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WO1995003067A1 - Agents pharmaceutiques a activite immunomodulatrice - Google Patents

Agents pharmaceutiques a activite immunomodulatrice Download PDF

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Publication number
WO1995003067A1
WO1995003067A1 PCT/RU1994/000162 RU9400162W WO9503067A1 WO 1995003067 A1 WO1995003067 A1 WO 1995003067A1 RU 9400162 W RU9400162 W RU 9400162W WO 9503067 A1 WO9503067 A1 WO 9503067A1
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WIPO (PCT)
Prior art keywords
glu
lys
cells
thymogen
cell
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PCT/RU1994/000162
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English (en)
Inventor
Vladimir Khatskelevich Khavinson
Sergei Vladimirovich Sery
Vladimir Viktorovich Malinin
Original Assignee
Khavinson Vladimir Khatskelevi
Sergei Vladimirovich Sery
Vladimir Viktorovich Malinin
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Application filed by Khavinson Vladimir Khatskelevi, Sergei Vladimirovich Sery, Vladimir Viktorovich Malinin filed Critical Khavinson Vladimir Khatskelevi
Priority to AU73922/94A priority Critical patent/AU7392294A/en
Publication of WO1995003067A1 publication Critical patent/WO1995003067A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • L-lysyl-L-glutamic acid L-Lys-L-Glu
  • L-glutamyl-L-tryptophane L-Glu-L-Tip
  • Tissue extracts that affect proliferation and/or differentiation of T-lymphocytes have been reported, including thymus extracts of animal origin, e.g., "thymosine" (1),
  • tissue extracts exist as complex mixtures that includes polypeptides. Widespread use of such biological mixtures in medical practice has been hindered by the lack of highly purified and characterized sources, as well as by the low yields obtainable, and, once isolated, the considerable variability of chemical and physical properties potentially effecting potency, stability, toxicity, and safety.
  • Thymogen is reportedly prepared as a synthetic L-Glu-L-Trp product whose design is in accord with a dipeptide in a fraction isolated a thymalin (2) extract of bovine thymus.
  • T-lymphocytes participate in cellular and humoral immune responses to foreign antigens that are triggered following binding of ligand to cell surface receptors.
  • the CD2 receptor on T-lymphocytes (previously known as the erythrocyte rosette receptor- abbreviated E-receptor) serves a dual role as both an adhesion molecule and a signal transducing molecule.
  • CD2 binding to LFA-3 may promote adhesive binding of T-lymphocytes to LFA3 (CD58) B-lymphocytes and thymic epithelial cells.
  • CD2 binding to CD59 and CD48 ligands may facilitate binding to other cells.
  • CD2 is a cell surface molecule bound by PHA and involved in PHA-induced lymphocyte blastogenesis.
  • CD4 and CD8 define MHC class specificity of T helper and cytotoxic T lymphocytes, respectively.
  • L-Lys-L-Glu dipeptide and Thymogen are capable of increasing expression of CD2 on T-lymphocytes and thymocytes, altering tissue distribution of lymphocyte subpopulations in experimental animal model systems, and stimulating immune cells involved in antimicrobial cellular immunity.
  • L-Lys-L-Glu and Thymogen also stimulated increased expression of CD4 (but not CD8) on peripheral blood lymphocytes isolated from patients with secondary immunodeficiency syndromes.
  • L-Lys-L-Glu and Thymogen altered the tissue distribution of T-lymphocytes and lymphocytes bearing cell surface Fc-receptors, and L-L, and L-Lys-L-Glu increased splenic weight and T-lymphocyte content.
  • L-Lys-L-Glu and Thymogen treatments increased the activation state of resident macrophages in experimental animal studies (as measured by NBT reduction); and, promoted neutrophil infiltration in response to a sterile inflammatory mediator (proteose peptone).
  • L-Lys-L-Glu and Thymogen treatments stimulated immune cells and reticuloendotheiial tissues participating in antimicrobrial mechanisms of cellular and humoral immunity, and indicate that the subject treatments heighten the state of antimicrobial immunity.
  • L-Lys-L-Glu and Thymogen are termed "immunomodulators" capable of heightening the state anti-microbial cellular or humoral immunity in a treated subject.
  • Embodiments of the invention provide methods of treatment with L-Lys-L-Glu (KE) and Thymogen to induce a heightened state of anti-microbial cellular or humoral immunity in a subject in need thereof.
  • KE L-Lys-L-Glu
  • T-lymphocytes from patients with secondary immunodeficiency syndromes following incubation with either dipeptide.
  • Increased expression of CD2 and CD4 accessory molecules on T-lymphocytes is compatible a heighten the state of innate or induced immunity to infection, e.g., by upregulating T-helper and cytotoxic T-lymphocytes to respond to lower levels of antigen.
  • L-Lys-L-Glu appeared to be more than 100-times more effective than Thymogen at increasing cell surface expression of CD2 and CD4 on lymphocytes. Neither dipeptide upregulated CD8 expression on lymphocytes.
  • CD2 is intended to mean the lymphocyte cell surface accessory molecule that is the homo- and heterotypic receptor mediating binding of heterologous erythrocytes (E; e.g., rabbit or sheep erythrocytes) to lymphocytes to form E-rosettes (as disclosed in Paul, W.E. Ed. "Fundamental Immunology, 3rd. Edition, Raven Press, N.Y. at page 562 ; incorporated herein by reference). Lymphocytes having cell surface CD2, when capable of forming rosettes with rabbit erythrocytes are referred to herein as E-rosette forming cells, abbreviated E-RFC.
  • E-RFC E-rosette forming cells
  • CD4 + lymphocyte is intended to mean a lymphocyte having a plurality of cell surface CD4 molecules as evidenced by binding of an antibody specific for CD4 to the cell surface, e.g., monoclonal antibody OKT4 (Ortho Diagnostics, Piscatawy, N.J.).
  • an antibody specific for CD4 e.g., monoclonal antibody OKT4 (Ortho Diagnostics, Piscatawy, N.J.).
  • CD8 + lymphocyte is intended to mean a lymphocyte having a plurality of cell surface CD8 molecules as evidenced by binding of an antibody specific for CD8 to the cell surface, e.g., monoclonal antibody OKT8 (Ortho Diagnostics, Piscatawy, NJ).
  • an antibody specific for CD8 e.g., monoclonal antibody OKT8 (Ortho Diagnostics, Piscatawy, NJ).
  • Reticuloendotheiial system is intended to mean immune tissues containing macrophages, lymphocytes, reticular cells, mast cells, basophils, eosinophils, neutrophils, and the like.
  • Representative components of the reticuloendotheiial system include lymph nodes, spleen, thymus, bone marrow, lung, liver, epithelial tissues, lymphatic vessels, blood, and the like.
  • PBL 'Teripheral blood leukocytes
  • lymphocytes e.g., lymphocytes, monocytes, eosinophils, basophils, neutrophils, plasma cells, mast cell precursors, and the like.
  • Immunocell is intended to encompass the cell types of the reticuloendotheiial system, e.g., lymphocytes, mononuclear phagocytes, neutrophils, basophils, eosinophils, mast cells, plasma cells, reticular cells in the spleen, Langerhans cells and ⁇ -bearing lymphocytes in the epithelia, Kupfer cells in the liver, and the like.
  • Lymphocytes is intended to encompass T-lymphocytes, (also referred to as T-cells), B-lymphocytes (also referred to as B-cells), natural killer-lymphocytes (NK-cells), cytotoxic T lymphocytes (CTL), T-helper lymphocytes, as well as precursors of the former and activated derivatives thereof.
  • Activated lymphocyte is intended to mean that subset of lymphocytes which has been i) exposed to a stimulus, and ii) has been triggered to change from a metabolically-quiescent cell into a cell with increased protein synthesis and/or DNA and RNA synthesis and possibly cell division.
  • Illustrative stimuli and triggering pathways leading to activated lymhocytes include interaction of Tcell receptors with antigens, interaction of interleukin receptors with interleukins, interaction of growth factor receptors with growth factors, interaction of lymphocyte cell surface determinants with antibody (e.g., anti-CD2) or mitogens (e.g., PHA), and the like.
  • Mononuclear phagocyte is intended to mean cells of the monocyte/macrophage lineage including e.g., monocytes, macrophages, dendritic cells, reticular cells, Langerhans cells, and the like.
  • activated macrophage is intended to mean a mononuclear phagocyte having an increased capacity for phagocytization and intracellular killing of a microbe.
  • activated macrophages may be recognized for example by biochemical markers (e.g., 5'-nucleotidase), increased oxidative metabolism (e.g., increased ability to reduce nitrotetrazolium blue dye, NBT), and the like.
  • Interle kin is intended to mean an agent released by a first immune cell that affectsa biological activity in a second immune cell.
  • Representative interleukins include cytokines such as tumor necrosis factors (e.g., TNF ⁇ and TNF ⁇ ), IL-1, IL-2 (also known as T cell growth factor), IL-3, IL-4, IL-5, IL-6, IFN- ⁇ and the like.
  • G-CSF granulocyte colony stimulating factor
  • M- CSF macrophage colony stimulating factor
  • GM-CSF granulocyte/macrophage colony stimulating factor
  • SCF stem cell factor
  • Interleukins that have growth stimulatory activity are included as a subcategory within this usage, (e.g., IL-2, IL-3, IL-6 and the like), as are compounds having a mitogenic effect on immune cells (i.e., PHA, Con-A, LPS, and the like).
  • Anti-microbial cellular and humoral immunity is intended to mean that immunity which ameliorates (makes better) or eliminates one or more clinical or laboratory indicia of disease produced by a microbe.
  • Merobe is intended to mean an agent that when multiplying in a subject causes a disease.
  • Representative microbes include viruses, bacteria, mycoplasma, mycobacteria, parasites, rickettsia, dengue fever agent, prion disease agent, kuru kuru disease agent, and the like.
  • Representative examples of clinical indicia of disease include (but are not limited to) redness and swelling, fever, malaise, septicemia, pyogenic exudates, and the like.
  • Laboratory indicia include (but are not limited to) abnormal values in measurements of: (i) neutrophil or lymphocyte counts in peripheral blood; (ii) hematocrit; (iii) serum alpha globulins or immunoglobulins; (iv) expression of one or more cell surface accessory molecules on an immune cell, for example, molecules indicating a maturation state or activation state of either a T-lymphocyte (e.g., CD2, CD3, CD4, CD28, and the like) or a B-lymphocyte (e.g., B220, surface Ig, expression of rearrangement-recombinase activating genes in Rag 1 and Rag 2, and the like) or a mononuclear phagocyte (e.g., Ia/Mac-1 expression, 5'-nucleotidase activity and the like); (v) synthesis of one or more interleukins (e.g., IL-2, IL-1, TNF, TGF- ⁇ , IFN- ⁇ , and the like);
  • Cellular immunity to a subject microbe is intended to mean that immunity which is conferred upon a host by immune cells and which e.g. in an experimental animal model system may be transferred from one animal to another using immune cells that are free of serum, and that is free of B-lymphocytes and/or plasma cells producing an immunoglobulin specifically reactive with the subject microbe.
  • Human immunity to a subject microbe is intended to mean that immunity which is conferred upon a host by antibody and which e.g. in an experimental animal model system may be transferred from one animal to another using immune serum that contains antibody that is free of cells, or alternatively, by B-lymphocytes and/or plasma cells that are producing antibody specifically reactive with the subject microbe.
  • “Innate anti-microbial cellular immunity” is used to mean that immunity which is preexistent in a host before exposure to a microbe such as e.g., that which is conferred by Langerhans cells and T-lymphocytes bearing ⁇ -T cell receptor molecules in epithelial tissues, or immune cells in biological fluids.
  • the term is intended to encompass anti-microbial activities of neutrophils, monocytes, platelets, macrophages, dendritic cells, and Langerhans cells, and not the activities of serum proteins such as coagulation factors, complement, lysozyme and the like.
  • Subject in need thereof is intended to mean a mammal, e.g., humans, domestic animals and livestock, having one or more clinical or laboratory indicia of infection with a microbe.
  • the subject may exhibit clnical disease activity or may have a subclinical or latent infection.
  • Polypeptide is intended to mean a serial array of amino acids of more than 16 and up to many hundreds of amino acids in length, e.g., a protein.
  • Embodiments of the invention provide methods for treating a subject to induce a heightened state of anti-microbial cellular or humoral immunity by administering a pharmaceutical preparation of L-Lys-L-Glu or L-Glu-L-Trp.
  • Preparations containing both L-Lys-L-Glu and L-Glu-L-Trp are also envisaged, as well as preparations containing one or both dipeptides in mixture with one or more antibiotics.
  • The is preferably a patient having a microbial infection.
  • the subject may optionally have an underlying immunodeficiency syndrome that occassions frequent, recurrent, or intermittent microbial infection.
  • the subject underlying syndromes include (but are not limited to) hereditary and acquired immunodeficiency syndromes such as primary immunodeficiency syndromes (e.g., DiGeorge Syndrome, Severe Combined Immunodeficiency Syndrome, Combined Immunodeficiency, X-linked lymphoproliferative disease); syndromes resulting from an underlying autoimmune disease (e.g., diabetes, systemic lupus erythematosus, Sjogrens syndrome); syndromes resulting from an underlying infection (e.g., HIN infection); syndromes resulting from a toxic reaction to a drug or a chemical; or secondary immunodeficiency disorders such as those disclosed in EXAMPLE 4, below).
  • primary immunodeficiency syndromes e.g., DiGeorge Syndrome, Severe Combined Immunodeficiency Syndrome, Combined Immunodeficiency, X-linked lymphoproliferative disease
  • syndromes resulting from an underlying autoimmune disease e.g., diabetes, systemic l
  • the subject treatment methods of the invention are preferably effective to ameliorate or eliminate one or more clinical or laboratory indicia of disease.
  • the subject treatment methods may be useful to decrease redness and swelling, fever, malaise, septicemia, pyogenic exudates, and the like, or to restore one or more measurements of a laboratory indicia of disease to a more normal value or to increase the subject value above the normal range to achieve increased anti-microbial immunity: for example, i) decreasing an elevated neutrophil or lymphocyte count in peripheral blood, ii) increasing an abnormally low hematocrit, iii) decreasing an elevated serum alpha globulin or immunoglobulin level, iv) increasing abnormally low expression of one or more cell surface accessory molecules on an immune cell, such as molecules indicating a maturation state or activation state of either a T-lymphocyte (e.g., CD2, CD3, CD4, CD28, and the like) or a B-lymphocyte (e.g., B220, surface Ig, expression of
  • a treatment course of about 10 ⁇ g kg to about 1 mg kg of a pharmaceutical preparation of L-Lys-L-Glu or L-Glu-L-Trp is administered to a subject daily over a period of about 3 days to about 10 days and optionally at the discretion of the attending physician the treatment course may be repeated after about 1 to about 6 months intermission.
  • L-Glu-L-Trp is administered by im injection daily to adults at 50-100 ⁇ g for 3-10 days and the attending physician prescribes a repeat course of therapy after 1-6 months if additional treatment is required.
  • the subject methods include those in which the L-Lys-L-Glu or L-Glu-L-Trp are administered by injection, e.g., by parenteral, intramuscular, intradermal, subcutaneous, and intraperitoneal injection.
  • the treatment dose is sufficient to increase a number or percentage of CD2 or Fc-receptor bearing lymphocytes in peripheral blood or in a reticuloendotheiial tissue, or to increase a number of immune cells in an inflammatory infiltrate, or to increase a proportion of phagocytically active cells in an inflammatory infiltrate.
  • L-Lys-L-Glu treatment or "L-Glu-L-Trp treatment” is intended to mean a method of delivering to a subject in need thereof a pharmaceutical preparation of L-Lys-L-Glu or L-Glu-L-Trp with the aim of treating or preventing one or more clinical or laboratory indicia of disease in the subject.
  • the subject methods include delivering the preparation to a patient i) before the disease has been diagnosed, e.g., prophylactic protocols delivered with the aim of preventing development of the disease, as well as, ii) after the disease has been diagnosed, e.g., therapeutic protocols.
  • the subject methods of the invention find a variety of prophylactic and therapeutic uses in treatment of immune pathophysiologic conditions in man and domestic animals.
  • the methods of the invention find use during in vitro maintenance and expansion of bone marrow, peripheral blood leukocytes, CD34 + lymphocytes, and other immune cells such as may occur prior to autologous or allogenic transplantation.
  • the KE- and EW-dipeptides of the invention may be administered in an intravenous bolus injection (or by perfusion) during (or after) surgery as a prophylactic treatment to prevent infection and the like
  • the dipeptides disclosed herein have the advantage of providing the desired effects at very low dosage levels and without toxicity.
  • a purpose of therapy in an acute setting may be to rapidly increase the concentration of an KE or EW in a tissue, e.g., by bolus intravenous injection or infusion. Alternatively, in other cases it may desirable to deliver the KE or EW over a longer period of time.
  • the compounds themselves are water-soluble at the low concentrations at which they are usually employed, they are preferably used in the form of their acid salts with pharmaceutically acceptable salts, e.g., acetic, citric, maleic, or succinic acid (as disclosed in greater detail below).
  • Freely-soluble salts of the KE or EW of this invention may also be converted to salts of low solubility in body fluids by modification with a slightly water-soluble pharmaceutically acceptable salt, e.g., tannic or palmoic acid, or by inclusion in a time-release KE or EW formulation such as covalently coupled to a larger carrier protein or peptide, or in timed-release capsules and the like.
  • a slightly water-soluble pharmaceutically acceptable salt e.g., tannic or palmoic acid
  • a time-release KE or EW formulation such as covalently coupled to a larger carrier protein or peptide, or in timed-release capsules and the like.
  • the acid addition salts of the subject KE and EW dipeptides with pharmaceutically acceptable acids will be biologically equivalent to the subject dipeptides themselves.
  • Multimers of KE and EW are also envisaged, such as easily hydrolyzable polypeptides (e.g., anhydrous chlorides or fluorides and the like) which when introduced into aqueous solution are hydrolized to monomeric KE.
  • "Multimer” as used herein is intended to mean a molecule containing two or more KE or EW.
  • Representative multimers include molecules in which two or more KE or EW covalently bonded to a common linker, or in which the two or more KE or EW dipeptides are non-covalently linked together through the linker (e.g., through ion or hydrophobic interactions).
  • the route of delivery of the KE or EW dipeptides and the like is determined by the disease and the site where treatment is required. For topical application it may be desirable to apply the KE or EW, analogs, agonists, and antagonists at the local site (e.g., by placing a needle into the tissue at that site) or by placing an impregnated bandage during surgery); while for more advanced diseases it may be desirable to administer the compositions systemically.
  • the KE or EW dipeptides, analogs, agonists, antagonists, derivatives and the like may be delivered by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, and intradermal injection, as well as, by intrabronchial instillation (e.g., with a nebulizer), transdermal delivery (e.g., with a lipid-soluble carrier in a skin patch), or gastrointestinal delivery (e.g., with a capsule or tablet).
  • the preferred therapeutic compositions for inocula and dosage will vary with the clinical indication.
  • the inocula is typically prepared from a dried peptide (or peptide conjugate) by suspending the peptide in a physiologically acceptable diluent such as water, saline, or phosphate-buffered saline.
  • a physiologically acceptable diluent such as water, saline, or phosphate-buffered saline.
  • Some variation in dosage will necessarily occur depending upon the condition of the patient being treated, and the physician will, in any event, determine the appropriate dose for the individual patient.
  • the effective amount of peptide per unit dose depends, among other things, on the body weight, physiology, and chosen inoculation regimen.
  • a unit does of peptide refers to the weight of peptide without the weight of carrier (when carrier is used).
  • KE or EW dipeptide at a tissue site in the microenvironment of the cells approaches a concentration of 10 "5 M to 10 "9 M.
  • Skilled practitioners can make use of clinical and laboratory indicia (above) to monitor patient response to the subject therapy and adjust the dosage accordingly. Since the pharmacokinetics and pharmacodynamics of KE and EW dipeptides, agonists, antagonists, and the like will vary in different patients, a most preferred method for achieving a therapeutic concentration in a tissue is to gradually escalate the dosage and monitor the clinical and laboratory indicia (above). The initial dose, for such an escalating dosage regimen of therapy, will depend upon the route of administration.
  • a KE or EW dipeptide with an approximate molecular weight of 200 to 400 daltons, an initial dosage of approximately 0.1 mg/kg body weight is administered and the dosage is escalated at 10-fold increases in concentration for each interval of the escalating dosage regimen.
  • L-Lys-L-Glu is commercially available (6), and is conveniently synthesize in solution reactions using classical methods of peptide synthesis such as the illustrative stepwise synthesis set forth in EXAMPLE 1, below.
  • the subject KE- and EW-dipeptides are synthesized by any of a number of automated techniques that are now commonly available. Generally speaking, these techniques involve stepwise synthesis by successive additions of amino acids to produce progressively larger molecules. The amino acids are linked together by condensation between the carboxyl group of one amino acid and the amino group of another amino acid to form a peptide bond. To control these reactions, it is necessary to block the amino group of one amino acid and the carboxyl group of the other.
  • the blocking groups should be selected for easy removal without adversely affecting the peptides, i.e., by racemization or by hydrolysis of the formed peptide bonds.
  • Amino acids with carboxyl- groups e.g., Asp, Glu
  • hydroxyl- groups e.g., Ser, homoserine, and tyrosine
  • peptides are synthesized by loading the carboxy- terminal amino acid onto an organic linker (e.g., PAM, 4-oxymethyl phenylacetamidomethyl) covalently attached to an insoluble polystyrene resin that is cross-linked with divinyl benzene. Blocking with t-Boc is used to protect the terminal amine, and hydroxyl- and carboxyl- groups are commonly blocked with O-benzyl groups. Synthesis is accomplished in an automated peptide synthesizer (Applied Biosystems, Foster City, CA, e.g., Model 430-A).
  • an organic linker e.g., PAM, 4-oxymethyl phenylacetamidomethyl
  • the product may be removed from the resin and blocking groups removed using hydrofluoric acid or trifluoromethyl sulfonic acid according to established methods (Bergot, B.J. and S.N. McCurdy, Applied Biosystems Bulletin, 1987).
  • a routine synthesis can produce 0.5 mmole of peptide-resin. Yield following cleavage and purification is approximately 60 to 70%.
  • Purification of the product peptides is accomplished for example by crystallizing the peptide from an organic solvent such as methyl-butyl ether, followed by dissolving in distilled water, and dialysis (if greater than about 500 molecular weight) or reverse HPLC (e.g., using a C18 column with 0.1% trifluoroacetic acid and acetonitrile as solvents) if less than 500 molecular weight.
  • Purified peptide is lyophilized and was stored in a dry state until use.
  • Pharmaceutically acceptable salts may be conveniently prepared from KE or EW dipeptides or analogs by conventional methods.
  • such salts may be, for example, prepared by treating a KE or EW dipeptide with an aqueous solution of the desired pharmaceutically acceptable metallic hydroxide or other metallic base and evaporating the resulting solution to dryness, preferably under reduced pressure in a nitrogen atmosphere.
  • a solution of a KE or EW dipeptide may be mixed with an alkoxide to the desired metal, and the solution subsequently evaporated to dryness.
  • the pharmaceutically acceptable hydroxides, bases, and alkoxides include those with cations for this purpose, including (but not limited to), potassium, sodium, ammonium, calcium, and magnesium.
  • compositions include hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valarate, oleate, laurate, borate, benzoate, lactate, phosphate, tosulate, citrate, maleate, furmarate, succinate, tartrate, and the like.
  • the compounds may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses.
  • suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions, and various nontoxic organic solvents.
  • the subject combination therapeutic agents are then readily administered in a variety of dosage forms such as tablets, lozenges, syrups, injectable solutions, and the like.
  • Combination therapeutic agents may also include both KE- and EW-dipeptide in the same unit dosage form.
  • Pharmaceutical carriers can, if desired, contain additional ingredients such as flavorings, binders, excipients, and the like.
  • tablets containing various excipients such as sodium citrate, calcium carbonate, and calcium phosphate may be employed along with various disintegrants such as starch, and preferably potato or tapioca starch, alginic acid, and certain complex silicates, together with binding agents such as polyvinylpyrolidone, sucrose, gelatin, and acacia.
  • disintegrants such as starch, and preferably potato or tapioca starch, alginic acid, and certain complex silicates
  • binding agents such as polyvinylpyrolidone, sucrose, gelatin, and acacia.
  • lubricating agents such as magnesium sterarate, sodium lauryl sulfate, and talc are often useful for tableting purposes.
  • Solid compositions of a similar type may also be employed as fillers in salt and hard-filled gelatin capsules. Preferred materials for this purpose include lactose or milk sugar and high molecular weight polyethylene glycols.
  • the essential active KE- or EW-dipeptide ingredients therein may be combined with various sweetening or flavoring agents, colored matter or dyes, and if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof.
  • solutions of the KE or EW, analog, or receptor fragment in sesame or peanut oil or in aqueous polypropylene glycol may be employed, as well as sterile aqueous saline solutions of the corresponding water soluble pharmaceutically acceptable metal salts previously described.
  • Such an aqueous solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal injection.
  • the sterile aqueous media employed are all readily obtainable by standard techniques well known to those skilled in the art. Additionally, it is possible to administer the aforesaid compounds topically (e.g., through a placed catheter) using an appropriate solution suitable for the purpose at hand.
  • shelf life stability is improved by adding excipients such as: a) hydrophobic agents (e.g., glycerol); b) sugars (e.g., sucrose, mannose, sorbitol, rhamnose, xylose); c) complex carbohydrates (e.g., lactose); and/or d) bacteriostatic agents.
  • hydrophobic agents e.g., glycerol
  • sugars e.g., sucrose, mannose, sorbitol, rhamnose, xylose
  • complex carbohydrates e.g., lactose
  • bacteriostatic agents e.g., lactose
  • Pharmacokinetic half-life of peptides is modified by coupling to carrier peptdes, polypeptides, and carbohydrates by chemical derivatization (e.g., by coupling side chain or N- or C-terminal residues), or chemically altering the amino acid to another amino acid (as above).
  • Pharmacokinetic half-life and pharmacodynamics may also be modified by: a) encapsulation (e.g., in liposomes); b) controlling the degree of hydration (e.g.,. by controlling the extent and type of glycosylation of the peptide); and, c) controlling the electrostatic charge and hydrophobicity of the peptide.
  • BM spleen and bone marrow
  • Phagocytic cells (%) 1.5 X t (p ⁇ 0.05) 1.7 X t p ⁇ 0.05)
  • EXAMPLES 2-6 The disclosed effects of L-Lys-L-Glu and Thymogen on macrophages and neutrophils (EXAMPLES 2-6) are compatible with the interpretation that the subject treatments of the invention heighten the state of innate immunity to infection by i) activating anti-microbial activity of resident macrophages; and, ii) promoting neutrophil infiltration into tissues in response to inflammatory agents.
  • KE and EW dipeptides are non-toxic to human and guinea pig lymphocytes in vitro, and non-toxic when in injected intraperitoneally into guinea pigs and mice as disclosed in the EXAMPLES, below.
  • KE and EW dipeptides also permits the preparation of appropriate nucleotide sequences (e.g., by standard techniques, and incorporation of these sequences into bacterial, yeast, and insect plasmid DNA's, as well as into mammalian cell viral vectors (e.g., retroviral vectors.)
  • Expression systems that may be used to produce the peptides of the invention include prokaryotic, eukaryotic, yeast, and insect cells. It is presently believed highly likely that KE and EW are cytomedines released from hydrolysis of tissue polypeptides, at a rate homeostatically determined (at least) by tissue pH and the enzyme activity in the tissues.
  • the present disclosure serves as a useful basis for constructing derivatized and covalently modified KE or EW analogs, antagonists, and the like.
  • the KE or EW may be used for preparation of an analog that is e.g. a) covalently modified by adenylation, methylation, acylatio ⁇ , phosphorylation, uridylation, fatty-acylation, glycosylation, and the like; b) a sterioisomer of an L-Lys-L-Glu or L-Glu-L-Trp, e.g., replacing a D- for an L- sterioisomer, and the like; c) a derivative of KE or EW, wherein one amino acid is substituted for another of like properties, i.e., a neutral nonpolar amino acid for another neutral nonpolar (e.g., W replaced by S, T, Y, N, Q, or C), an acidic amino acid for another acid (e.g., E replaced by D
  • Bifunctional KE or EW dipeptides may also be synthesized to combine advantageous properties of both KE and EW into a single molecule (e.g., a molecule according to Formula ⁇ , below), or as a mixture of KE and EW.
  • the KE and EW dipeptides disclosed herein also provide basis for organic synthesis of molecules that include derivatives, analogues, agonists and the like, that have immunomodulatory activity.
  • the subject organic molecules conform to the following general Formula I: namely,
  • R ⁇ is a neutral polar, neutral nonpolar, or basic residue selected from a functional group containing a halogen atom such as an amine, amide, amido- and the like, or alternatively, R ⁇ is selected from a functional group containing a straight or branched peptide chain, or straight or branched chain alkyl-, alkoxy-, or cyclic hydrocarbon, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, cyclohexyl-, phenoxy-, glycol- and the like, or alternatively, R 5 is selected from a hydrophobic or amphipathic residue such as a glycerol-phosphatide or fatty-acyl chain (e.g., phosphatidyl-ehtanolamine, phosphatidyl-choline, phosphatidyl-inositol,
  • R 2 and R 4 are straight or branched alkyl chains selected from methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-; isopropyl-, isobutyl-, isopentyl-, isohexyl-, and the like;
  • R 3 is a positively charged residue such as an amine, amide, amido, residue and the like, or alternatively, R 3 is a negatively charged residue such as a modified carbonyl- residues such as carboxylic acids, carboxylic acid amides, acyl-modified carboxylic acids, carbamates, di-alcohols, aldehydes, and the like;
  • R 5 and R $ are electron donor residues such as acetyl-, alkyl-, or modified carbonyl- residues such as carboxylic acids, carboxylic acid amides, acyl-modified carboxylic acids, carbamates, di-alcohols, aldehydes, or alternatively, one or more cyclic or heterocyclic hydrocarbon rings such as phenyl-, phenoxy-, and the like;
  • Derivatives may also be constructed as lipopeptides constructed with multiple KE or EW residues (e.g., di-, tri-, and tetra-lipopeptides and the like).
  • a glycerol-phosphatide multimer is provided in Formula II, below, as a phosphatidyl fatty acid (abbreviated fatty acyl in Formula II) of glycerol having one "aa ⁇ aa 2 " dipeptide (e.g., KE) and a second "aa 3 aa 4 " dipeptide (e.g., EW).
  • R x and R 2 are "non-interfering residues", as set forth below, that may used to stabilize or enhance the biological activity of compound of Formula II.
  • Analog is intended to mean a chemical compound according to Formula I, above, that mimics or improves on the electronic, steric, hydrophobic, and 3-dimensional space filling requirements of the constituent residues in KE or EW that are involved in binding to the ligand-receptor (e.g., a mimetic chemical KE or EW compositions).
  • Representative analogs include chemical mimetic compounds that are capable of antagonizing binding of KE or EW to a ligand-receptor, i.e., antagonists (as defined below),and other ligands that are capable of binding to a ligand receptor and exerting effects similar to KE or EW, i.e., agonists (as defined below).
  • Antist as used herein means a chemically modified KE, EW, or organic chemical molecule according to Formula I or Formula II, above, that is capable of spacially conforming to the molecular space filled by a KE or EW ligand and that is further capable of combining with the subject ligand receptors to initiate an action that is initiated by KE or EW following binding to their specific ligand receptor(s) on cells in vitro or in vivo.
  • Representative examples of actions initiated by KE and EW may be: i) upregulation of lymphocyte cell surface determinants such as CD2, CD4, CD28, CD45, CD58, CD59,- LFA 1, ICAM 1, ICAM 2, ICAM 3, and the like; ii) macrophage activation; iii) stimulating (or inhibiting) lymphocyte blastogenesis in response to antigen (or mitogen), iv) stimulating release of interleukins from lymphocytes (e.g., IL2, IL4, IL5, IL6, IL10, IFN ⁇ , TGF ⁇ and the like); v) mobilization of intracellular calcium (e.g., through membrane calcium channels) and cyclic AMP; vi) stimulating cell interactions between T-helper lymphocytes, B-lymphocytes and antigen presenting cells in initiation of primary and secondary humoral (antibody-mediated) immune responses; vii) stimulation of macrophages to increase their antibacterial and cytotoxic activities to microbial and mammalian target cells, and the like.
  • Antagonist as used herein means a chemical molecule according to Formula I or Formula ⁇ , above, that spacially conforms to the molecular space filled by KE or EW and is further capable of combining with the subject ligand rece ⁇ tor(s), as set forth above, to inhibit, neutralize, impede or reverse, at least in part, an action initiated following binding of EW or KE to the subject ligand receptor on a lymphocyte or a macrophage.
  • Non-interfering residue as used herein means any chemical residue that when present in position R ! or 1 ⁇ of Formula I, or position Rj or R 2 of Formula II does not interfere with binding of the subject ligand of the respective formula to a ligand receptor.
  • non-interfering amino acid residues or glycosyl residues may be useful in positions Rj and Rg of Formula I (or positions R j or R 2 of Formula II) to stabilize or enhance the biological activity of the subject compound.
  • the biological activities of chemical analogs according to Formula I or Formula II may be compared with those of KE or EW in vivo in experimental animal model systems, or alternatively, it may prove convenient to use in vitro assays to monitor intracellular second messenger pathways triggered in the test cells by interaction of a ligand with a ligand receptor.
  • Biological activity of a compound according to Formula I or Formula II may be determined by testing for second messenger pathways triggered by receptor binding the subject ligand. Second messenger pathways may be monitored e.g.
  • KE and EW by testing intracellular levels of Ca * *, cAMP, cGMP, adenyl cyclase, tyrosine kinase, guanylate cyclase, Protein kinase C; or, cellular release of interleukins, or mediators such as arachidonic acid metabolites, prostaglandins, and the like.
  • the present disclosure of significant biological effects of KE and EW on immune cells also serves as basis for isolating cell surface receptors binding KE and EW, and for identifying other ligands in biological fluids which may bind to the subject receptors.
  • Ligand refers to a compound that is capable of filling the three- dimensional space in a receptor binding site so that electrostatic repulsive forces are minimized, electrostatic attractive forces are maximized, and hydrophobic and hydrogen bonding forces are maximized.
  • Representative ligands include EW and KE.
  • Ligands bind to their specific receptors in a specific and saturable manner, e.g., specificity may be determined by the ability of the subject ligand to bind to the ligand receptor in a manner that is not competitively inhibited in the presence of an excess (e.g., 1000-fold molar excess) of KE or EW.
  • Ligand receptor is used herein to refer to a receptor capable of binding a ligand, according to the definition above, in a specific and saturable manner.
  • a labeled ligand e.g., radiolabeled or biotin-labeled KE or EW at a concentration selected from within the range of O.lnM to lOmM
  • the cells are washed (e.g., by centrifugation through an isobutylpthalate or sucrose cushion) and the amount of labeled ligand associated with the cell pellet is determined (e.g., by quantifying radioactivity or reacting the sample with enzymatically-labeled avidin, washing to remove unbound avidin, and then adding substrate to visualize the enzyme-bound-avidin-biotin receptor complex).
  • the data obtained in this manner may be used to conduct a Scatchard binding analysis of the data from which association constant and the relative binding affinity of the ligand-receptor for the ligand may be determined.
  • the association constant for binding of a ligand e.g., KE or EW
  • the association constant for binding of a ligand is about O.OlnM to about ImM, and most preferably about InM to about O.lmM.
  • Ligand receptors may also be conveniently prepared and may be used as pharmacological inhibitors, antagonists, and agonists of KE or EW binding to lymphocyte receptors.
  • receptor or its binding domains
  • receptor may conveniently be isolated by affinity chromatography of receptor preparations (and fragments) in tissue and cell extracts on chromatographic resins containing bound KE or EW, or alternatively, by binding the receptor to such a resin and then treating with one or more selected proteases to create receptor fragments that remain bound to the resin.
  • the receptor (or fragments) may be eluted from the resin either at high salt and/or low pH, or by competing the binding of the receptor (or fragments) to the resin with excess soluble KE or EW ligand.
  • the amino acid sequence of the receptor is conveniently determined by automated amino acid sequencing, and the sequence may be used to construct synthetic peptides, and nucleotide probes (e.g., degenerate probes) for cloning the subject receptor.
  • Ligand-receptor fragments is a term used to refer to portions of the subject ligand receptor that are smaller in size than a ligand receptor isolated from a natural source, e.g., tissue, biological fluids and the like. Fragments may be prepared from a ligand receptor isolated from a tissue and then subjected to proteolytic degradation or treatment with a chemical such as cyanogen bromide. In the latter case, the subject fragments of the receptor are conveniently purified before use, e.g., by reverse-phase HPLC or immoaffinity chromatography.
  • fragments of the ligand receptor may be prepared by expressing a portion of a nucleotide sequence of a genomic or cDNA clone capable of expressing the subject ligand receptor, e.g., a portion of ligand receptor nucleotide sequence in an expression plasmid or vector introduced into a cell, wherein the cell manufactures the subject receptor fragment and the fragment can be purified (as above).
  • Fragments of the subject ligand receptor may be soluble in biological fluids and aqueous solutions and may bind KE or EW with a greater or lesser KD than a complete (non-degraded) ligand receptor.
  • Substantially purified refers to a preparation from a natural source that contains a "peptide” (i.e., intended to mean about 2 to about 9 amino acids), a ligand according to criteria set forth above, or a ligand receptor or receptor fragment that is i) enriched greater than about 100-fold from a natural source material, e.g., a membrane preparation of a cell, and that ii) contains less than 5% peptide or polypeptide impurities detectable by reverse-phase HPLC.
  • a natural source material e.g., a membrane preparation of a cell
  • KE-peptidase and "EW-peptidase” are used herein to refer to dipeptidylpeptidases capable of catalyzing hydrolysis of a Lysyl-Glutamic acid peptide bond in KE or a Glutamyl-Tryptophane peptide bond in EW.
  • the subject enzymes inactivate KE and EW peptides and render them biologically inactive.
  • KE- and EW-peptidases may conveniently be purified from tissues and cells using conventional purification methods and colorimetric or fluorescent peptide substrates in protease test assays.
  • inhibitors of KE- and EW-peptidases may be identified in protease test assays.
  • the subject inhibitors of KE- and EW-peptidases may have biological effects similar to KE or EW, since they may decrease proteolytic degradation and increase the biological half life of an endogenous (i.e., natural) KE or EW in a tissue.
  • the present disclosure further provides basis for diagnostic immunoassays, antibody reagents and the like for measuring levels of KE and EW in biological fluids. While KE and EW are relatively nonimmunogenic, (falling into the class of low molecular weight haptens), when conjugated to a carrier (e.g., KLH) antibodies may be induced in experimental animals. Polyclonal and monoclonal antibodies to KE and EW find uses in a variety of immunoassay formats for diagnostic monitoring the levels of the subject dipeptides in bodily fluids in health and disease.
  • a carrier e.g., KLH
  • Representative immunoassay formats include: enzyme linked immunoabsorbent assays (ELISA), radioimmunoassays (RIA), fluorescence immunoassay (FIA), time-resolved fluorescence assays (TRF), and cascade assay formats that are routinely used in the art for increasing low-end sensitivity of assays.
  • ELISA enzyme linked immunoabsorbent assays
  • RIA radioimmunoassays
  • FFA fluorescence immunoassay
  • TRF time-resolved fluorescence assays
  • cascade assay formats that are routinely used in the art for increasing low-end sensitivity of assays.
  • Individuals with high levels of the KE or EW dipeptide may be at decreased relative risk of infectious disease, while those with decreased levels may be at an increased relative risk.
  • the KE and EW dipeptides find uses as positive and negative controls in the subject diagnostic assays.
  • the subject assays may be assembled for use in a reagent
  • biological fluids are used herein to mean tissue fluids (such as joint fluid, cerebrospinal fluid and the like), plasma and serum, fluids in body cavities (i.e., peritoneal fluid, lung lavage fluid, urogenital mucus secretions, and the like), urine, feces, sputum, sweat, and the like.
  • Thymogen L-Glu-L-Trp
  • L-Lys-L-Glu may exert their effects in stimulating expression of CD2 on thymocytes by direct ligand-receptor interaction of cell surface CD2 receptors.
  • N ⁇ N ⁇ -dibenzyloxicarbonyllysyl- ⁇ -benzylglutamic acid 0.154 g (0.65mmol) of ⁇ -dibenzylglutamic acid was suspended in 3 ml of dimethylformamide. While mixing, 0.091 ml (0.65 mol.) of triethylamine was added, followed by 0.300 g (0.59 mmol.) of N-oxisuccinimide ether N°, IsT-dibenzyloxicarbonyllysine. The reaction was allowed to proceed for 12 hours at room temperature, with mixing., after which solvent was evaporated under vacuum at 40°C.
  • Thymocytes Thymus cells isolated from male guinea pigs (180-200 g) under standard conditions of tissue mincing and differential centrifugation in medium 199. Isolated thymocytes were treated with trypsin to remove cell surface E-receptors capable of binding rabbit erythrocytes (E). The percentage of cells capable forming E-rosettes (E-RFC) was decreased by about two-fold and under these conditions. Thymogen has been reported to restore T-cell receptors and E-RFC following incubation at 37°C. Studies were conducted in which the activity of L-Lys-L-Glu dipeptide and Thymogen were compared in the latter assay.
  • Thymocytes were prepared from 48 guinea pigs, treated with trypsin, and tested for
  • T-Lymphocyte Subsets in Human Peripheral Blood The effects of L-Lys-L-Glu on human peripheral blood T-lymphocytes was investigated using indirect immunofluorescence microscopy and OKT4 and OKT8 monoclonal antibodies (Ortho, USA) directed to lymphocyte cell surface differentiation antigens.
  • lymphocytes were isolated from heparinized peripheral blood
  • Lymphocytes were prepared by centrifugation on Ficoll-Hypaque, washed, and then incubated in vitro for 45 minutes at 37°C in the presence (or absence) of L-Lys-L-Glu or
  • Thymogen at concentrations of 1 mg/ml, 0.01 mg/ml, 0.0001 mg/ml. Following incubation the cells were washed with medium 199 and the percentage of OKT4
  • T-helper and OKT8 + lymphocytes determined by indirect immunofluorescent microscopy.
  • the OKT4 + /OKT8 + ratios for this group of patients is also shown in TABLE 3.
  • the results increased the percentage of detectable OKT4 T-helper lymphocytes after only 45 minutes at 37°C at all concentrations tested.
  • Thymogen increased the percentage of detectable T-helpers at concentrations of 1 and 0.01 mg/ml.
  • Neither Thymogen nor L-Lys-L-Glu altered the percentage of detectable OKT8 T-suppressor cells.
  • L-Lys-L-Glu was about 100-times more effective than Thymogen in inducing the observed in vitro increase in detectable T-helper lymphocytes.
  • Test agents were administered to animals once daily by the intramuscular (im) route over a period of 5 days and at the following doses: namely, dipeptide L-Lys-L-Glu-
  • Physiological saline was administered im to the animals of the control group. The effects of these treatments was determined on day 10 (i.e., 5 days after the last injection) by preparing cells from peripheral blood (PBL), thymus (TYM), lymph nodes (LN), spleen (SPL), and red pulp of bone marrow (BM). Cells were tested for antibody Fc receptors (EA-RFC), complement receptors (EAC-RFC), T-lymphocytes, "active" T-lymphocytes (E-RFC), B-lymphocytes (EA-RFC and EAC-RFC; according to the method of citation #10). The results of these analyses are shown in TABLES 4A-4C, below, where data are expressed either as the number of RFC xlO 9 per liter of blood, or RFC x 10 3 per milligram (mg) of tissue.
  • TABLES 4A-4C show that animals treated for 5 days with dipeptide L-Lys-L-Glu or Thymogen had 3-fold more T-lymphocytes in peripheral blood (i.e., PBL; E-RFC) than control animals treated with saline.
  • T-lymphocytes in LN and BM decreased (i.e., 17% for Lys-Glu and 50% for Thymogen) while splenic and thymus T-cells increased by about 11-16% for Lys-Glu and 2-9% for Thymogen.
  • animals who received L-Glu-L-Trp showed an increased number of T-lymphocytes in peripheral blood, spleen and marrow.
  • L-Lys-L-Glu increased in a statistically significant manner the number of Fc-receptor bearing cells isolated from blood, thymus, spleen, and bone marrow (TABLE 4B). Numbers of complement receptor bearing cells (EAC-RFC) were increased 2-fold in blood by L-Glu-L-Trp treatment, but not by L-Lys-L-Glu treatment. However, L-Lys-L-Glu stimulated a statistically significant increase in splenic complement receptor bearing cells (TABLE 4C).
  • each group of animals was sub-divided into two subgroups of 10 animals each: i.e., one subgroup of animals was injected ip with 10% sterile proteose peptone to induce neutrophils (induced), while the other subgroup were non-induced (non-induced).
  • the animals of the four induced-subgroups were sacrificed 2.5 hours after the ip injection.
  • Thymus and spleen were weights were determined in the non-induced animals; cell suspensions were prepared from each thymus and spleen; and, resident peritoneal exudate cells (PEC) were harvested by lavage with medium 199 from each animal.
  • PEC resident peritoneal exudate cells
  • B-lymphocytes in the splenic cell populations from the non-induced animals was determined by phase-contrast and indirect immunofluorescence (11) using rabbit antisera specific for murine Thy-1 and immunoglobulin (Ig).
  • Rabbit anti-Thy-1 was raised by immunization with a murine brain homogenate (12) and anti-Ig by immunization with an ammonium sulfate precipitate of murine serum.
  • the percentage macrophages in the resident PEC populations were determined in Romanowsky stained smears of cells, the cells then collected by centrifugation at 150 x g/10 minutes, and macrophage activity measured by reduction of nitroblue tetrazolium (NBT; 12) before and after induction with complement-opsonized zymosan (guinea pig complement activated by zymosan; 14). NBT reduction was quantified spetrophotometrically at 540 nm. Pinocytic activity of the resident peritoneal macrophages was assessed by measuring uptake of neutral red dye
  • the percentage of neutrophils in the PEC population was determined using phase contrast microscopy, and in response to induction with complement-opsonized zymosan. Phagocytic activity of neutrophils was determined by incubating the cells with 2.5 x 10 Staphylococcus aureus per milliliter (S. aureus from a
  • the data are expressed as both the percentage of phagocytic cells (% of the total cell population phagocytizing 1 or more staphylococci and the phagocytic index
  • Thymogen induced a statistically significant decrease in thymic weight at both dosages, and had opposing effects on splenic weight at different dosages.
  • the 1 mg/kg dosage of Thymogen administered in these studies approximates a clinically effective dosage in humans for immune stimulation.
  • Thymogen 10 1.0 34.8*2.5 4- 0.9 52.4 ⁇ 1.71 1.0 0.1 0.01 41.2 ⁇ 2.1* t l.l 50.8 ⁇ 3.0 1.0
  • Thymogen 1 0.059 ⁇ 0.003 0.116 ⁇ 0.008 0.01 0.101 ⁇ 0.002** 0.205 ⁇ 0.012**
  • Thymogen (0.01 mg/kg) treated animals were induced with complement-opsonized zymosan, 1.75-fold and 2-fold increases, respectively, in NBT reducing activity were observed.
  • peritoneal macrophages from L-Lys-L-Glu treated animals appeared to be about 1.3-times more pinocytically active than those from
  • the results presented in TABLE 8 show a significant 1.6-fold elevation of neutral red uptake by resident peritoneal macrophages from animals treated with ip injections of
  • Thymogen (0.1 mg/kg) doubled the number of neutrophils that could be induced following injection of proteose peptone.
  • Thymogen 10 1 31.5 ⁇ 0.4* 1.89 ⁇ 0.03 0.1 0.1 17.8 ⁇ 1.13 1.90 ⁇ 0.05
  • Thymogen pretreatment at a dose of 0.01 mg/kg did not appear to stimulate neutrophil emigration in response to the sterile proteose peptone inflammatory stimulus (TABLE 9), nor the phagocytic activity of the cells so induced (TABLE 10).
  • L-Lys-L-Glu appears to have a 100-1000 fold greater effect on lymphocyte tissue distribution than Thymogen, with a possible mobilization of thymic and bone marrow lymphocytes and increased T-lymphocytes in the spleen at the conclusion of the 6 day ip treatment regimen.
  • the combined results indicate that treatment with L-Lys-L-Glu and
  • Thymogen can induce changes in distribution of lymphocyte cell populations, and changes in distribution and activities of macrophages and neutrophils, both of which changes favor a heightened state of innate immunity in the treated animals.
  • L-Lys-L-Glu and Thymogen are termed "immunomodulators".
  • Brain-associated antigen reactivity rabbit anti-mouse brain with mouse lymphoid cells. Cell. Immunol. 2: 353-361.

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Abstract

Préparations peptidiques pharmaceutiques destinées à augmenter l'état d'immunité cellulaire ou humorale antimicrobienne chez un sujet le nécessitant, consistant essentiellement en une préparation L-Lys-L-Glu ou L-Glu-L-Trp et en un vecteur pharmaceutiquement acceptable.
PCT/RU1994/000162 1993-07-21 1994-07-21 Agents pharmaceutiques a activite immunomodulatrice WO1995003067A1 (fr)

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EP0818462A1 (fr) * 1995-03-02 1998-01-14 Vladislav Isakovich Deigin Peptide et son procede d'obtention
WO1998009985A2 (fr) * 1996-09-03 1998-03-12 Yeda Research And Development Co. Ltd. Peptides anti-inflammatoires et leurs utilisations
EP0832900A1 (fr) * 1995-06-07 1998-04-01 Vladislav Isakovich Deigin Peptide et procede de production
WO1999066948A2 (fr) * 1998-06-23 1999-12-29 Obschestvo S Ogranichennoi Otvetstvennostiju ''klinika Instituta Bioregulyatsii I Gerontologii'' Utilisation d'un dipeptide pour stimulation de processus de regeneration
US6060452A (en) * 1996-03-13 2000-05-09 Cytran, Inc. Analogs of L-Glu-L-Trp having pharmacological activity
WO2000042994A2 (fr) * 1999-01-21 2000-07-27 North Shore-Long Island Jewish Research Institute Inhibition de la dissemination de bacteries
WO2002062371A1 (fr) * 2001-01-25 2002-08-15 Sankt-Peterburgskaya Obschestvennaya Organizatsia 'sankt-Peterburgskiy Institut Bioregulyatsii I Gerontologii Szo Ramn' Dipeptide inhibant l'angiogenese utilise en ophthalmopathie
WO2003078383A1 (fr) * 2002-03-20 2003-09-25 Yonsei University Nouveaux lipides positivement charges et liposomes comprenant ces lipides positivement charges
EP2604264A1 (fr) * 2010-12-23 2013-06-19 Obshestvo S Ogranichennoj Otvetstvennostju "citoNIR" Composition pharmaceutique pour traiter les affections virales
EP2918282A1 (fr) * 2014-03-14 2015-09-16 OOO "CytoNIR" Produit pharmaceutique destiné au traitement de maladies virales, comme la grippe

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US6911431B1 (en) 1989-08-30 2005-06-28 Melmotte, Inc. Pharmaceutical angiostatic dipeptide compositions and methods of use thereof
EP0818462A1 (fr) * 1995-03-02 1998-01-14 Vladislav Isakovich Deigin Peptide et son procede d'obtention
EP0818462A4 (fr) * 1995-03-02 2000-05-31 Immunotech Dev Inc Peptide et son procede d'obtention
EP0832900A1 (fr) * 1995-06-07 1998-04-01 Vladislav Isakovich Deigin Peptide et procede de production
EP0832900A4 (fr) * 1995-06-07 1999-10-06 Immunotech Dev Inc Peptide et procede de production
US5902790A (en) * 1995-10-03 1999-05-11 Cytran, Inc. Pharmaceutical angiostatic dipeptide compositions and method of use thereof
CZ298345B6 (cs) * 1995-10-03 2007-09-05 Cytran, Ltd. Farmaceutické dipeptidické prostredky s angiostatickým úcinkem a zpusoby jejich použití
AU714846B2 (en) * 1995-10-03 2000-01-13 Cytran Ltd. Pharmaceutical angiostatic dipeptide compositions and methods of use thereof
WO1997012625A1 (fr) * 1995-10-03 1997-04-10 Cytran, Ltd. Compositions pharmaceutiques a base de dipeptide angiostatique et leur procede d'utilisation
US6096713A (en) * 1995-10-03 2000-08-01 Cytran, Inc. Pharmaceutical angiostatic dipeptide compositions and methods of use thereof
US6060452A (en) * 1996-03-13 2000-05-09 Cytran, Inc. Analogs of L-Glu-L-Trp having pharmacological activity
WO1998009985A3 (fr) * 1996-09-03 1998-05-07 Yeda Res & Dev Peptides anti-inflammatoires et leurs utilisations
US6126939A (en) * 1996-09-03 2000-10-03 Yeda Research And Development Co. Ltd. Anti-inflammatory dipeptide and pharmaceutical composition thereof
WO1998009985A2 (fr) * 1996-09-03 1998-03-12 Yeda Research And Development Co. Ltd. Peptides anti-inflammatoires et leurs utilisations
US6642201B1 (en) * 1998-06-23 2003-11-04 Obschestvo S Ogranichennoi Otvetstven Nostiji “Klinika Instituta Bioregulyatsii Gerontologii” Use of a dipeptide for stimulating repair processes
AU776693B2 (en) * 1998-06-23 2004-09-16 Obschestvo S Ogranichennoi Otvetstvennostiju "Klinika Instituta Bioregulyatsii I Gerontologii" Use of a dipeptide for stimulating repair processes
WO1999066948A3 (fr) * 1998-06-23 2000-03-23 Obschestvo S Ogranichennoi Otv Utilisation d'un dipeptide pour stimulation de processus de regeneration
WO1999066948A2 (fr) * 1998-06-23 1999-12-29 Obschestvo S Ogranichennoi Otvetstvennostiju ''klinika Instituta Bioregulyatsii I Gerontologii'' Utilisation d'un dipeptide pour stimulation de processus de regeneration
WO2000042994A3 (fr) * 1999-01-21 2003-08-28 Long Island Jewish Res Inst Inhibition de la dissemination de bacteries
WO2000042994A2 (fr) * 1999-01-21 2000-07-27 North Shore-Long Island Jewish Research Institute Inhibition de la dissemination de bacteries
WO2002062371A1 (fr) * 2001-01-25 2002-08-15 Sankt-Peterburgskaya Obschestvennaya Organizatsia 'sankt-Peterburgskiy Institut Bioregulyatsii I Gerontologii Szo Ramn' Dipeptide inhibant l'angiogenese utilise en ophthalmopathie
WO2003078383A1 (fr) * 2002-03-20 2003-09-25 Yonsei University Nouveaux lipides positivement charges et liposomes comprenant ces lipides positivement charges
EP1485346A1 (fr) * 2002-03-20 2004-12-15 Yonsei University Nouveaux lipides positivement charges et liposomes comprenant ces lipides positivement charges
EP1485346A4 (fr) * 2002-03-20 2006-04-12 Univ Yonsei Seoul Nouveaux lipides positivement charges et liposomes comprenant ces lipides positivement charges
EP2604264A1 (fr) * 2010-12-23 2013-06-19 Obshestvo S Ogranichennoj Otvetstvennostju "citoNIR" Composition pharmaceutique pour traiter les affections virales
EP2604264A4 (fr) * 2010-12-23 2013-08-07 Obshestvo S Ogranichennoj Otvetstvennostju Citonir Composition pharmaceutique pour traiter les affections virales
EP2918282A1 (fr) * 2014-03-14 2015-09-16 OOO "CytoNIR" Produit pharmaceutique destiné au traitement de maladies virales, comme la grippe

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