claim 61, wherein the peptide has a sequence as set forth in one of sequences 1-25.

65. A method for preventing or treating hyperlipidemia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

66. The method of claim 65, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

67. The method of claim 65, wherein said peptide is a synthetic peptide.

68. The method of claim 65, wherein the peptide has a sequence as set forth in one of sequences 1-25.

69. A method for preventing or treating hypercholesterolemia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

70. The method of claim 69, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

71. The method of claim 69, wherein said peptide is a synthetic peptide.

72. The method of claim 69, wherein the peptide has a sequence as set forth in one of sequences 1-25.

73. A method for preventing or treating glucosuria, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

74. The method of claim 73, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

75. The method of claim 73, wherein said peptide is a synthetic peptide.

76. The method of claim 73, wherein the peptide has a sequence as set forth in one of sequences 1-25.

77. A method for preventing or treating diabetes, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

78. The method of claim 77, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

79. The method of claim 77, wherein said peptide is a synthetic peptide.

80. The method of claim 77, wherein said peptide has a sequence as set forth in one of SEQ ID Nos. 1-25.

81. A method for preventing or treating aids, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

82. The method of claim 81, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

83. The method of claim 81, wherein said peptide is a synthetic peptide.

84. The method of claim 81, wherein the peptide has a sequence as set forth in one of sequences 1-25.

85. A method for preventing or treating HIV infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

86. The method of claim 85, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

87. The method of claim 85, wherein said peptide is a synthetic peptide.

88. The method of claim 85, wherein the peptide has a sequence as set forth in one of sequences 1-25.

89. A method of preventing or treating a condition associated with myeloablative doses of radiotherapy and chemotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT), comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

90. The method of claim 89, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

91. The method of claim 89, wherein said peptide is a synthetic peptide.

92. The method of claim 89, wherein said peptide has a sequence as set forth in one of SEQ ID Nos. 1-25.

93. A method of treating an erythropoietin-treatable condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

94. The method of claim 93, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

95. The method of claim 93, wherein said peptide is a synthetic peptide.

96. The method of claim 93, wherein the peptide has a sequence as set forth in one of sequences 1-25.

97. A method of increasing the effect of erythropoietin, which method comprises administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

98. The method of claim 97, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

99. The method of claim 97, wherein said peptide is a synthetic peptide.

100. The method of claim 97, wherein said peptide has a sequence as set forth in one of sequences 1-25.

101. A method of treating a thrombopoietin-treatable condition, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

102. The method of claim 101, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

103. The method of claim 101, wherein said peptide is a synthetic peptide.

104. The method of claim 101, wherein the peptide has a sequence as set forth in one of sequences 1-25.

105. A method of increasing the effect of thrombopoietin, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

106. The method of claim 105, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

107. The method of claim 105, wherein said peptide is a synthetic peptide.

108. The method of claim 105, wherein the peptide has a sequence as set forth in one of sequences 1-25.

109. A method of enhancing peripheral stem cell mobilization, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide derived from an N terminus portion of α S1 casein.

110. The method of claim 109, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

111. The method of claim 109, wherein said peptide is a synthetic peptide.

112. The method of claim 109, wherein the peptide has a sequence as set forth in one of sequences 1-25.

113. A pharmaceutical composition for preventing or treating an autoimmune disease, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

114. The pharmaceutical composition of claim 113, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

115. The pharmaceutical composition of claim 113, wherein said peptide is a synthetic peptide.

116. The pharmaceutical composition of claim 113, wherein said peptide has a sequence as set forth in one of sequences 1-25.

117. A pharmaceutical composition for preventing or treating viral diseases, which comprises, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

118. The pharmaceutical composition of claim 117, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

119. The pharmaceutical composition of claim 117, wherein said peptide is a synthetic peptide.

120. The pharmaceutical composition of claim 117, wherein the peptide has a sequence as set forth in one of sequences 1-25.

121. A pharmaceutical composition for preventing viral infection, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

122. The pharmaceutical composition of claim 121, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

123. The pharmaceutical composition of claim 121, wherein said peptide is a synthetic peptide.

124. The pharmaceutical composition of claim 121, wherein the peptide has a sequence as set forth in one of sequences 1-25.

125. A pharmaceutical composition for inducing hematopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

126. The pharmaceutical composition of claim 117, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

127. The pharmaceutical composition of claim 117, wherein said peptide is a synthetic peptide.

128. The pharmaceutical composition of claim 117, wherein the peptide has a sequence as set forth in one of sequences 1-25.

129. A pharmaceutical composition for inducing hematopoietic stem cell proliferation, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

130. The pharmaceutical composition of claim 121, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

131. The pharmaceutical composition of claim 121, wherein said peptide is a synthetic peptide.

132. The pharmaceutical composition of claim 121, wherein the peptide has a sequence as set forth in one of sequences 1-25.

133. A pharmaceutical composition for inducing proliferation and differentiation of hematopoietic stem cells, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

134. The pharmaceutical composition of claim 133, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

135. The pharmaceutical composition of claim 133, wherein said peptide is a synthetic peptide.

136. The pharmaceutical composition of claim 133, wherein the peptide has a sequence as set forth in one of sequences 1-25.

137. A pharmaceutical composition for inducing megakaryocytopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

138. The pharmaceutical composition of claim 137, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

139. The pharmaceutical composition of claim 137, wherein said peptide is a synthetic peptide.

140. The pharmaceutical composition of claim 137, wherein said peptide has a sequence as set forth in one of sequences 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 α S1 casein, and a pharmaceutically acceptable carrier.

142. The pharmaceutical composition of claim 141, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

143. The pharmaceutical composition of claim 141, wherein said peptide is a synthetic peptide.

144. The pharmaceutical composition of claim 141, wherein the peptide has a sequence as set forth in one of sequences 1-25.

145. A pharmaceutical composition for inducing leukopoiesis, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

146. The pharmaceutical composition of claim 145, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

147. The pharmaceutical composition of claim 145, wherein said peptide is a synthetic peptide.

148. The pharmaceutical composition of claim 145, wherein said peptide has a sequence as set forth in one of sequences 1-25.

149. A pharmaceutical composition for inducing thrombopoiesis, which comprises, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

150. The pharmaceutical composition of claim 149, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

151. The pharmaceutical composition of claim 149, wherein said peptide is a synthetic peptide.

152. The pharmaceutical composition of claim 149, wherein the peptide has a sequence as set forth in one of sequences 1-25.

153. A pharmaceutical composition for inducing proliferation of plasma cells, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

154. The pharmaceutical composition of claim 153, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

155. The pharmaceutical composition of claim 153, wherein said peptide is a synthetic peptide.

156. The pharmaceutical composition of claim 153, wherein said peptide has a sequence as set forth in one of sequences 1-25.

157. A pharmaceutical composition for inducing proliferation of dendritic cells, the pharmaceutical composition comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

158. The pharmaceutical composition of claim 157, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

159. The pharmaceutical composition of claim 157, wherein said peptide is a synthetic peptide.

160. The pharmaceutical composition of claim 157, wherein the peptide has a sequence as set forth in one of sequences 1-25.

161. A pharmaceutical composition for inducing macrophage proliferation, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

162. The pharmaceutical composition of claim 161, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

163. The pharmaceutical composition of claim 161, wherein said peptide is a synthetic peptide.

164. The pharmaceutical composition of claim 161, wherein the peptide has a sequence as set forth in one of sequences 1-25.

165. A pharmaceutical composition for preventing or treating thrombocytopenia, which comprises, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein and a pharmaceutically acceptable carrier.

166. The pharmaceutical composition of claim 165, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

167. The pharmaceutical composition of claim 165, wherein said peptide is a synthetic peptide.

168. The pharmaceutical composition of claim 165, wherein the peptide has a sequence as set forth in one of sequences 1-25.

169. 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 α S1 casein and a pharmaceutically acceptable carrier.

170. The pharmaceutical composition of claim 169, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

171. The pharmaceutical composition of claim 169, wherein said peptide is a synthetic peptide.

172. The pharmaceutical composition of claim 169, wherein the peptide has a sequence as set forth in one of sequences 1-25.

173. 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 α S1 casein and a pharmaceutically acceptable carrier.

174. The pharmaceutical composition of claim 173, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

175. The pharmaceutical composition of claim 173, wherein said peptide is a synthetic peptide.

176. The pharmaceutical composition of claim 173, wherein the peptide has a sequence as set forth in one of sequences 1-25.

177. A pharmaceutical composition for preventing or treating hyperlipidemia, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

178. The pharmaceutical composition of claim 177, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

179. The pharmaceutical composition of claim 177, wherein said peptide is a synthetic peptide.

180. The pharmaceutical composition of claim 177, wherein said peptide has a sequence as set forth in one of sequences 1-25.

181. A pharmaceutical composition for preventing or treating hypercholesterolemia, which comprises, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

182. The pharmaceutical composition of claim 181, wherein said peptide is a fragment derived by fragmentation of α S1 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 set forth in one of sequences 1-25.

185. A pharmaceutical composition for preventing or treating glucosuria, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

186. The pharmaceutical composition of claim 185, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

187. The pharmaceutical composition of claim 185, wherein said peptide is a synthetic peptide.

188. The pharmaceutical composition of claim 185, wherein the peptide has a sequence as set forth in one of sequences 1-25.

189. A pharmaceutical composition for preventing or treating diabetes, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

190. The pharmaceutical composition of claim 189, wherein said peptide is a fragment derived by fragmentation of α S1 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 HIV infection, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

194. The pharmaceutical composition of claim 193, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

195. The pharmaceutical composition of claim 193, wherein said peptide is a synthetic peptide.

196. The pharmaceutical composition of claim 193, wherein said peptide has a sequence as set forth in one of sequences 1-25.

197. A pharmaceutical composition for preventing or treating HIV infection, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

198. The pharmaceutical composition of claim 197, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

199. The pharmaceutical composition of claim 197, wherein said peptide is a synthetic peptide.

200. The pharmaceutical composition of claim 197, wherein the peptide has a sequence as set forth in one of sequences 1-25.

201. A pharmaceutical composition for preventing or treating a condition associated with myeloablative doses of radiotherapy and chemotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT), comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein and a pharmaceutically acceptable carrier.

202. The pharmaceutical composition of claim 201, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

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 sequences 1-25.

205. A pharmaceutical composition for treating a thrombopoietin-treatable condition, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

206. The pharmaceutical composition of claim 205, wherein said peptide is a fragment derived by fragmentation of α S1 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 sequences 1-25.

209. A pharmaceutical composition for increasing the effect of thrombopoietin, comprising, as an active ingredient, a peptide derived from an N terminus portion of α S1 casein and a pharmaceutically acceptable carrier.

210. The pharmaceutical composition of claim 209, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

211. The pharmaceutical composition of claim 209, wherein said peptide is a synthetic peptide.

212. The pharmaceutical composition of claim 209, wherein the peptide has a sequence as set forth in one of sequences 1-25.

213. A pharmaceutical composition for enhancing peripheral stem cell mobilization comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

214. The pharmaceutical composition of claim 213, wherein said peptide is a fragment derived by fragmentation of α S1 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 sequences 1-25.

217. A pharmaceutical composition for inducing hematopoiesis, which comprises, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

218. The pharmaceutical composition of claim 217, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

219. The pharmaceutical composition of claim 217, wherein said peptide is a synthetic peptide.

220. The pharmaceutical composition of claim 217, wherein the peptide has a sequence as set forth in one of sequences 1-25.

221. A pharmaceutical composition for inducing hematopoietic stem cell proliferation, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

222. The pharmaceutical composition of claim 221, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

223. The pharmaceutical composition of claim 221, wherein said peptide is a synthetic peptide.

224. The pharmaceutical composition of claim 221, wherein the peptide has a sequence as set forth in one of sequences 1-25.

225. A pharmaceutical composition for inducing proliferation and differentiation of hematopoietic stem cells, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

226. The pharmaceutical composition of claim 225, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

227. The pharmaceutical composition of claim 225, wherein said peptide is a synthetic peptide.

228. The pharmaceutical composition of claim 225, wherein said peptide has a sequence as set forth in one of sequences 1-25.

229. A pharmaceutical composition for inducing megakaryocytopoiesis, comprising, as active ingredients, thrombopoietin and a peptide derived from an N-terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

230. The pharmaceutical composition of claim 229, wherein said peptide is a fragment derived by fragmentation of α S1 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 sequences 1-25.

233. A pharmaceutical composition for inducing erythropoiesis, the pharmaceutical composition comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

234. The pharmaceutical composition of claim 233, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

235. The pharmaceutical composition of claim 233, wherein said peptide is a synthetic peptide.

236. The pharmaceutical composition of claim 233, wherein said peptide has a sequence as set forth in one of sequences 1-25.

237. A pharmaceutical composition for inducing leukopoiesis, which comprises, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

238. The pharmaceutical composition of claim 237, wherein said peptide is a fragment derived by fragmentation of α S1 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 set forth in one of sequences 1-25.

241. A pharmaceutical composition for inducing thrombopoiesis, which comprises, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

242. The pharmaceutical composition of claim 241, wherein said peptide is a fragment derived by fragmentation of α S1 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 sequences 1-25.

245. A pharmaceutical composition for preventing or treating thrombocytopenia, which comprises, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

246. The pharmaceutical composition of claim 245, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

247. The pharmaceutical composition of claim 245, wherein said peptide is a synthetic peptide.

248. The pharmaceutical composition of claim 245, wherein the peptide has a sequence as set forth in one of sequences 1-25.

249. 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 α S1 casein, and a pharmaceutically acceptable carrier.

250. The pharmaceutical composition of claim 249, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

251. The pharmaceutical composition of claim 249, wherein said peptide is a synthetic peptide.

252. The pharmaceutical composition of claim 249, wherein said peptide has a sequence represented by one of sequences 1-25.

253. A pharmaceutical composition for preventing or treating granulocytopenia, comprising, as active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

254. The pharmaceutical composition of claim 253, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

255. The pharmaceutical composition of claim 253, wherein said peptide is a synthetic peptide.

256. The pharmaceutical composition of claim 253, wherein said peptide has a sequence as set forth in one of sequences 1-25.

257. A pharmaceutical composition for treating or preventing an indication selected from the group consisting of: an autoimmune disease or condition, a viral disease, a viral infection, a hematologic disease, a hematologic defect, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, glucosuria, hyperglycemia, diabetes, aids, HIV-1, helper T cell disorders, dendritic cell defects, macrophage defects, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, human immune system disorders resulting from disease treatment for immunodeficiency and bacterial infection, comprising, as an active ingredient, a peptide derived from the N terminus portion of α S1 casein, and a pharmaceutically acceptable carrier.

258. The pharmaceutical composition of claim 257, wherein said peptide is a fragment derived by fragmentation of α S1 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 depicted in one of sequences 1-25.

261. A pharmaceutical composition for treating or preventing an indication selected from the group consisting of: a hematologic disease, a defect in the blood system, thrombocytopenia, pancytopenia, granulocytopenia, dendritic cell deficiency, macrophage deficiency, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, myelodysplastic syndrome, aplastic anemia, and bone marrow deficiency, comprising, as active ingredients, a peptide derived from the N-terminal portion of thrombopoietin and α S1 casein, and a pharmaceutically acceptable carrier.

262. The pharmaceutical composition of claim 261, wherein said peptide is a fragment derived by fragmentation of α S1 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 sequences 1-25.

265. A purified peptide having an amino acid sequence selected from the group consisting of sequences 1-25.

266. A pharmaceutical composition comprising a purified peptide having an amino acid sequence selected from the group consisting of sequences 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 sequences 1-25 and a pharmaceutically acceptable carrier.

268. A method of enhancing colonization of a donated blood stem cell in a myeloablative recipient, the method comprising treating a donor of the donated blood stem cell with a peptide derived from an N terminus portion of α S1 casein prior to donation and implantation of the donated blood stem cell in the recipient.

269. The method of claim 268, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

270. The method of claim 268, wherein said peptide is a synthetic peptide.

271. The method of claim 268, wherein the peptide has a sequence set forth in one of sequences 1-25.

272. A method of enhancing colonization of a donated blood stem cell in a myeloablative recipient, the method comprising treating the donated blood stem cell with a peptide derived from an N terminus portion of α S1 casein prior to implanting the donated blood stem cell in the recipient.

273. The method of claim 272, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

274. The method of claim 272, wherein said peptide is a synthetic peptide.

275. The method of claim 272, wherein the peptide has a sequence set forth in one of sequences 1-25.

276. A method of enhancing the colonization of blood stem cells in a myeloablative recipient, the method comprising treating the blood stem cells with a peptide derived from an N terminus portion of α S1 casein prior to implantation of the blood stem cells in the recipient.

277. The method of claim 276, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

278. The method of claim 276, wherein said peptide is a synthetic peptide.

279. The method of claim 276, wherein the peptide has a sequence set forth in one of sequences 1-25.

280. A method of enhancing colonization of a donated blood stem cell in a myeloablative recipient, the method comprising treating a donor of the donated blood stem cell with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to donation and implantation of the donated blood stem cell in the recipient.

281. The method of claim 280, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

282. The method of claim 280, wherein said peptide is a synthetic peptide.

283. The method of claim 280, wherein the peptide has a sequence as set forth in one of sequences 1-25.

284. A method of enhancing colonization of a donated blood stem cell in a myeloablative recipient, the method comprising treating the donated blood stem cell with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to implanting the donated blood stem cell in the recipient.

285. The method of claim 284, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

286. The method of claim 284, wherein said peptide is a synthetic peptide.

287. The method of claim 284, wherein the peptide has a sequence set forth in one of sequences 1-25.

288. A method of enhancing the colonization of blood stem cells in a myeloablative recipient, the method comprising treating the blood stem cells with a peptide derived from an N terminus portion of α S1 casein and thrombopoietin prior to implantation of the blood stem cells in the recipient.

289. The method of claim 288, wherein said peptide is a fragment derived by fragmentation of α S1 casein.

290. The method of claim 288, wherein said peptide is a synthetic peptide.

291. The method of claim 288, wherein the peptide has a sequence set forth in one of sequences 1-25.

292. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of an autoimmune disease.

293. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of a viral disease.

294. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention of viral infection.

295. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing hematopoiesis.

296. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing hematopoietic stem cell proliferation.

297. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing hematopoietic stem cell proliferation and differentiation.

298. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing megakaryocytopoiesis.

399. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing erythropoiesis.

300. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing leukopoiesis.

301. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing thrombopoiesis.

302. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing plasma cell proliferation.

303. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing dendritic cell proliferation.

304. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for inducing macrophage proliferation.

305. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of thrombocytopenia.

306. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of pancytopenia.

307. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of granulocytopenia.

308. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of hyperlipidemia.

309. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of hypercholesterolemia.

310. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of glucosuria.

311. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of diabetes.

312. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of aids.

313. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of HIV infection.

314. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the prevention or treatment of a condition associated with myeloablative doses of radiotherapy and chemotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT).

315. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for the treatment of a thrombopoietin-treatable condition.

316. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for increasing the effect of thrombopoietin.

317. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for enhancing peripheral stem cell mobilization.

318. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for enhancing the colonization of provided blood stem cells in vivo at a myeloablative receptor.

319. Use of a peptide derived from an N terminus portion of α S1 casein in the manufacture of a medicament for enhancing blood stem cell colonization in a myeloablative receptor.

320. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating an autoimmune disease.

321. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating viral diseases.

322. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating viral infection.

323. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing hematopoiesis.

324. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cell proliferation.

325. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cell proliferation and differentiation.

326. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing megakaryocytopoiesis.

327. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing erythropoiesis.

328. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing leukopoiesis.

329. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing thrombopoiesis.

330. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing proliferation of plasma cells.

331. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing dendritic cell proliferation.

332. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing macrophage proliferation.

333. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating thrombocytopenia.

334. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating pancytopenia.

335. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia.

336. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating hyperlipidemia.

337. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating hypercholesterolemia.

338. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating glucosuria.

339. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating diabetes.

340. The use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for the prevention or treatment of aids.

341. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating HIV infection.

342. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating a condition associated with myeloablative doses of radiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic Bone Marrow Transplantation (BMT).

343. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for treating a thrombopoietin-treatable condition.

344. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for increasing the effect of thrombopoietin.

345. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing colonization of provided blood stem cells in a myeloablative recipient.

346. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing blood stem cell colonization in a myeloablative recipient.

347. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing peripheral stem cell mobilization.

348. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing hematopoiesis.

349. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing hematopoietic stem cell proliferation.

350. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, together with a pharmaceutically acceptable carrier, for inducing proliferation and differentiation of hematopoietic stem cells.

351. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing megakaryocytopoiesis.

352. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing erythropoiesis.

353. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing leukopoiesis.

354. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for inducing thrombopoiesis.

355. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating thrombocytopenia.

356. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating pancytopenia.

357. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating granulocytopenia.

358. Use of a pharmaceutical composition comprising, aS an active ingredient, a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating an indication selected from the group consisting of: autoimmune diseases or conditions, viral diseases, viral infections, hematologic diseases, hematologic defects, thrombocytopenia, pancytopenia, granulocytopenia, hyperlipidemia, hypercholesterolemia, diabetes mellitus, hyperglycemia, diabetes mellitus, aids, HIV-1, helper T cell disorders, dendritic cell defects, macrophage defects, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, immune system disorders resulting from chemotherapy or radiation therapy, human immune system disorders resulting from disease treatment of immune deficiencies and bacterial infections.

359. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for preventing or treating an indication selected from the group consisting of: hematologic disorders, hematologic deficiencies, thrombocytopenia, pancytopenia, granulocytopenia, dendritic cell deficiencies, macrophage deficiencies, hematopoietic stem cell disorders including platelet, lymphocyte, plasma cell and neutrophil disorders, pre-leukemic conditions, myelodysplastic syndromes, aplastic anemia, and bone marrow deficiencies.

360. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing colonization of provided blood stem cells in a myeloablative recipient.

361. Use of a pharmaceutical composition comprising, aS active ingredients, thrombopoietin and a peptide derived from an N terminus portion of α s1 casein, and a pharmaceutically acceptable carrier for enhancing blood stem cell colonization in a myeloablative recipient.

362. The use of any one of claims 292-361, wherein the peptide is a fragment derived by fragmentation of α S1 casein.

363. The use of any one of claims 292-361, wherein the peptide is a purified peptide having a sequence as shown in one of sequences 1-25.

364. The use of any one of claims 292-361, wherein the peptide is a synthetic peptide.

365. The use of any one of claims 292-361, wherein the peptide has a sequence as shown in one of sequences 1-25.