EP0215805A1 - Peptides de regulation immunitaire - Google Patents

Peptides de regulation immunitaire

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Publication number
EP0215805A1
EP0215805A1 EP19860900764 EP86900764A EP0215805A1 EP 0215805 A1 EP0215805 A1 EP 0215805A1 EP 19860900764 EP19860900764 EP 19860900764 EP 86900764 A EP86900764 A EP 86900764A EP 0215805 A1 EP0215805 A1 EP 0215805A1
Authority
EP
European Patent Office
Prior art keywords
lys
ser
arg
ala
thr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19860900764
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German (de)
English (en)
Inventor
Gary Scott Hahn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Patent GmbH
Immunetech Pharmaceuticals
Original Assignee
Merck Patent GmbH
Immunetech Pharmaceuticals
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Publication date
Application filed by Merck Patent GmbH, Immunetech Pharmaceuticals filed Critical Merck Patent GmbH
Publication of EP0215805A1 publication Critical patent/EP0215805A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • 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
    • 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
    • C07K5/06113—Asp- or Asn-amino acid
    • 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
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06147—Dipeptides with the first amino acid being heterocyclic and His-amino acid; Derivatives thereof
    • 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
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • 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
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • 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
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • 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
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • 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
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the mammalian immune system consists of a collection of cells and molecules which contribute to the defense of the host against foreign and other undesirable agents including infectious agents and cancer.
  • the molecular portion of the immune system includes immunoglobulins, complement, histocompatibility antigens, and a variety of chemicals and peptide hormones which may either stimulate or inhibit the growth, development and activation of portions of .the cellular immune system.
  • the cellular immune system consists of white blood cells (leukocytes) and includes lymphocytes, monocytes, macrophages, neutrophils, eosinophils, basophils and mast cells, among others.
  • leukocytes may circulate in blood or other body fluids, or may be fixed in various lytnphoid organs including the thymus, bone marrow, lymph nodes and other organs.
  • the cellular and molecular immune systems In order to provide an effective immune defense, the cellular and molecular immune systems must interact in a highly intricate and complex manner. Optimally, the immune defense response should be precisely balanced to provide sufficent destructive force to eliminate infectious or foreign agents or cancer cells without unnecessarily destroying healthy cells. A suboptimal immune response may result in uncontrolled growth of an infectious agent or cancer which may lead to the death of the host. A excessive immune response, by contrast, may destroy not only the intended target, but substantial quantities of surrounding healthy tissue as well. The many molecules of the molecular immune system play a major role in regulating and "fine tuning" the cellular immune response to maximize destruction of infectious agents and cancer cells while minimizing destruction of surrounding healthy cells.
  • Autoimmune diseases are an example of conditions in which a substantial portion of an immune response is directed toward healthy host cells.
  • autoimmune diseases tolerance for host cells and molecules is reduced or eliminated resulting in significant destruction of otherwise healthy cells and organs.
  • MHC major histocompatibility complex
  • a particular MHC haplotype may substantially increase the risk of self-tolerance loss and subsequent autoimmunity.
  • infection by certain viruses or bacteria is believed to trigger the loss of self-tolerance which, in the setting of an appropriate MHC haplotype, results in an autoimmune disease.
  • the triggering events which lead to a loss of self-tolerance remain unknown.
  • Autoimmune diseases may affect every organ of the body.
  • diseases thought to have an autoimmune pathogenesis include, but are not limited to, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, ankylosing spondylitis, Reiter's syndrome, Sjogren's syndrome, polymyositis-dermatomyositis, thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, regional enteritis (Crohn's disease), chronic active hepatitis, primary biliary cirrhosis, idiopathic interstitial pulmonary fibrosis, Goodpasture's syndrome, postviral encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis,
  • Grave's disease Hashimoto's thyroiditis, juvenile onset insulindependent diabetes, Addison's disease, pernicious anemia, pemphigus, bullous pemphigoid and other diseases and conditions.
  • the immune response responsible for the rejection of transplanted organs among genetically non-identical animals or humans in many ways resembles an autoimmune disease in that an otherwise healthy transplanted organ may be destroyed by the recipient's immune system. Such destruction occurs because the recipient's immune system recognizes the "foreign" histocompatibility antigens present on cells of the transplanted organ and trigger a destructive immune response.
  • the complex regulation of immune responsiveness results from interactions between all classes of leukocytes, molecules secreted by leukocytes and cells and molecules from other organ systems.
  • One class of leukocytes in particular, termed thymusderived lymphocytes or T-lymphocytes (T cells) is considered to be critically important to the coordination and regulation of most immune responses.
  • T cells may be divided into various subsets which have distinct immune functions.
  • Helper T cells for example, are critical for the growth and development of B cells into antibody-secreting plasma cells.
  • Helper T cells are also critical for the growth and development of other T cell subsets such as Killer T cells which can directly destroy infectious agents, cancer cells, transplanted organs and, in autoimmune disease, healthy cells.
  • other T cell subsets termed suppressor T cells actively suppress the growth and development of B cells.
  • Suppressor T cells are also critical for the development and maintenance of immune tolerance that prevents the development of autoimmune disease and organ transplantation rejection.
  • Thymulin has been shown to have hormonal properties related to the promotion, of T-lymphocyte differentiation which include the induction of various antigenic markers on murine and human T-lymphocyte precursors, the delaying of allogeneic skin graft rejection in mice, enhancement of the generation of alloantigen reactive cytotoxic T-lymphocytes in thymectomized mice, modulation of thymic natural killer (NK) cell activity in tumor-bearing and NZB mice, inhibition of antibody-bearing lymphocyte production at late stages of differentiation, and suppression of Experimental Allergic Encephalomyelitis in guniea pigs.
  • NK thymic natural killer
  • U.S. Patent No. 4,190,646 discloses the pentapeptide Arg-Lys-AspVal-Tyr and certain derivatives capable of inducing the differentiation of bone marrow cells to T-lymphocytes.
  • the dipeptide Gln-Lys and certain derivatives thereof are reported in U.S. Patent No. 4,215,111 to be capable of inducing differentiation of both T- and B-lymphocytes.
  • Other pentapeptides capable of inducing T- and/or B-lymphocyte differentiation are described in U.S. Patent Nos. 4,261,886 and 4,505,853.
  • tuftsin isolated by Najjar and described in U.S. Patent No. 3,778,426, has the sequence Thr-Lys-ProArg. Tuftsin has been shown to stimulate i n vitro phagocytosis by granulocytes, monocytes and macrophages. Other studies have shown tuftsin to be active in nanomolar concentrations in many species including humans, cows, dogs, rabbits, guinea pigs and mice.
  • tuftsin has been shown to stimulate such immune response functions as antibody-dependent cell-mediated cytotoxicity (ADCC) and Natural Killer cell activity in mouse splenic cells, anti-bacterial activity of PMN-leukocytes and tissue macrophages, antigenspecific macrophage-dependent T-lymphocyte education and antibody synthesis to T-lymphocyte-dependent and independent antigens in vivo and in vitro.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • N-lymphocyte-dependent T-lymphocyte education antigenspecific macrophage-dependent T-lymphocyte education and antibody synthesis to T-lymphocyte-dependent and independent antigens in vivo and in vitro.
  • Fridkin et al. Molecular and Cellular Biochemistry 41:73 (1981); Najjar et al., in Pick (ed.), Lymphokine Reports (Academic Press 1980), p. 157; Nishioka, Life Sciences 28:1081 (1981).
  • Rigin a tetrapeptide analog of tuftsin having the peptide sequence Gly-Gln-Pro-Arg, has been shown to have phagocytosis-stimulating properties similar to those of tuftsin.
  • peptides and peptide derivatives have a surprising ability to regulate T cell functions, and in particular to suppress T cell proliferation, and provide thereby potentially significant therapeutic benefits in the treatment of human or animal disease.
  • the peptides are capable of suppressing the growth of various neoplastic cells. Due to their short length, these peptides are relatively easy and inexpensive to manufacture. In addition, they are effective at non-toxic pharmacological dosage levels. Accordingly, it is one object of the present invention to provide new peptides with important biological and pharmacological activities, as well as related and novel methods for using these peptides in suppressing the mammalian immune system in vitro and in vivo.
  • a further object of the invention is to provide new peptides and methods related to the therapeutic treatment of mammalian autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, and other diseases.
  • a further object of the invention is to provide new peptides and methods related to the therapeutic treatment of diseases involving a deficient, excessive or otherwise malfunctioning mammalian immune system response, including such conditions as organ transplantation rejection and bone marrow trans lantation rejection.
  • a still further object of the invention is to provide new peptides and methods related to the treatment of neoplastic diseases.
  • X and Y are residues of amino acids or amino acid derivatives with positively charged side chains, preferably lysine, ornithine, arginine, or histidine, or D-enantiomers of any of these residues;
  • a and C are any substituents, including amino acid residues, that serve to preserve or augment the immunoregulatory activity of the peptide;
  • B is a residue of an amino acid or amino acid derivative that preserves or augments the immunoregulatory activity of the peptide; and
  • n is 0 or 1.
  • the peptides of the present invention can be characterized either by formula A-X-C (la) or by formula A-X-B-Y-C (lb). Compounds of formula la are preferred.
  • X will be chosen from among the amino acids lysine, ornithine, D-lysine and D-ornithine; in lb, X will be most preferably lysine, arginine, histidine or ornithine, while Y will most preferably be lysine, arginine or histidine.
  • A may be, for example, hydrogen or an N-acetyl, N-acyl, N-aliphatic, or N-aromatic group; or A may be an amino acid such as alanine, glycine, leucine, valine, phenylalanine, tyrosine, asparagine, aspartic acid, sarcosine, glutamine, glutamic acid, serine, or threonine, or an amino acid derivative including D-enantiomers of these amino acids, such as D-alanine or D-aspartic acid.
  • B may be, for example, a residue of an amino acid such as valine, leucine, glutamic acid, glutamine, serine, threonine, glycine, alanine, proline, asparagine, aspartic acid, arginine, histidine, isoleucine, tyrosine, phenylalanine, lysine, ornithine, or tryptophan, or of a D-enantiomer or other derivative of an amino acid, such as D-valine or D-serine.
  • an amino acid such as valine, leucine, glutamic acid, glutamine, serine, threonine, glycine, alanine, proline, asparagine, aspartic acid, arginine, histidine, isoleucine, tyrosine, phenylalanine, lysine, ornithine, or tryptophan, or of a D-enantiomer or other derivative of an amino
  • C preferably will be a small uncharged chemical constituent, for example -NH 2 , -NHR or -OR, where R is preferably H or a lower alkyl; or C may be a residue of an amino acid such as glycine, alanine, leucine, valine, serine, cysteine, sarcosine, threonine, proline, glutamine, phenylalanine, tyrosine, homoserine or a D-enantiomer or other derivative of an amino acid.
  • R is preferably H or a lower alkyl
  • C may be a residue of an amino acid such as glycine, alanine, leucine, valine, serine, cysteine, sarcosine, threonine, proline, glutamine, phenylalanine, tyrosine, homoserine or a D-enantiomer or other derivative of an amino acid.
  • immunoregulatory activity will be achieved for a large number of diverse substituents in the A, B and C positions, so long as the constituents X and Y are as defined above.
  • immunoregulatory activity may be retained where either or both of the terminal groups A and C comprise multiple amino acids that do not interfere substantially with the immunoregulatory activity associated with the central tripeptide sequence X-B-Y.
  • the present invention specifically contemplates that derivatives, including homologs, analogs, enantiomers, extensions, or other substitutions not specifically disclosed herein are within the scope and spirit of the present invention.
  • N ⁇ -acylated amino acid RCO-(amino acid) N ⁇ -acetyl amino acid Ac-(amino acid) N ⁇ -methyl amino acid N-Me-(amino acid) des- ⁇ -amino acid desamino-(amino acid) Amino acid amide (Amino acid)-NH 2 , -NHR, -NR 2
  • amino acid derivative refers to homologs, analogs, D-enantiomers, and chemically substituted or otherwise modified forms of amino acids such as those listed above.
  • amino acid derivatives may be substituted for naturally-occurring amino acids in the peptides of the present invention while still preserving or augmenting immunoregulatory activity.
  • chemical substitution of acyl, alkyl, and other substituents at the N-terminus, or formation of esters or amides at the C-terminus will frequently yield peptide derivatives with preserved or augmented immunoregu- latory activity.
  • amino acids within the disclosed peptides may be substituted by amino acids which are chemically similar by virtue of similar side chain size, charge, shape, solubility, or other chemical characteristics while still retaining the peptide's immunoregulatory activity.
  • Amino acids with such chemical similarity are termed "functionally conserved.” Functional classes of the common genetically-coded amino acids have been specified by Dayhoff, et al. in Atlas of Protein Sequence and Structure, volume 5 (National Biomedical Research Foundation, 1972), page 98.
  • Derivatives of the naturally-occurring amino acids such as ornithine, homoserine, homolysine, des-N ⁇ -amino lysine and homoarginine, have chemical structures and properties comparable to their naturally-occurring analogs or homologs, and thus may, in proper cases, be substituted in the peptides of the present invention to preserve or even increase immunoregulatory activity.
  • immunoregulatory activity of the subject peptides resides in the capability of the peptides to suppress autoimmune responses, to suppress organ transplantation rejection responses, or to suppress neoplastic cell growth.
  • immunoregulatory activity may be realized in in vitro systems that are modeled after i n vivo mammalian disease conditions, as well as in in vivo systems.
  • X and Y are residues of amino acids or amino acid derivatives with positively charged side chains at physiological pH (i.e., pH 6 to 8) or D-enantiomers of any of these residues;
  • a and C are amino acid residues or other substituents, that preserve or augment the immunoregulatory activity of the peptide;
  • B is a residue of an amino acid or amino acid derivative that preserves or augments the immunoregulatory activity of the peptide; and
  • n is 0 or 1.
  • X may be chosen from among residues of such naturally-occurring positively charged amino acids as Lys, Arg and His.
  • residues of positively charged amino acids other than those naturally-occurring acids listed above may be used successfully in the X position.
  • substitution of the D-enantiomeric form of the amino acid X will, in some cases, augment, and, in many cases, substantially preserve the immunoregulatory activity of the subject peptide.
  • residues of desamino forms of the central amino acid X, wherein the N ⁇ -amino group of X is absent may preserve or augment the immunoregulatory activity of the peptide.
  • amino acid derivatives including homologs, analogs, enantiomers and otherwise modified forms of naturally occurring positively charged amino acids, can also be expected to yield significant immunoregulatory activity.
  • ornithine which is closely homologous to lysine, demonstrates significant activity despite the fact the ornithine does not occur naturally in proteins or in other molecular constituents of the immune system.
  • Especially preferred for X are Lys and Orn.
  • D-enantiomeric form of these amino acids (D-Lys, D-Orn) as well as the deaminated forms ( desamino-Lys, desamino-Orn) may be substituted for the naturally occurring L-enantiomer in proper cases, with the result that immunoregulatory activity or the peptide is substantially preserved or augmented.
  • X and Y are any positively-charged amino acids separated by any single amino acid, B, which tripeptide has immunoregulatory activity.
  • terminal constituent A is hydrogen and terminal constituent C is a hydroxyl group.
  • X and Y may be the same or different positively charged amino acids, preferably chosen from among Lys, Arg, His and Orn. Lys and Arg are particularly preferred in the X and Y positions.
  • the following tripeptide sequences are particularly preferred in the present invention:
  • immunoregulatory activity may be achieved through the use of His or Orn in the X or Y positions. His or Orn may be substituted in the X position, while His is preferred in the Y position.
  • the present invention contemplates that modified, homologous, analogous or artificial forms of positively charged amino acids may be utilized in the X and Y positions of lb.
  • D-enantiomeric forms of positively charged amino acids may, in appropriate combination with other constituents of the subject polypeptides, substantially preserve or augment the immunoregulatory activity of the peptide.
  • Constituent B comprises the residue of any amino acid or amino acid derivative which preserves or augments the immunoregulatory activity of the subject polypeptides. It has been discovered that a very broad range of amino acids may be incorporated into the B position to yield polypeptides with significant immunoregulatory activity.
  • B will be chosen from among the residues of the following amino acids or their D-enantiomers : Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, He, Tyr, Phe, Lys, Orn, Trp.
  • B will be chosen from among the following amino acids or their D-enantiomers: Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala.
  • the B position amino acids listed are preferably used in conjunction with the positively charged amino acids Lys or Arg in the X und Y positions.
  • the amino acids Val, Glu and Gin are especially preferred in the B position.
  • Asp and Ser are highly active substituents in the B position.
  • Ser and Thr are examples of suitable B substituents when X is Lys and Y is Arg, while Gly, Ala, Ser and Thr are all active when X is Arg and Y is Lys.
  • terminal constituent A which is substituted at the N ⁇ -position of X, may be chosen from a number of chemical groups or substituents.
  • terminal constituent A may be hydrogen (H-) alone, yielding a peptide of the general formula X-(B-Y) n -C.
  • constituent A may be an N ⁇ -acetyl substituent, thus yielding a peptide of the general formula
  • substituents in the A position that may preserve or augment immunoregulatory activity include aliphatic and aromatic acyl substituents of the form RCO-, as well as substituents of the form R-, where R is preferably an unbranched or branched alkyl group of one to eight carbons, and may also be C 2 -C 8 alkenyl, C 2 -C 8 alkynyl,
  • constituent A may be an amino acid.
  • Ala and D-Ala are especially preferred amino acids for constituent A.
  • amino acids for terminal constituent A are the following: Gly, Leu, Val, Phe, Tyr, Asn, Asp, Sar, Gin, Glu, Ser and Thr as well as the D-enantiomers of these amino acids.
  • substitutions of more than one amino acid have been shown to yield active polypeptides, particularly of formula lb.
  • a terminal constituents of the form Val-Asp-, Gly-Asp-, Ala-Asp-, Ser-Asp-, Thr-Val-Asp- and Leu-Thr-Val-Asp- have been successfully used in the A position.
  • the immunoregulatory activity of the present polypeptides may be preserved with a large number of diverse substituents, including sequences containing multiple amino acids, in the A position as well as in the C terminal position.
  • Terminal constituent C also may be chosen from a number of chemical groups or substituents which preserve or augment the immunoregulatory activity of the subject peptides. In general, it is preferred that C be a relatively small, uncharged species (including a hydroxyl (-OH) group ). Furthermore, amidation, esterification and addition of one or more amino acids is possible in the terminal group C.
  • one preferred embodiment of C is of the form
  • R is hydrogen or a lower alkyl, such as C 1 -C 8 alkyl substituent.
  • R is ethyl
  • X is Lys and A is hydrogen
  • Lys-NHCH 2 CH 3 is representative.
  • R groups such as C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 6 -C 14 aryl, C 7 -C 14 alkaryl, C 7 -C 14 aralkyl and C 3 -C 14 cycloalkyl may be utilized in C terminal substituents of the form -NHR or -OR to yield amides and esters, respectively. Secondary amino groups of the form -NR 2 may also be expected to yield active peptide amides.
  • Particularly significant immunoregulatory activity may be obtained by using lower alkyl R groups in the polypeptide amides described above. C 1 -C 3 lower alkyl groups are particularly preferred. In particular, significant activity has been obtained in tripeptide amides of the form Lys-B-Lys-NH 2 wherein B is Val, Leu, Pro, Glu, Gin or Gly. Contrarily, amidation where the B position amino acid is Asp, Ala, Thr, Arg, or His yields peptides with lower, although still significant, immunoregulatory activity.
  • Amino acids and derivatives thereof may also be used as C terminal constituents.
  • amino acids may also be used as C terminal constituents.
  • it is particularly preferred that such amino acids have relatively small, uncharged side chains.
  • Ser, Gly, Ala, Leu, Val and Gys are particularly preferred as C terminal constituents.
  • Ser has been shown to yield a particularly high immunoregulatory activity, particularly in the combination
  • Lys-Ser Gly and Ala are especially preferred in the combinations Orn-Gly, Lys-Gly and Orn-Ala.
  • the D-enantiomeric form of amino acids in the C cerminal position may demonstrate significant immunoregulatory activity.
  • the combination Orn-D-Ala is especially preferred.
  • amino acids having relatively small, uncharged side chains are particularly preferred as C constituents in the present invention
  • significant immunoregulatory activity is also achieved using amino acids with relatively larger uncharged side chains as C terminal constituents.
  • C terminal constituents are the following amino acids, as well as their D-enantiomers: Thr, Pro, Gin, Phe, Tyr.
  • Other amino acids, or derivatives thereof, including homologs, analogs, enantiomers, or combinations thereof, as well as other non-amino acid chemical constituents may likewise be incorporated into the C terminal position to substantially preserve or augment the immunoregulatory activity of the subject peptides.
  • HomoSer and Sar respectively may be utilized successfully in the present invention as C terminal constituents.
  • amides or esters of C terminal amino acids may be so used.
  • the peptide Lys-Ser-NH 2 is particularly preferred.
  • combinations or sequences of amino acids which substantially preserve or augment. the immunoregulatory activity of the subject peptides may be incorporated as C terminal constituents. It is believed that it is within the skill of the applicable art to identify and utilize as C terminal constituents those chemical substituents, including amino acids and their derivatives, that substantially preserve or augment the immunoregulatory activity of the subject peptides, and which are not disclosed specifically herein.
  • a peptide of the present invention may incorporate one, two, three, or more amino acids.
  • the present invention contemplates a single, positively charged amino acid (the central amino acid, X) flanked by non-amino acid terminal constituents A and C. Certain of these "monopeptides" demonstrate extremely high immunoregulatory activity. Particularly preferred are lower alkyl amides of Lys, Orn, D-Lys and D-Orn.
  • D-Orn-NHCH 2 CH 3 The present invention countenances a large number of highly active dipeptides. Many of these are characterized by an A terminal constituent that comprises a non-amino acid species and a C terminal constituent that comprises an amino acid or a homolog, analog, or other derivative thereof. In particular, it is particularly preferred that the C terminal constituent be Ser, Lys, Ala or Cys, and that the central amino acid
  • X be Lys or Orn.
  • the A terminal constituent is preferably hydrogen (H-) or an acetyl group.
  • the C terminal constituent may be amidated to form a lower alkyl amide.
  • dipeptides of the following general form are particularly preferred:
  • Lys-Ser, Lys-Gly, Lys-Ser-NH 2 , Orn-Gly and Orn-Ala are especially preferred.
  • D-enantiomeric forms of amino acids in the X and C positions has been shown, to substantially preserve or augment immunoregulatory activity in a number of cases.
  • Particularly preferred D-enantiomeric substitutions include, in the central amino acid X position, D-lysine and D-ornithine, and, in the C terminal position, D-alanine.
  • D-Lys-Ser and Orn-D-Ala have been shown to have particularly high immunoregulatory activity.
  • the present invention contemplates that class of dipeptides charac- terized by an amino acid A terminal constituent in conjunction with a non-amino acid C terminal constituent.
  • the C terminal constituent be a group other tha hydroxyl group (-OH), although dipeptides such as Asp-Lys and Ser-Lys, in which C is a hydroxyl group, have been shown to exhibit immunoregulatory activity.
  • a C terminal substituent of the form -NHR, wherein R is preferably hydrogen or a lower alkyl, accompanied by an A-position amino acid as discussed below, will yield a dipeptide amide of the central positively charged amino acid X. Amides of this central amino acid have been shown to have particularly significant immunoregulatory activity. Particularly preferred as A terminal amino acids are
  • Ala is especially preferred as an A terminal constituent in peptides of the form
  • X is preferably Lys, D-Lys, Orn, or D-Orn
  • C is preferably Gly, Ala, D-Ala, or Ser.
  • D-Ala as an A terminal constituent is preferred, yielding, f.e. a peptide of the form
  • peptides of formulae laa to lam, lama to lamz and Ian to lat which correspond to formula la, but wherein in laa: X is a residue of an amino acid or amino acid derivative with a positively charged side chain;
  • A is H, R, RCO, or a residue of an amino acid or amino acid derivative
  • C is NH 2 , NHR, NR 2 , OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of the form NH 2 , NHR, NR 2 , or OR; and R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X is a residue of an amino acid or amino acid derivative with a positively charged side chain
  • A is H, acetyl, Ala, Gly, Val, Phe, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-Val, D-Phe, D-Tyr, D-Asp, D-Ser or D-Thr; and C is any chemical substituent, residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide;
  • X is a residue of an amino acid or amino acid derivative with a positively charged side chain
  • A is any chemical substituent, residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is NH 2 , NHR, OR, Gly, Ala, Ser, Cys, Sar, Thr, Pro, Gln, Phe, Tyr, HomoSer, D-Ala, D-Ser, D-Cys, D-Thr, D-Pro, D-Gln, D-Phe, D-Tyr, D-HomoSer, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR or OR, where R is a C 1 -C 8 alkyl;
  • X is a residue of an amino acid or amino acid derivative with a positively charged side chain
  • A is H, R, RCO, Ala, Gly, Val, Phe, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-Val, D-Phe, D-Tyr, D-Asp, D-Ser or D-Thr;
  • C is NH 2 , NHR, OR, Gly, Ala, Ser, Cys, Sar, Thr,
  • X is a residue of an amino acid or amino acid derivative with a positively charged side chain
  • A is H, acetyl, Ala, and D-Ala
  • C is NHR, Gly, Ala, Ser, D-Ala, D-Ser, Gly-NHR, Ala-NHR, Ser-NHR, D-Ala-NHR, or D-Ser-NHR, where R is a C 1 -C 8 alkyl;
  • X is Lys, Orn, Arg, His, D-Lys, D-Orn, D-Arg, or D-His;
  • A is any chemical substituent, residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is any chemical substituent, residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • X is Lys, Orn, Arg, His, D-Lys, D-Orn, D-Art or D-His;
  • A is H, R, RCO, a residue of an amino acid or an amino acid derivative
  • C is NH 2 , NHR, NR 2 , -OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of the form NH 2 , NHR, NR 2 , or OR;
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl; in Iah: X is Lys, Orn, Arg, His, D-Lys, D-Orn, D-Arg, or D-His;
  • A is H, R, RCO, Ala, Gly, Val, Phe, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-Val, D-Phe, D-Tyr, D-Asp, D-Ser and D-Thr;
  • C is NH 2 , NHR, OR, Gly, Ala, Ser, Cys, Sar, Thr, Pro, Gin, Phe, Tyr, HomoSer, D-Gly, D-Ala, D-Ser, D-Cys, D-Ser, D-Thr, D-Pro, D-Gln, D-Phe, D-Tyr, D-HomoSer, or a residue of an amino acid amide and or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR, or OR; and
  • R is a C 1 -C 8 lower alkyl
  • X is Lys, Orn, Arg, His, D-Lys, D-Orn, D-Arg, or D-His;
  • A is H, acetyl, Ala, or D-Ala
  • C is NHR, Gly, Ala, Ser, D-Ala, D-Ser, Gly-NHR,
  • X is Lys, Orn, D-Lys or D-Orn;
  • A is any chemical substituent, residue of an amino acid, or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is any chemical substituent, residue of an amino acid, or amino acid derivative that preserves the immunoregulatory activity of said peptide; in Iak: X is Lys, Orn, D-Lys or D-Orn;
  • A is H, R, RCO, or a residue of an amino acid or amino acid derivative
  • C is NH 2 , NHR, NR 2 , OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of the form NH 2 , NHR, NR 2 , or OR;
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X is Lys, Orn, D-Lys or D-Orn;
  • A is H, R, RCO, Ala, Gly, Val, Phe, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-Val, D-Phe, D-Tyr, D-Asp, D-Ser or D-Thr;
  • C is NH 2 , NHR, Gly, Ala, Ser, Cys, Sar, Thr, Pro, Gin, Phe, Tyr, HomoSer, D-Ala, D-Ser, D-Cys, D-Thr, D-Pro, D-Gln, D-Phe, D-Tyr, D-HomoSer, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR, or OR; and R is a C 1 _ C 8 alkyl;
  • X is Lys, Orn, D-Lys or D-Orn;
  • A is H, acetyl, Ala, or D-Ala
  • C is NHR, Gly, Ala, Ser, D-Ala, D-Ser, Gly-NHR,
  • A is H
  • C is -NHR, where R is H, methyl or ethyl
  • C is NHR, where R is H, methyl or ethyl
  • A is H or acetyl
  • C is Gly or Gly-NHR, where R is H, methyl or ethyl;
  • A is Ala
  • C is Gly or Gly-NHR, where R is H, methyl or ethyl;
  • X is D-Lys
  • A is H or acetyl; and C is Gly or Gly-NHR, where R is H, methyl or ethyl;
  • A is H or acetyl
  • C is Gly or Gly-NHR, where R is H, methyl or ethyl;
  • X is D-Orn;
  • A is H or acetyl;
  • C is Gly or Gly-NHR, where R is H, methyl or ethyl;
  • A is H or acetyl
  • C is Ala or Ala-NHR, where R is H, methyl or ethyl; in Iami: X is D-Lys;
  • A is H or acetyl
  • C is Ala or Ala-NHR, where R is H, methyl or ethyl;
  • A is H or acetyl
  • C is D-Ala or D-Ala-NHR, where R is H, methyl or ethyl;
  • A is H or acetyl
  • C is D-Ala or D-Ala-NHR, where R is H, methyl or ethyl;
  • A is H or acetyl
  • C is Ala or Ala-NHR, where R is H, methyl or ethyl;
  • A is H or acetyl
  • C is Ala or Ala-NHR, where R is H, methyl or ethyl;
  • A is H or acetyl
  • C is D-Ala or D-Ala-NHR, where R is H, methyl or ethyl; in I amo : X i s D-Orn ;
  • A is H or acetyl
  • C is D-Ala or D-NHR, where R is H, methyl or ethyl;
  • X is Lys
  • A is H or acetyl
  • C is Ser or Ser-NHR, where R is H, methyl or ethyl
  • X is D-Lys
  • A is H or acetyl
  • C is Ser or Ser-NHR, where R is H, methyl or ethyl
  • A is H or acetyl; and C is D-Ser or D-Ser-NHR, where R is H, methyl or ethyl;
  • X is D-Lys
  • A is H or acetyl
  • C is D-Ser or D-Ser-NHR, where R is H, methyl or ethyl;
  • A is H or acetyl
  • C is Ser or Ser-NHR, where R is H, methyl or ethyl
  • A is H or acety
  • C is Ser or Ser-NHR, where R is H, methyl or ethyl; in lav: X is Orn;
  • A is H or acetyl
  • C is D-Ser or D-Ser-NHR, where R is H, methyl or ethyl;
  • A is H or acetyl
  • C is D-Ser or D-Ser-NHR, where R is H, methyl or ethyl;
  • X is Lys or D-Lys
  • A is Ala or D-Ala; and C is Gly;
  • X is Lys or D-Lys
  • A is Ala or D-Ala
  • C is Ala or D-Ala
  • X is Lys or D-Lys
  • A is Ala or D-Ala
  • C is Ser or D-Ser; in Ian: X is a residue of an amino acid or amino acid derivative with a positively charged side chain;
  • A is Ala, Val, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-Val, D-Phe, D-Tyr, D-Asp, D-Ser or D-Thr;
  • C is Cys, Sar, Thr, HomoSer, D-Ala, D-Ser, D-Cys, D-Thr, D-Pro, D-Gln, D-Phe, D-Tyr, D-HomoSer, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR or OR; and R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X is Lys, Orn, Arg, His, D-Lys, D-Orn, D-Arg, or D-His;
  • A is Ala, Val, Tyr, Asp, Sar, Ser, Thr, D-Ala,
  • C is Cys, Sar, Thr, HomoSer, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR or OR;
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X is Lys or D-Lys
  • A is R, RCO, Ala, Gly, Val, Phe, Tyr, Asp, Sar, Ser, Thr, D-Ala, D-Val, D-Phe, D-Tyr, D-Asp, D-Ser, or D-Thr;
  • C is NH 2 , NHR, OR, Gly, Ala, Ser, Cys, Sar, Thr, Pro, Gin, Phe, Tyr, HomoSer, D-Ala, D-Ser, D-Cys, D-Thr, D-Pro, D-Gln, D-Phe, D-Tyr, D-HomoSer, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR or OR; and R is a C 1 -C 8 lower alkyl;
  • X is Lys or D-Lys
  • A is acetyl, Ala or D-Ala
  • C is NH 2 , NHR, Gly, Ala, Ser, D-Ala, D-Ser, Gly-NHR, Ala-NHR, Ser-NHR, D-Ala-NHR, or D-Ser-NHR, where R is a C 1 -C 8 alkyl;
  • X is Orn or D-Orn;
  • A is H, R, RCO, Ala, Gly, Val, Phe, Tyr, Asp,
  • C is NH 2 , NHR, NR 2 , OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of the form NH 2 , NHR, NR 2 , or OR;
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl; provided that Orn-Ala and Orn-NH 2 are not included;
  • X is Orn or D-Orn
  • A is H, R, RCO, Ala, Gly, Val, Phe, Tyr, Asp, Sar, D-Ala, D-Val, D-Phe, D-Tyr or D-Asp;
  • C is NH 2 , NHR, Gly, Ala, Ser, Cys, Sar, Thr, Pro, Gln, Phe, Tyr, HomoSer, D-Ala, D-Ser, D-Cys, D-Thr, D-Pro, D-Gln, D-Phe, D-Tyr, D-HomoSer, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR or OR;
  • R is a C 1 -C 8 alkyl; provided that Orn-Ala and Orn-NH 2 are not included; in l at : X is Orn or D-Orn ;
  • A is H, acetyl, Ala or D-Ala; and C is NH 2 , NHR, Gly, Ala, Ser, D-Ala, D-Ser, Gly-NHR, Ala-NHR, Ser-NHR, D-Ala-NHR or D-Ser-NHR, wehre R is a C 1 -C 8 alkyl; provided that Orn-Ala and Orn-NH 2 are not included.
  • X and Y are residues of amino acids or amino acid derivatives with positively charged side chains
  • A is H, R, RCO, amino acids, and amino acid derivatives formed by N -s ⁇ bstitution of an amino acid with a substituent of the form R or RCO;
  • B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is OH, NH 2 , NHR, NR 2 , OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of the form NH 2 , NHR, NR 2 , or OR;
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X and Y are residues of amino acids or amino acid derivatives with positively charged side chains;
  • A is H, R, RCO, or a residue of any amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide;
  • B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is OH, NH 2 , NHR, NR 2 , OR, or a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide;
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X and Y are each residues of amino acids or amino acid derivatives with positively charged side chains;
  • A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn,
  • Glu Ser, Thr, D-Asp, D-Ala, D-Val, D-Leu, D-Asn, D-Glu, D-Ser, D-Thr, or a residue of a derivative of one of the foregoing amino acids formed by N ⁇ - ubstitution with a substituent of the form R or RCO;
  • B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala,
  • C is OH, NH 2 , NHR, NR 2 , OR, Gly, Ser, Ala, Val, Leu, D-Ser, D-Ala, D-Val, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR, NR 2 , or OR; and
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X and Y are each residues of amino acids or amino acid derivatives with positively charged side chains;
  • A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn,
  • B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, lie, Tyr, Phe, Lys, Orn, Trp, D-Val, D-Ser, or D-Ala;
  • C is OH, NH 2 , NHR, Gly, or Ser;
  • R is a C 1 - C 8 alkyl
  • X and Y are each Lys, Arg, His, Orn, D-Lys, D-Arg, D-His, or D-Orn;
  • A is a chemical substituent, residue of an amino acid or amino acid derivative, or sequence of amino acids or amino acid derivatives, that preserves the immunoregulatory activity of said peptide;
  • B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is a chemical substituent, residue of an amino acid or amino acid derivative, or sequence of amino acids or amino acid derivatives, that preserves the immunoregulatory activity of said peptide;
  • X an Y are each Lys, Arg, His, Orn, D-Lys, D-Arg, D-His, or D-Orn;
  • A is H, R, RCO, or a residue of an amino acid or amino acid derivative formed by N ⁇ -substitution of an amino acid with a substituent of the form R or RCO;
  • B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is OH, NH 2 , NHR, NR 2 , OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of the form NH 2 , NHR, NR 2 , or OR; and R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X and Y are each Lys, Arg, His, Orn, D-Lys,
  • A is H, R, RCO, or a residue of amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is OH, NH 2 , NHR, NR 2 , OR, or a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X and Y are each Lys, Arg, His, Orn, D-Lys, D-Arg, D-His, or D-Orn;
  • A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, Thr, D-Asp, D-Ala, D-Val, D-Leu, D-Asn, D-Glu, D-Ser, D-Thr, or a residue of a derivative of one of the foregoing amino acids formed by N ⁇ -substitution with a substituent of the form R or RCO;
  • B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, lle, Tyr, Phe, Lys, Orn, Trp, or a residue of one of the D-enantiomers of the foregoing amino acids;
  • C is OH, NH 2 , NHR, NR 2 , OR, Gly, Ser, Ala, Val, Leu, D-Ser, D-Ala, D-Val, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a sub st it u ent of the form NH 2 , NHR, NR 2 , or OR; and
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X and Y are each Lys, Arg, His, Orn, D-Lys, D-Arg, D-His, or D-Orn;
  • A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, Thr, D-Asp or a residue of a derivative of one of the foregoing amino acids formed by N -substitution with a substituent of the form R or RCO;
  • B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, lie, Tyr, Phe, Lys, Orn, Trp, D-Val, D-Ser, or D-Ala;
  • C is OH, NH 2 , NHR, Gly or Ser; and R is a C 1 -C 8 alkyl;
  • X and Y are each Lys, Arg, His, or Orn;
  • A is a chemical substituent, a residue of an amino acid or amino acid derivative, or sequence of amino acids or amino acid derivatives, that preserves the immunoregulatory activity of said peptide;
  • B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is a chemical substituent, a residue of an amino acid or amino acid derivative, or sequence of amino acids or amino acid derivatives, that preserves the immunoregulatory activity of said peptide;
  • X and Y are each Lys, Arg, His, or Orn;
  • A is H, R, RCO, or a residue of an amino acid or amino acid derivative formed by N ⁇ -substitution of an amino acid with a substituent of the form R or RCO;
  • B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is OH, NH 2 , NHR, NR 2 , OR, or a residue of an amino acid or amino acid amide or ester formed by substitution of an amino acid with a substituent of the form NH 2 , NHR, NR 2 , or OR;
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X and Y are each Lys, Arg, His, or Orn;
  • A is hydrogen, R, RCO, or a residue of amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • B is a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide
  • C is OH, NH 2 , NHR, NR 2 , OR, or a residue of an amino acid or amino acid derivative that preserves the immunoregulatory activity of said peptide; and R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X and Y are each Lys, Arg, His, or Orn;
  • A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn,
  • Glu Ser, Thr, D-Asp, D-Ala, D-Val, D-Leu, D-Asn, D-Glu, D-Ser, D-Thr, or a residue of a derivative of one of the foregoing amino acids formed by N -substitution with a substituent of the form R or RCO;
  • B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala,
  • C is OH, NH 2 , NHR, NR 2 , OR, Gly, Ser, Ala, Val, Leu, D-Gly, D-Ser, D-Ala, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR, NR 2 , or OR; and
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or C 3 -C 14 cycloalkyl;
  • X and Y are each Lys, Arg, His, or Orn;
  • A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, Thr, D-Asp or a residue of a derivative of one of the foregoing amino acids formed by N -substitution with a substituent of the form R or RCO;
  • B is Val, Leu, Glu, Gln, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, lie, Tyr, Phe, Lys, Orn, Trp, D-Val, D-Ser, or D-Ala;
  • C is OH, NH 2 , NHR, Gly or Ser;
  • R is a C 1 -C 8 alkyl
  • X is Lys, Arg, His, or Orn
  • Y is Lys, Arg, or His
  • A is Ac, Gly, Ala, Val, Asp, Asn, Glu, Arg, Ser, Thr, D-Asp, Ac-Asn, Val-Asp-, Gly-Asp-, Ala-Asp-, Ser-Asp-, Thr-Val-Asp-, or Leu-Thr-Val-Asp-;
  • B is Val, Ser, Thr, Gly, Ala, Asp, Lys, or D-Ser;
  • C is OH, or NHR, where R is H or C 1 -C 8 alkyl; in Ibp: X and Y are each Lys;
  • A is H
  • B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Pro, Asn, Asp, Arg, His, lle, Tyr, Phe, Lys, Orn, D-Ala, or D-Ser;
  • X and Y are each Lys; A is H;
  • B is Val, Leu, Glu, Gin, Ser, Gly, Pro, Tyr, Phe, Lys, Trp, or D-Val;
  • C is NHR, where R is H or a C 1 -C 8 alkyl
  • Y is Arg; A is H, B is Ser, Thr, Gly, or Ala; and
  • C is OH or NHR, where R is hydrogen or a C 1 -C 8 alkyl
  • Y is Lys; A is H;
  • B is Val, Ser, Thr, Gly, or Ala
  • C is OH or NHR, where R is H or a C 1 -C 8 alkyl
  • Y is Lys; A is H;
  • B is Asp or Ser
  • C is OH or NHR, where R is H or a C 1 -C 8 alkyl; in Ibu : X i s Lys ;
  • Y is Lys or Arg ;
  • A is Gly, Ala, Ser, Thr, Val, or Ac-Asn; B is Val; and C is OH or NHR, where R is H or a C 1 -C 8 alkyl;
  • A is Ala, Val, Ser, Thr, or Ac-Asn; B is Thr; and C is OH or NHR, where R is H or a C 1 C 8 alkyl;
  • Y is Lys or Arg
  • A is Ac-, Ala, Asn, Glu, Gly, Arg, Ac-Asn, Gly-Asp-, Ala-Asp-, Val-Asp-, Ser-Asp-, or Thr-Val-Asp-;
  • B is Ser;
  • C is OH or NHR, where R is H or a C 1 -C 8 alkyl
  • X and Y are residues of amino acids or amino acid derivatives with positively charged side chains;
  • A is H, R, RCO, a residue of an amino acid or amino acid derivative formed by N ⁇ -substitution of an amino acid with a substituent of the form R or RCO, provided that A is not Thr if X is Lys and Y is Arg or a derivative of Arg;
  • B is Val, Leu, Gin, Ser, Thr, Gly, Ala, Asp, Arg, His, Ile, Tyr, Phe, Lys, Sar, D-Thr, or D-Pro;
  • C is OH, NH 2 , NHR, NR 2 , OR, Gly, Ser, Ala, Val, Leu, D-Ser, D-Ala, D-Val, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR, NR 2 , or OR; and
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or a C 3 -C 14 cycloalkyl;
  • X and Y are residues of amino acids or amino acid derivatives with positively charged side chains:
  • A is H, R, RCO, a residue of an amino acid or amino acid derivative formed by N ⁇ -substitution of an amino acid with a substituent of the form R or RCO;
  • B is Glu, Asn, Orn, Trp, D-Val, D-Ser, D-Ala, D-Leu, D-Glu, D-Gln, D-Ala, D-Asn, D-Asp, D-Arg, D-His, D-Ile, D-Tyr, D-Phe, D-Lys, D-Orn, D-Trp, or a residue of a derivative of one of the foregoing amino acids;
  • C is OH, NH 2 , NHR, NR 2 , OR, Gly, Ser, Ala, Val,
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or a C 3 -C 14 cycloalkyl;
  • X and Y are residues of amino acids or amino acid derivatives with positively charged side chains;
  • A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, D-Asp or a residue of a derivative of one of the foregoing amino acids formed by N ⁇ -substitution with a substituent of the form R or RCO;
  • B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Asp, Arg, His, Ile, Tyr, Phe, Lys, Orn, Trp, D-Val, D-Ser, or D-Ala;
  • C is OH, NH 2 , NHR, Gly or Ser; and R is a C 1 -C 8 alkyl;
  • X and Y are each Lys, Arg, His, Orn, D-Lys, D-Arg, D-His or D-Orn;
  • A is H, R, RCO, a residue of an amino acid or amino acid derivative formed by N ⁇ -substitution of an amino acid with a substituent of the form R or RCO, provided that A is not Thr if X is Lys and Y is Arg or a derivative of Arg;
  • B is Val, Leu, Gin, Ser, Thr, Gly, Ala, Asp, Arg, His, Ile, Tyr, Phe, Lys, Sar, D-Thr, or D-Pro;
  • C is OH, NH 2 , NHR, NR 2 , OR, Gly, Ser, Ala, Val, Leu, D-Ser, D-Ala, D-Val, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR, NR 2 , or OR; and
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or a C 3 -C 14 cycloalkyl;
  • X and Y are each Lys, Arg, His, Orn, D-Lys, D-Arg, D-His or D-Orn;
  • A is H, R, RCO, a residue of an amino acid or amino acid derivative formed by N ⁇ -substitution of an amino acid with a substituent of the form R or RCO;
  • B is Glu, Asn, Orn, Trp, D-Val, D-Ser, D-Ala, D-Leu, D-Glu, D-Gln, D-Ala, D-Asn, D-Asp, D-Arg, D-His, D-lle, D-Tyr, D-Phe, D-Lys, D-Orn, D-Trp, or a residue of a derivative of one of the foregoing amino acids;
  • C is OH, NH 2 , NHR, NR 2 , OR, Gly, Ser, Ala, Val, Leu, D-Ser, D-Ala, D-Val, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR, NR 2 , or OR; and
  • R is a C 1 - C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or a C ,3 -C 14 cycloalkyl;
  • X and Y are each Lys, Arg, His, Orn, D-Lys, D-Arg, D-His, or D-Orn;
  • A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, D-Asp or a residue of a derivative of one of the foregoing amino acids formed by N -substitution with a substituent of the form R or RCO;
  • B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Asn, Asp, Arg, His, Ile, Tyr, Phe, Lys, Orn, Trp, D-Val, D-Ser, or D-Ala;
  • C is OH, NH 2 , NHR, Gly or Ser
  • R is a C 1 -C 8 alkyl
  • X and Y are each Lys, Arg, His, or Orn;
  • A is H, R, RCO, a residue of an amino acid or amino acid derivative formed by N ⁇ -substitution of an amino acid with a substituent of the form R or RCO, provided that A is not Thr if X is Lys and Y is Arg or a derivative of Arg;
  • B is V al , Le u , Gin , Ser , Thr , Gly , Ala , Asp , Ar g , His , I le , Tyr , Phe , Lys , Sar , D- Thr , or D-Pro ;
  • C is OH, NH 2 , NHR, NR 2 , OR, Gly, Ser, Ala, Val, Leu, D-Ser, D-Ala, D-Val, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NR 2 , or OR; and
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or a C 3 -C 14 cycloalkyl;
  • X and Y are each Lys, Arg, His, or Orn;
  • A is H, R, RCO, a residue of an amino acid or amino acid derivative formed by N ⁇ -substitution of an amino acid with a substituent of the form R or RCO;
  • B is Glu, Asn, Orn, Trp, D-Val, D-Ser, D-Ala, D-Leu, D-Glu, D-Gln, D-Ala, D-Asn, D-Asp, D-Arg, D-His,
  • C is OH, NH 2 , NHR, NR 2 , OR, Gly, Ser, Ala, Val, Leu, D-Ser, D-Ala, D-Val, D-Leu, or a residue of an amino acid amide or ester formed by substitution of one of the foregoing amino acids with a substituent of the form NH 2 , NHR, NR 2 , or OR; and
  • R is a C 1 -C 8 aliphatic, C 6 -C 14 aryl, aralkyl or alkaryl, or a C 3 -C 14 cycloalkyl; in Ibzf: X and Y are each Lys, Arg, His, or Orn;
  • A is H, R, RCO, Asp, Gly, Ala, Val, Leu, Asn, Glu, Ser, D-Asp or a residue of a derivative of one of the foregoing amino acids formed by N -substitution with a substituent of the form R or RCO;
  • B is Val, Leu, Glu, Gin, Ser, Thr, Gly, Ala, Asn, Asp, Arg, His, He, Tyr, Phe, Lys, Orn, Trp, D-Val, D-Ser, or D-Ala;
  • C is OH, NH 2 , NHR, Gly or Ser
  • R is a C 2 -C 8 alkyl
  • X is Lys, Arg, His, or Orn
  • Y is Lys, Arg, or His
  • A is Ac, Gly, Ala, Val, Asp, Asn, Glu, Arg, Ser, Thr, D-Asp, Ac-Asn, Val-Asp-, Gly-Asp-, Ala-Asp-, Ser-Asp-, Thr-Val-Asp-, or Leu-Thr-Val-Asp-;
  • B is Val, Ser, Thr, Gly, Ala, Asp, Lys, or D-Ser;
  • C is OH, or NHR, where R is H or C 1 -C 3 alkyl
  • X and Y are each Lys; A is H;
  • B is Val, Leu, Glu, Gin, Ser, Thr, Ala, Asn, Arp, Arg, His, Ile, Tyr, Phe, Lys, Orn, D-Ala, or D-Ser;
  • X and Y are each Lys; A is H; B is Val, Leu, Glu, Gin, Ser, Gly, Pro, Tyr, Phe, Lys, Trp, or D-Val; and
  • C is NHR, where R is H or a C 1 -C 3 alkyl
  • A is H
  • B is Ser, Thr, Gly, or Ala; and C is OH or NHR, where R is H or a C 1 -C 3 alkyl;
  • A is H
  • B is Val, Ser, Thr, Gly or Ala
  • C is OH or NHR, where R is hydrogen or a C 1 -C 3 lower alkyl
  • Y is Lys
  • B is Asp or Ser
  • C is OH or NHR, where R is hydrogen or a C 1 -C 3 alkyl
  • Y is Lys or Arg
  • A is Gly, Ala, Ser, Val, or Ac-Asn;
  • C is OH or NHR, where R is hydrogen or a C 1 -C 3 alkyl; in Ibzn: X is Lys;
  • Y is Lys or Arg
  • A is Ala, Val, Ser, or Ac-Asn
  • C is OH or NHR, where R is H or a C 1 -C 3 alkyl; in Ibzo: X is Lys;
  • Y is Lys or Arg
  • A is Ac, Ala, Asn, Glu, Gly, Arg, Ac-Asn, Gly-Asp-, Ala-Asp-, Val-Asp-, Ser-Asp-, or Thr-Val-Asp-;
  • C is OH or NHR, where R is H or a C 2 -C 3 alkyl.
  • the peptides of formula I can be prepared by usual methods of peptide synthesis.
  • a peptide of formula I is prepared by liberating it from its corresponding functional derivative which may be bound by a covalent bond to a solid resin by treatment under acidic or basic conditions, and, if desired, the peptide thus obtained is esterified, amidated and/or acylated to yield a corresponding ester, N- and/or O-acyl derivative or is transformed into one of its pharmacologically acceptable salts by treatment with a base or an acid.
  • T-lymphocytes play a powerful role in the in vivo induction, suppression, and regulation of immune responses leading to a number of autoimmune and other disease conditions.
  • in vitro assays have been developed which are predictive of in vivo T cell function.
  • lymphoid cells from peripheral blood or from the spleen, lymph nodes or other organs are isolated and cultured with or without potential immunoregulatory drugs. The extent to which lymphocyte function in general and T cell function in particular are regulated may be assessed by many measurement techniques.
  • the most common techni- que measures the amount of a radioactive precursor of DNA (triti- ated thymidine) incorporated into newly synthesized DNA of lymphocytes. Since only "activated" cells that are preparing to divide synthesize new DNA, the amount of tritiated thymidine incorporated in cells and the resultant incorporated radioactivity is directly related to the extent of cell activation and resultant proliferation. In order to predict whether a potential immunoregulatory drug will be useful in treating a particular disease or class of diseases, it is important that it be tested in in vitro or in vivo system that are reflective of the particular disease or diseases in question. Several in vitro assays of T cell function in particular are highly predictive of in vivo efficacy in treating organ transplantation rejection and autoimmune disease.
  • the mixed lymphocyte response is an in vitro analog of the in vivo organ transplantation rejection response.
  • MLR mixed lymphocyte response
  • T cell-containing lymphocyte preparations from two humans or animals are cultured together for five to seven days under standard cell culture conditions.
  • T cells from both subjects recognize histocompatibility molecules of the other subject. If both subjects are genetically identical, their histocompatiblity molecules will also be identical and no activation or cell proliferation will occur. The radioactivity incorporated into cells will therefore be very low since few cells are stimulated to divide.
  • both subjects are genetically non-identical, both subjects cells will recognize the foreign histocompatibility molecules of the other and will divide and proliferate.
  • This assay is termed a two-way MLR since each subject's cells respond to the other's proliferative stimuli. If one of the subject's cells is prevented from dividing by, for example, exposure to gamma ir radiation or raitomycin C, then the observed proliferation and incorporated radioactivity will be due only to the other subject's cellular proliferation. This assay is termed a one-way MLR since each subject's cells respond to the other's proliferative stimuli. If one of the subject's cells is prevented from dividing by, for example, exposure to gamma ir radiation or raitomycin C, then the observed proliferation and incorporated radioactivity will be due only to the other subject's cellular proliferation. This assay is termed a one-way
  • MLR MLR-derived cytotoxic or Killer T cells
  • T cells from a transplant recipient are sensitized to the histocompatibility antigens of a transplanted organ in vitro and then injected into the recipient, the sensitized T cells will cause the organ to be rejected.
  • Sensitized T cells alone are therefore both necessary and sufficient to cause organ transplant rejection.
  • the peptides of the present invention can substantially suppress the MLR without significantly affecting cell viability. This indicates that such peptides have therapeutic usefulness since they can suppress the immune response responsible for the immnunologic rejection of transplanted organs and autoimmune diseases.
  • suppression of T cell activities in vitro by peptides of the present invention is but one manifestation of the complex immunoregulation which occurs in vitro and in vivo and which leads to reduction or prevention of autoimmune disease or of the rejection of transplanted organs.
  • the peptides of the present invention can suppress a MLR at concentrations listed in table 1, lower peptide concentrations typically result in stimulation of tritiated thymidine uptake which is reflective of the peptides' ability to activate certain immunoregulatory processes.
  • Administration of the subject peptides to animals for example, can in certain cases result in stimulation of certain T lymphocyte subsets, which can in turn lead to net suppression of a clinical disease or condition. It is thus entirely within the scope of the peptides of the present invention that certain immunostimulatory properties, in addition to the suppressive properties described herein, will be expressed in alternate in vitro and in vivo tests of the subject peptides.
  • the MLR is a specific example of a more general T cell response toward "foreign" antigens.
  • T cells recognize and proliferate in response primarily to antigens located on histocompatibility molecules of the stimulator cells. Molecules unrelated to histocompatibility antigens may also serve as potent stimulators of T cell proliferative and killing responses. In fact virtually any molecule, if presented to T cells in an appropriate manner, may stimulate T cell proliferative, regulatory and killing responses directed toward the stimulating antigen.
  • Such antigen-specific T cell responses in humans are thought to be responsible for many of the autoimmune diseases listed earlier when immune tolerance is reduced or lost.
  • an antigen-specific T cell proliferation assay which uses mice that have been immunized with a particular antigen such as Bovine Serum Albumin (BSA).
  • BSA Bovine Serum Albumin
  • T cells in lymph nodes which drain the injection site are activated and stimulated to proliferate.
  • regulatory and Killer T cells which specifically recognize BSA are generated.
  • the draining lymph nodes are removed and cells eluted from them are placed into a standard culture.
  • BSA or a second, unrelated antigen is then added to the cultures with or without the immunoregulatory peptides of the present invention.
  • Quanti- tation of antigen-induced T cell activation and proliferation is assessed by cellular tritiated thymidine incorporation at the end of the culture incubation in a manner similar to that used in MLR cultures.
  • Cultures containing BSA will produce high levels of tritiated thymidine incorporation because the BSA-sensitized T cells recognize the BSA molecules and become stimulated to proli- ferate.
  • Cultures containing an unrelated antigen by contrast, have low stimulation levels because they lack receptors directed toward the unrelated antigen.
  • the peptides of the present invention substantially suppress the T cell proliferative response toward specific molecular or cellular antigens without significantly affecting cellular viability. This observation is of therapeutic importance because it indicates that such peptides can suppress an abnormal T cell immune response directed toward a wide range of unrelated antigens different from those found on histocompatibility antigens. As previously discussed, abnormal Killer T cell responses directed against either foreign or normal "self" antigens on various organs are thought to be responsible for many autoimmune diseases. Other autoimmune diseases may result from an abnormal regulatory T cell response towards specific antigens that result in an imbalanced response of othe r T cell-regulated portions of the immune system.
  • T cells isolated from such an immunized animal may be very specific for molecules and cells of the injected organ showing that the T cells have receptors for organ-specific antigens.
  • organs or their preparations as immunizing agents as described, it is possible to develop animal models of autoimmune diseases involving virtually any organ system, some of which were previously enumerated.
  • FIGS 1 and 2 demonstrate the efficacy of one peptide and one of its peptidomimetic analogs in treating an animal model of human multiple sclerosis (MS).
  • MS multiple sclerosis
  • EAE Experimental Allergic Encephalomyelitis
  • SJL/J mice were immunized with a homogenate of spinal cord from SJL/J mice in a Complete Freund's Adjuvant medium. This homogenate contains cells and molecules representative of the many potential antigens which are present in the central nervous system (CNS).
  • CNS central nervous system
  • mice typically develop vestibular and occular disturbances, weakness, paralysis, and other signs of central nervous system destruction characteristic of both EAE and MS ten to fifteen days after initial immunization.
  • the EAE mod is discussed in Brown et al., Lab. Invest. 45(3) :278-284 (1981).
  • the disease sever- ity in this animal MS model waxes and wanes during the disease course.
  • Mice receiving only saline injections two to three times per week exhibit high peaks and troughs of disease severity.
  • mice receiving peptide or peptidomimetic demonstrated very mild disease initially and were clinically disease free by the study's end. Microscopic analysis of brain sections from both peptide and saline-treated mice is presented in Table 3.
  • the peptides may also be used to regulate the growth of neoplastic (cancer) cells in vivo. All cancer cells have in common the property of unregulated cell replication. Whereas normal cells replicate at a rate consistent with the normal functioning of the organ in which the cells reside, cancerous cells replicate in an unregulated manner, which leads to tumor formation and possible destruction of adjacent healthy tissue.
  • the peptides of the present invention can suppress or prevent the growth of a variety of neoplastic cells in vitro.
  • Figure 3 demonstrates the anticancer properties of one of the peptides of the present invention in mice with lymphoma/leukemia of T cell origin. Whereas saline treated mice had a median survival of 23.5 days after injection of lymphoma/leukemia cells, peptide-treated mice had a median survival of 30.5 days. Additionally, all saline-treated mice were dead by day 31 while one peptide-treated mouse was tumor-free at day 50.
  • the peptides of the present invention may be administered to humans or other mammals by a variety of means commonly utilized with respect to other therapeutic agents.
  • a dosage range of from about 25 ⁇ g/kg to about 50 mg/kg of body weight may be employed, with a preferred range of about 2.5 to 50 mg/kg.
  • Sterile saline, aqueous dextrose and glycols are preferred liquid carriers, particularly (when isotonic) for injectable solutions.
  • Intravenous, intraperitoneal, and intramuscular injections may also be used at dosages comparable to those of subcutaneous injections.
  • the peptides may be administered orally at dosages generally ranging from one to 100 times that of an injected dose, although greater dosages may be required depending on factors such as food intake.
  • the peptides may be combined with appropriate pharmaceutical carriers in the form of pills, capsules, elixers, suspensions, aerosols, powers, or in other forms.
  • Intranasal administration may be achieved by using an appropriate amphipathic vehicle such as, for example, glycocholic acid or EDTA.
  • Administration may be achieved topically or transdermally, using an appropriate carrier in the form of, for example, an ointment.
  • Sublingual, rectal, or topical ocular administration using appropriate pharmaceutical media may be employed.
  • Sustained-release formulations using, for example, liposome delivery media are also appropriate vehicles for administering the subject peptides.
  • Lys-Ser-NH 2 50 99 100 99 desamino-Lys-Ser 200 98 400 99
  • Lys-Leu-Lys-NH 2 50 99 100 89
  • Lys-Tyr-Lys-NH 2 100 99 400 98
  • Lys-D-Val-Lys-NH 2 400 99 400 63 Lys-Ser-Arg 100 92 100 49
  • Val-Lys-Val-Lys-NH 2 100 99 200 99
  • concentrations of the subject peptides which produce inhibition or stimulation of proliferation may occasionally vary from the concentrations listed depending on many factors including age of the donor, time of day of blood drawing, food intake and other factors.
  • Lys-NH 2 100 98 D-Lys-NH 2 100 99 D-Lys-NH 2 CH 2 CH 3 100 36
  • Lys-Ser-NH 2 25 desamino-Lys-Ser 100 68 Ac-Lys-Ser 50 98 Ac-Lys-Ser-NH 2 50 99 N-Ch 3 -Lys-D-Ser 100 99
  • Lys-Cys 100 74 Lys-Phe 50 99 Lys-Tyr 100 75 Lys-Asp 100 92
  • Ala-Lys-Ser 50 98 Ala-Lys-Gly 100 99 D-Ala-D-Lys-D-Ser 100 -31 Phe-Lys-D-Ala 100 99
  • concentrations of the subject peptides which produce inhibition or stimulation of proliferation may occasionally vary from the concentrations listed depending on many factors including age of the donor, time of day of blood drawing, food intake and other factors.
  • mice were injected subcutaneously with mouse spinal cord homogenate in Complete Freund's Adjuvant on days 0 and 7 and injected with saline or 1 mg peptide 3 times per week from day 11 to 32. The number of mice in each group is enclosed within parentheses. Tissue was sectioned, the sample identity was coded and was examined microscopically by an uninformed observer. The percent inhibition of lesions is given in the last column in parentheses.
  • Orn-D-Ala 100 89 100 86 Orn-Gly 100 95 100 86
  • Lys-Val-Lys 100 64 200 85 Lys-Glu-Lys 200 43 200 43 Lys-Gln-Lys 200 99 200 94 Lys-Arg-Lys 200 73 100 50 Lys-His-Lys 200 99 200 94 Lys-D-Ala-Lys 25 -19 100 -83
  • Lys-Val-Lys-NH 2 200 93 100 82 Lys-Leu-Lys-NH 2 200 96 200 96 Lys-Pro-Lys-NH 2 100 95 100 90 Lys-Tyr-Lys-NH 2 200 85 200 81 Lys-D-Val-Lys-NH 2 200 28 100 24
  • concentrations of the subject peptides which produce inhibition may occasionally vary from the concentrations listed depending on many factors including age of the cells, culture conditions and other factors.
  • Lys-Ser-NH 2 100 65 100 78 desamino-Lys-Ser 200 20 200 25
  • Orn-D-Ala 200 88 100 52 Orn-Gly 200 72 200 48
  • Lys-Ser-Tyr 100 37 100 -9 Lys-Ser-Ala 200 46 200 58 Lys-Arg-Lys 100 70 50 72
  • concentrations of the subject peptides which produce inhibition may occasionally vary from the concentrations listed depending on many factors including age of the cells, culture conditions and other factors.
  • the dicyclohexylammonium salt (73 g) was dissolved in icewater (800 ml), the pH adjusted to 3 with l N sodium bisulfate and the product extracted into ethyl acetate (500 ml, 200 ml, 100 ml). The combined organic extracts were dried over sodium sulfate and the solution evaporated to give N,N'.-bis-t-butyloxycarbonyl-L-lysyl-L-serine (46.3 g). Deprotection to bis-trifluoroacetate salt.
  • N,N'-bis-t ⁇ butyloxycarbonyl-L-lysyl-L-serine 17.8 g was dissolved in dichloromethane (70 ml), anhydrous trifluoroacetic acid (50 ml) added and the mixture stirred at room temperature for 0.5 hours. The solution was evaporated under reduced pressure and the residue triturated three times with dichloromethane and three times with ether to give the bis-trifluoroacetate salt of L-lysyl-L-serine in quantitative yield as a colorless, hygroscopic solid.
  • N,N'-Bis-t-butyloxycarbonyl-L-lysyl-L-serine (20 g) was dissolved in an anhydrous solution of hydrogen chloride in ethyl acetate (3.5 N, 200 ml) and the mixture stirred at room temperature for 0.5 hours. The solution was evaporated under reduced pressure and the residue triturated four times with ether to give the dihydrochloride salt of L-lysyl-L-serine in quantitative yield as a white hygroscopic solid.
  • N,N'-bis-t-butyloxycarbonyl-L-lysyl-L-serine 25 g was dissolved in dioxane (50 ml) and treated with a solution of hydrogen chloride in anhydrous dioxane (4 N, 250 ml). The mixture was stirred at room temperature for 0.5 hours, evaporated under reduced pressure and the residue triturated four times with ether to give the dihydrochloride salt of L-lysyl-L-serine in quantitative yield as a colorless, glassy hygroscopic solid.
  • N,N'-Bis-benzyloxycarbonyl-L-lysyl-L-serine (3 g) was dissolved in a mixture of methanol (20 ml) and water (20 ml) containing acetic acid (0.35 ml, 2 equivalents) and hydrogenated over 10% palladium on carbon (1 g) at atmospheric pressure overnight. After removing the catalyst by filtration, the solution was concentrated to approximately half the volume under reduced pressure, and the residue lyophilized, to give the acetate salt of L-lysyl-L-serine in quantitative yield as a colorless, hygroscopic solid.
  • Steps 1-7 constitute one complete coupling cycle.
  • the peptide was cleaved from the resin by treatment with liquid hydrogen fluoride (10 ml per gram of dry resin) in the presence of anisole (1 ml per gram of resin) and 0 ⁇ C for one hour. After removal of the hydrogen fluoride under vacuum, ether (10 ml per gram of resin) was added, the resin transferred to a sintered glass funnel and washed alternately with ether (4 x 40 ml) and chloroform (3 x 40 ml). The resin was sucked dry and the peptide extracted with 1 N aqueous acetic acid (4 x 30 ml). The combined extracts were lyophilized, redissolved in distilled water (7 ml) and relyophilized to give 816 mg of the acetate salt of the crude peptide, Lys-Ser.
  • Lys-Val-Lys is obtained from tert.butoxycarbonyl- ⁇ -2-chlorobenzoxy-carbonyl-L-lysine-resin with t-butoxycarbonyl-L-valine and t-butoxycarb ⁇ nyl- ⁇ -benzoxycarbonylL-lysine.
  • Example 4 Purification of Lys-Ser using carboxymethyl Sephadex chromatography. Following the general procedure described in Example 4, the crude peptide from Example 2 (2 g) was dissolved in distilled water (1 1), the pH adjusted to 4.5-6.5 (1 M aqueous ammonia) and the solution applied to a column (2.5 x 30 cm) of CM-Sephadex (Pharmacia, Inc.) which had been equilibrated with 0.01 M ammonium acetate, pH 4.5-6.5. The column was then eluted with a linear gradient of 0.01 M ammonium acetate, pH 4.5-6.5 (350 ml) to 0.30 M ammonium acetate, pH 6.5 (350 ml).
  • Lys-Ser using Sephadex G-10 chromatography.
  • the peptide from Example 2 (1.0 g) was dissolved in a minimum volume of distilled water (5 ml) and applied to a column (2.5 x 100 cm) of Sephadex G-10 (Pharmacia) which had been equilibrated with 3% aqueous acetic acid.
  • the column was eluted with the same solvent and the fractions which contained the pure product combined and lyophilized to give L-lysyl-L-serine acetate salt as a colorless, glassy, hygroscopic solid.
  • Lys-Val-Lys is purified.
  • Lys-NH 2 The crude peptide, Lys-NH 2 , was cleaved from the resin using liquid hydrogen fluoride, following the procedure described in Example 3. Yield: 693 mg. Analogously, Lys-Val-Lys-NH 2 is prepared.
  • Peptide amides may be synthesized by an identical procedure, using 4-methylbenzhydrylamine resin in place of benzhydrylamine resin.
  • the resin was collected on a sintered glass funnel, washed successively with ethanol and dichloromethane and dried under vacuum.
  • the peptide was cleaved from the resin using liquid hydrogen fluoride at 0°C, as described in Example 3 and purified by chromatography on C-18 silica, as described in Example 6 to give N ⁇ -acetylL-lysyl-L-serine acetate as a colorless, glassy hygroscopic solid. Yield: 304.7 mg.
  • the mixture is heated with agitation at 80oC using an oilbath for two hours and then allowed to stand at room temperature overnight.
  • the resin is transferred to a sintered glass funnel, washed with dimethylsulfoxide (3 times), alternately with methanol and dichloromethane (3 times each) and finally with methanol (2 times) and dried under vacuum. Degree of substitution is calculated from the weight gain of the resin.
  • N,N-bis-benzyloxycarbonyl-L-lysine (35.6 g, 0.086 mole) is dissolved in tetrahydrofuran (350 ml), the solution cooled to -15°C and treated with N-methylmorpholine (9.02 ml, 0.082 mole) followed by. isobutyl chloroformate (10.64 ml, 0.082 mole).
  • Lys-Glu-Lys 3 Boc- ⁇ -Cbz-L-Lys Boc- ⁇ -Bzl-L-Glu; 7 Boc- ⁇ -Cbz-L-Lys Lys-Gln-Lys 3 Boc- ⁇ -Cbz-L-Lys Boc-X-L-Gln; 7
  • Lys-Ser-Arg 3 Boc-N g - Boc-OBzl-L-Ser; 7 tosyl-L-Arg Boc- ⁇ -Cbz-L-Lys Lys-Thr-Arg 3 BOC-N9- Boc-OBzl-L-Thr 7 tosyl-L-Arg Boc- ⁇ -Cbz-L-Lys Arg-Gly-Lys Boc- ⁇ -Cbz-L-Lys Boc-Gly; 4,7
  • ** P refers to the purification protocol followed, as described in the Example number(s) given in this column. Where two protocols are listed, these were utilized in the order listed.
  • Trifluoroacetic Acid was distilled from phosphorus pentoxide at atmospheric pressure. Dicyclohexylamine was dried over solid potassium hydroxide and distilled under vacuum.
  • Dioxane was dried over metallic sodium pieces (freshly cut) and distilled at atmospheric pressure.
  • Dimethylformamide was dried over solid potassium hydroxide, decanted and distilled from ninhydrin (1 g per liter of solvent) under vacuum.
  • Pyridine was dried over potassium hydroxide, decanted and distilled from ninhydrin (1 g per liter of solvent) at atmospheric pressure.
  • Products were visualized by spraying the plates with ninhydrin (1% solution in ethanol) or chlorine peptide spray (1% solution of t-butyl hypochlorite in cyclohexane, followed by a 1% solution of o-toluidine in 10% acetic acid).
  • High performance liquid chromatography was carried out on C-18 reverse-phase columns (5 ⁇ m particle size, 4.6 mm i.d. x 25 cm) using gradients of acetonitrile in 5 mM aqueous hexanesulfonic acid or octanesulfonic acid. Flow-rates were typically 1 ml/min., chromatograms were monitored at 215 nm and 10-20 ⁇ g loads of samples were injected.
  • Murine Mixed Lymphocyte Response Assay Procedure The murine MLR assay procedure is described in Dutton, R.W., J. Exp. Med., 123:655-671 (1966).
  • the spleen is removed from mice of two different strains, and the spleen cells from each strain are disbursed and separately suspended in human serum albumin in RPMI1640 (2% HSA-RMPI, Irvine Scientific).
  • the cell suspensions are then centrifuged at 400 G for ten minutes at 15oC, and the cell pellets resuspended in 2% HSA-RPMI to a total volume of 50 ml.
  • the cell concentration is ascertained using, e.g., a hemacytometer.
  • a stimulator cell suspension is prepared by incubating the cells of one strain with 25 ⁇ g mitomycin C per ml for 30-60 minutes at 37oC or irradiation with a total of 3200 roentgens in order to inhibit DNA synthesis. After washing the stimulator cells three times with 2% HSA-RPMI, both the stimulator cells and the untreated responder cells are suspended in KC 2000 (KC Biologicals) to a concentration of 4 x 10 6 cells/ml.
  • KC 2000 KC Biologicals
  • Equal portions of each suspension are then combined, and a control group is prepared in a microtiter plate well by transferring 0.1 ml aliquots each of the cell mixture and KC 2000.
  • An experimental group is prepared by transferring to a microtiter plate well 0.1 ml of the cell mixture and 0.1 ml of the test peptide diluted to twice the experimental concentration.
  • the microtiter plates are incubated in a humidified 37°C incubator with an atmosphere of 5% CO 2 for seven days, whereupon 1 ⁇ Ci 3 H-thymidine is added in 0.025 ml/well.
  • the control and experimental groups are incubated for six hours and then harvested using a 12-channel SCATRON cell harvester or the equivalent.
  • venous blood is extracted from two unrelated human subjects (A and B) in sterile syringes containing 0.15 ml heparin (10,000 units/ml) per 60 cc blood.
  • the mononuclear cells are isolated by centrifuging 40 ml of a 1:1 mixture of blood and sterile RPMI-1640 (Irvine Scientific), underlaid with 8 ml Ficoll-Paque (Pharmacia Fine Chemicals), for 30 minutes at 400 G and 15°C. The plasma is discarded and the mononuclear cell band adjusted to 40 ml with 2% human serum albumin in RPMI-1640 (2% HSA-RPMI, Irvine Scientific). The cell mixture is centrifuged for 8 minutes at 40 G and 15°C, residual plasma discarded, and the cell band rediluted and recentrifuged as immediately above.
  • the cell pellet is resuspended in 10.0 ml KC 2000 (KC Biologicals), and the mononuclear cells counted using a Coulter counter or the equivalent.
  • Stimulator cells (A* and B*) are prepared by treating 20 X 10 6 mononuclear cells with irradiation or mitomycin C as described above in the murine MLR assay procedure.
  • Responder cells (A and B) are prepared by washing the untreated cells once with 2% HSA-RPMI. Both responder and stimulator cells are adjusted to a final concentration of 2 X 10° mononuclear cells/ml in KC 2000.
  • experimental and control groups are assayed by incubating equal-volume mixtures of, e.g., responder cells A and stimulator cells B* for seven days with and without the subject peptide, as described in the murine MLR procedure. 3 H-thymidine is then added to each sample, followed by incubation for six hours, harvesting, and counting of the incorporated labelled DNA in the newly replicated cells as in the murine assay.
  • the antigen-specific T-cell proliferation assay consists of injecting a live mouse with a specific antigen for a period sufficient to allow sensitization to the antigen, culturing the sensitized lymph node cells,, and then reexposing the lymph node cells to the antigen after treatment with the subject peptide. Following an appropriate incubation period, the amount of immunoresponsive T-cell proliferation is measured using labelled-DNA uptake methods, e.g., tritiated thymidine incorporation, thus providing a measure of the immunoregulatory activity of the subject peptide with respect to a specific antigen.
  • the data in Table 2 reflect peptide-induced inhibition of T-cell proliferation in response to the antigen ovalbumin. Similar assays may be performed using such antigens as bovine serum albumin, human IgG, sheep, chicken or bovine erythrocytes, or haptens such as dinitrophenol.
  • the antigen-specific T cell proliferation assay is described in "Antigen Induced Proliferation Assay for Mouse TLymphocyte Response to Monovalent Antigens," Eur. J. Immuno. 8:112-118 (1978).
  • a mouse is injected subcutaneously at the base of the tail with 0.1 mg ovalbumin in 0.05 ml Complete Freund's
  • Adjuvant After seven days, the mouse is sacrificed and its superficial inguinal lymph nodes removed aseptically. Excess fat is removed, and the lymph node cells are dispersed into 15 ml of 2% human serum albumin (HSA) in phosphate-buffered saline (PBS) by, e.g., gentle teasing between the frosted surfaces of two sterile microscope slides. The dispersed single cells are then gently resuspended three to five times using a sterile plastic pipet, transferred to a sterile centrifuge tube, and the supernatent transferred to a new centrifuge tube, free of settled debris, after three minutes.
  • HSA human serum albumin
  • PBS phosphate-buffered saline
  • the single cell suspension is centrifuged for ten minutes at 400 G and 15oC, and the pellet resuspended and tested for cell viability by measuring the exclusion of trypan blue dye (0.5% solution, 0.020 ml) from cells of the cell suspension (0.100 ml). The cells should measure at least about 95% viable. The cells are then washed twice in 2% HSA-PBS and resuspended at approximately 8 x 10° lymph node cells/ml in Complete Click's Medium. The cell count is determined and the lymph node cell solution diluted to a final concentration of 4 x 10 6 cells/ml with Complete Click's Medium.
  • Control groups receive Complete Click's Medium. After at least 15 minutes, 100 ⁇ g/ml of ovalbumin is added to groups with and without the subject peptide. Control groups are prepared without antigen and with 100 ⁇ g/ml of anti gens unrelated to ovalbumin (e.g., human IgG) or with mitogens such as concanavalin A, phytohemagglutinen, and polkweed mitogen. The plates are incubated at 37°C and 5% CO 2 . On day four to day five, 1 ⁇ Ci of 3 H-thymidine is added in 0.025 ml/well, and the plates are incubated for 24 hours before harvesting and counting as described with respect to the murine MLR assay procedure.
  • EXAMPLE 18 Leukemia and Tumor Cell Proliferation Assay Mouse leukemia/lymphoma and human tumor cell lines are available from, e.g., American Type Culture Collection, 12301 Parklawn Drive, Rockville MD 20852-1776. The individual cells are gently and sterilely dispersed to suspension in PBS. Cell viability is ascertained using the trypan blue dye exclusion method described in Example 16, and should be at least about 85%. The cells are washed 2-3 times in PBS and then twice in RPMI 1640. The cells are then resuspended in a sufficient quantity of approximately 10% FCS-RPMI or HSA-RPMI to yield a final cell concentration of 1 x 10 6 cells/ml.
  • 0.1 ml aliquots of the tumor cell suspension are transferred to microtiter plate wells.
  • 0.1 ml aliquots of appropriately-diluted peptide in RPMI 1640 are added as indicated in Tables 4 and 5.
  • the cells are pulsed for six hours with 1 ⁇ Ci 3 H-thymidine, harvested, and counted as described in the foregoing examples.
  • General procedures relating to the measurement of neoplastic cell proliferation may be found in Sample et al.,

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Abstract

Des peptides et des dérivés de peptides, ainsi que leurs procédés d'utilisation dans le système immunitaire de mammifères afin de supprimer des réactions d'auto-immunité, des réactions de rejet lors de la transplantation d'organes, et la croissance cellulaire néoplastique. Ces peptides se caractérisent par la formule A-X-(B-Y)n-C dans laquelle X et Y sont des acides aminés résiduels ou de dérivés d'acides aminés avec des chaînes latérales positivement chargées, par exemple Lys, Orn, Arg, His, D-Lys, D-Orn, D-Arg, ou D-Wis, ou des émantiomères -D d'un de ces acides aminés résiduels; A et C sont des substituants qui entretiennent ou augmentent l'acitivité de régulation immunitaire de ces peptides, et n est égal à 0 ou 1. L'activité de ces peptides comprend la suppression de la prolifération de lymphocytes-T dans des systèmes in vitro analogues à des états pathologiques in vivo de mammifères, la régulation de la prolifération de cellules cancéreuses in vitro et in vivo, et la réduction in vivo de lésions associées à des maladies auto-immunitaires. Ces peptides ont un potentiel thérapeutique bénéfique pour des êtres humains pour ce qui est du traitement de maladies auto-immunitaires et néoplastiques, de rejets de greffes et d'organes, et d'autres maladies.
EP19860900764 1985-01-18 1986-01-15 Peptides de regulation immunitaire Withdrawn EP0215805A1 (fr)

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US692711 1985-01-18
US80345285A 1985-11-29 1985-11-29
US80550485A 1985-11-29 1985-11-29
US803452 1985-11-29
US805504 1985-11-29

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WO1989009225A1 (fr) * 1988-03-28 1989-10-05 The Regents Of The University Of California Peptides du facteur de croissance des neurones du systeme nerveux peripherique
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EP0490379A3 (fr) * 1990-12-13 1992-06-24 BERLIN-CHEMIE Aktiengesellschaft Dérivés de diamino-acides et compositions pharmaceutiques
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US6126939A (en) * 1996-09-03 2000-10-03 Yeda Research And Development Co. Ltd. Anti-inflammatory dipeptide and pharmaceutical composition thereof
AU8912698A (en) 1997-08-19 1999-03-08 University Of Medicine And Dentistry Of New Jersey Inhibition of hiv-1 replication using d-amino acid peptides
US6989435B2 (en) * 1997-09-11 2006-01-24 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
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US7238711B1 (en) 1999-03-17 2007-07-03 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
EP2386565A3 (fr) * 1999-01-12 2013-11-20 Cambridge Enterprise Ltd. Composés et procédés pour inhiber ou augmenter une réponse inflammatoire
JP2003501393A (ja) * 1999-06-05 2003-01-14 ザ・ボード・オブ・トラスティーズ・オブ・ザ・レランド・スタンフォード・ジュニア・ユニバーシティ 心臓血管細胞増殖を阻害するための方法および組成物
ATE510852T1 (de) 2001-10-30 2011-06-15 Dsm Ip Assets Bv Dermopharmazeutisch und kosmetisch wirksame oligopeptide
US20050084967A1 (en) 2002-06-28 2005-04-21 Xcyte Therapies, Inc. Compositions and methods for eliminating undesired subpopulations of T cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation
US9616011B2 (en) 2005-04-27 2017-04-11 The Procter & Gamble Company Personal care compositions
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