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WO1999037315A1 - Derives chimiques d'autoantigenes et de peptides suppresseurs d'auto-immunisation, et preparations pharmaceutiques les contenant - Google Patents

Derives chimiques d'autoantigenes et de peptides suppresseurs d'auto-immunisation, et preparations pharmaceutiques les contenant Download PDF

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Publication number
WO1999037315A1
WO1999037315A1 PCT/US1999/001884 US9901884W WO9937315A1 WO 1999037315 A1 WO1999037315 A1 WO 1999037315A1 US 9901884 W US9901884 W US 9901884W WO 9937315 A1 WO9937315 A1 WO 9937315A1
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Prior art keywords
autoimmune
fatty acid
delivery
group
peptide
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PCT/US1999/001884
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English (en)
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Jane Pei-Fan Bai
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Bai Jane Pei Fan
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Priority to AU25667/99A priority Critical patent/AU2566799A/en
Publication of WO1999037315A1 publication Critical patent/WO1999037315A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • 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 invention relates to the fatty acid acylated conjugates of an autoantigen, fatty acid acylated conjugates of immunologically reactive peptide fragments of said autoantigen, fatty acid acylated conjugates of analogs of said autoantigen, and fatty acid acylated conjugates of analogs of said peptide fragments, and to their medical use.
  • Autoimmune diseases are characterized by an abnormal immune response involving either cells or antibodies, that are in either case directed against normal autologous tissues.
  • Autoimmune diseases in mammals can generally be classified in one of two different categories: cell-mediated disease (i.e. T - 2 - cells) or antibody-mediated disorders.
  • Antibody- mediated production is usually T-cell dependent.
  • Non-limiting examples of cell-mediated autoimmune diseases include multiple sclerosis, rheumatoid arthritis, autoimmune thyroiditis, insulin-dependent diabetes mellitus (IDDM) , autoimmune uveoretinitis.
  • Non-limiting examples of antibody-mediated autoimmune disorders include myasthenia gravis and systemic lupus erythematosus (or SLE) .
  • An autoimmune reaction is one mounted by the body against its own antigens (known as "autoantigens") . It is widely believed that a specific class of active T cells lymphocytes is produced which recognizes autoantigens.
  • the current treatments for both categories of autoimmune diseases involve administration of drugs which non-specifically suppress the immune response.
  • drugs are methotrexate, cyclophosphamide, Imuran (azathioprine) and cyclosporin A.
  • Steroid compounds such as prednisone and methylprednisilone are also employed in many instances.
  • These drugs have limited efficacy against both cell- and antibody-mediated autoimmune diseases.
  • Use of such drugs is limited by virtue of their toxic side effects and also because they induce "global" immuno-suppression in a patient receiving prolonged treatment with the drug, e.g. the normal protective immune response to pathogenic microorganisms is down- regulated thereby increasing the risk of infections caused by these pathogens.
  • a further drawback is that there is an increased risk that malignancies will develop in patients receiving prolonged global immuno-suppression.
  • autoimmune diseases such examples include administration of MBP, proteolipoprotein (PLP) for multiple sclerosis (MS) , administration of gluta ic acid decarboxylase 65 (GAD 65 ) for IDDM, administration of S- antigen for uveoretinitis, administration of alloantigen and MHC (major histocompatibility complex) peptide for rejection of transplantation, administration of thyroglubulin for thyroiditis, administration of collagen for rheumatoid arthritis (RA) , and administration of acetylcholine receptor for myasthenia gravis (Chem . Immunol . Basel . ,
  • Autoantigen or its peptide fragments is generally administered orally utilizing non- enteric coated or enteric coated tablet or capsule, or is administered nasally or pulmonarily using aerosol or inhalation, as disclosed in U.S. Pat. No. 5,399,347, 5,475,086, 5,571,499, 5,571,500, 5,594,100, 5,641,473, 5,641,474, 5,643,868, and 5,645,820 and in publication ( J. Exp. Med . (1996) 183:1561-1567) .
  • a palmityl derivative of herpes simplex virus peptide at the lysine amino group when injected intraperitoneally, reportedly enhanced the effect of eliciting antibody production and cytotoxic T cells (J. Virol . (64) 680-685, 1990).
  • immunoadjuvants are not required for such derivatives to induce immunological defensive attacks, humoral and cellular attack (Peptide in Immunology (1996) , John Wiley & Sons, New York ) .
  • a construct of linking fatty acid, myristic acid, to a ino acid or other compound is disclosed in U.S. Patent Nos. 5,744,631 and 5,599,947, and such construct is claimed to be a good substrate for N- myristoyl-transferase and/or its acyl coenzyme and useful as anti-viral and anti-fungal agents.
  • This prior art does not provide useful instruction for treating autoimmune diseases using fatty acid conjugates.
  • fatty acid derivatives of pharmacological active peptides were used for enhancing absorption of pharmacologically active peptides from the intestine to the blood circulation, from the circulation to the brain, for increasing resistance to intestinal and plasma enzymatic degradation, and for improving physical properties.
  • TRH thyrotropin-releasing hormone
  • tetragastrin reportedly lost partial pharmacological activities (Pharm . Res . (1993) 10:1488-1492; J. Pharm . Pharmcol . (1992) 44:717-721).
  • pivalate ester (a branched C5 alkyl chain) with high stability against chymotrypsin activity is the most desirable and esters with a straight chain fatty acid are not desirable due to their rapid degradation by chymotrypsin.
  • the prior art does not provide any useful instruction for treating autoimmune diseases using fatty acid conjugates.
  • U.S. Pat. 5,631,347 discloses a method for processing N-palmitoyl Lys B29 human insulin in the production process and eliminate adverse gelling problem of the conjugated insulin, and describes such conjugate is useful for administration by injection for treating diabetes by its hypoglycemic effect.
  • This prior art does not disclose that fatty acid- acylated insulin can achieve better clinical attenuation of IDDM before the onset of IDDM through the induction of immunological tolerance and arrest of T cell-mediated attack.
  • the prior art does not provide any useful specific instruction about the structural characteristics of fatty acid-acylated conjugates of autoantigen, fatty acid acylated conjugates of immunologically reactive peptide fragments of said autoantigen, fatty acid acylated conjugates of - 10 - analogs of said autoantigen, and fatty acid acylated conjugates of analogs of said peptide fragments, for better therapeutic benefit of ameliorating autoimmune diseases.
  • fatty acid-acylated autoantigen fatty acid-acylated autoimmune-suppressive peptide fragments of said autoantigen, fatty acid-acylated conjugates of analogs of said peptide and fatty acid-acylated conjugates of analogs of said autoantigen are useful for better modulation of the immune system and better clinical attenuation of autoimmune diseases.
  • the present invention is the only invention disclosed so far which provides specific instruction to ensure a clinically useful efficacy by enhancing secretion of inhibitory cytokines and suppressing secretion of inflammatory cytokines, by inducing regulatory memory T cells, via fatty acid-acylated conjugates of autoantigen, fatty acid-acylated conjugates of auto-immune suppressive peptide fragments of said autoantigen, fatty acid-acylated conjugates of analogs of said peptide, and fatty acid-acylated conjugates of analogs of said autoantigens.
  • the present invention is the only invention disclosed so far which provides the specific structural characteristics of fatty acid-acylated conjugates of autoantigen, fatty acid-acylated conjugates of auto-immune suppressive peptide fragments of said autoantigen, fatty acid-acylated conjugates of analogs of said peptide, and fatty acid-acylated conjugates of analogs of said autoantigen for detecting and ameliorating autoimmune diseases.
  • PBS phosphate-buffer saline
  • PLP 139-151)
  • BA 103 lauric acid-conjugated PLP
  • Post immunization treatment was given intra-nasally at a dose of PLP (50 ⁇ g) or BA 103 at an equal amount in PBS.
  • PBS PBS
  • PLP 139-151)
  • BA 104 oleic acid -conjugated PLP
  • Post immunization treatment was given intra-nasally at a dose of PLP (50 ⁇ g) or conjugated peptide at an equal molar amount in PBS.
  • PBS PBS
  • PLP 139-151)
  • BA 102 acetic acid-conjugated PLP
  • 139- 151) BA 102
  • PBS PBS
  • PLP PLP
  • 139-151 lauric acid -conjugated PLP
  • BA 103 lauric acid -conjugated PLP
  • PBS PBS
  • PLP PLP
  • 139-151 butyric acid -conjugated PLP
  • BA 105 butyric acid -conjugated PLP
  • PBS PBS
  • PLP 139-151
  • BA 106 octanoic acid -conjugated PLP
  • FIG. 7 shows the T cell proliferative responses to PBS, PLP (139-151) , acetyl-Tyr-PLP (139- 151) using T cells from draining lymph nodes of PLP (139-151) primed SJL mice.
  • FIG. 8 ELIOTSPOT analysis for interleukin 4 (IL-4) of draining lymph node, inguinal and poplineal, cells (LNC) obtained from different treatment groups of SJL mice at day 41 post- immunization.
  • LNC cells from PBS-treated mice, BA 101 (PLP (139-159) -treated mice, and BA 105-treated mice were stimulated in culture for 24 hr with PLP (139-151) (50 ⁇ g) .
  • PLP 139-151
  • Con A was added to maximize the stimulation to validate the in vitro system.
  • the data are the ratio of the number of IL-4 secreting cells from each treatment group versus - 13 - the control and expressed as mean ⁇ S.D. of three experiments for each treatment group.
  • the control is the culture without PLP (139-151) using LNC from PBS- treated group.
  • FIG. 9 ELIOTSPOT analysis for interferon (INF- ⁇ ) of draining lymph node, inguinal and poplineal, cells (LNC) obtained from different treatment groups of SJL mice at day 62 post- immunization.
  • LNC cells from PBS (phosphate-buffered saline) -treated mice, BA 101 (PLP (139-159) -treated mice, BA 102-treated mice, and BA 103-treated mice were stimulated in culture for 24 hr with PLP (139- 151) (50 ⁇ g) .
  • PLP 139- 151
  • Con A was added to maximize the stimulation.
  • the data are the ratio of the number of INF- ⁇ secreting cells from each treatment group versus the control and expressed mean ⁇ S.D. of three experiments for each treatment group.
  • the control is the culture without PLP (139- 151) using LNC from PBS-treated group.
  • FIG. 10 ELIOTSPOT analysis for transforming growth factor jS (TGF- ) of draining lymph node, inguinal and poplineal, cells (LNC) obtained from different treatment groups of SJL mice at day 41 post-immunization ( Figure 10 a) and at day 62 post- immunization ( Figure 10b) .
  • LNC cells from PBS- treated mice, BA 101 (PLP (139-159) -treated mice, BA 102-treated mice, BA 103-treated mice, and BA 105- treated mice were stimulated in culture for 24 hr with PLP (139-151) (50 ⁇ g) .
  • PLP 139-151
  • the data are the ratio of the number of TGF- ⁇ secreting cells from each treatment group versus the control and expressed mean ⁇ S.D. of three experiments for each treatment group.
  • the control is the culture without PLP (139- 151) using LNC from the PBS-treated group. - 14 -
  • FIG. 11 ELIOTSPOT analysis for tumor necrosis factor ⁇ (TNF- ⁇ ) of draining lymph node, inguinal and poplineal, cells (LNC) obtained from different treatment groups of SJL mice at day 41 post- immunization.
  • LNC cells from PBS-treated mice, BA 101 (PLP (139-159) -treated mice, BA 103-treated mice, BA 105-treated mice were stimulated in culture for 24 hr with PLP (139-151) (50 ⁇ g) .
  • PLP 139-151
  • Con A was added to maximize the stimulation.
  • the data are the ratio of the number of TNF- ⁇ secreting cells from each treatment group versus the control and expressed mean ⁇ S.D. of three experiments for each treatment group.
  • the control is the culture without PLP (139- 151) using LNC from the PBS-treated group.
  • the autoimmune disease is remarkably alleviated.
  • said conjugate enhances the in vitro proliferation of T cells, providing a means to more effectively identify those who have cell-mediated attack yet without any clinical manifestations.
  • the conjugate is formed via a covalent bond between the carboxyl group of fatty acid and a free functional group of amino acid residue of autoantigen, between the carboxyl group of fatty acid and a free functional group of amino acid residue of disease-suppressive peptides of said autoantigen, - 1 5 - between the carboxyl group of fatty acid and a free functional group of amino acid residue of analogs of said peptide, between the carboxyl group of fatty acid and a free functional group of analog of said amino acid residue of said peptide analog, between the carboxyl group of fatty acid and a free functional group of amino acid residue or analogs of said amino acid of analogs of said autoantigen.
  • the conjugate uses one or more saturated and non-saturated fatty acids.
  • the conjugate fatty acids containing at least 2 carbons.
  • said conjugate in the diagnosis and treatment of patients having, or at risk of having, autoimmune diseases. Not only is it possible to use said fatty acid conjugates to more effectively ameliorating people with autoimmune disease, but it is also now possible to more accurately classify patients with such autoimmune diseases as insulin-dependent diabetes mellitus.
  • an autoimmune disease occurs when the immune system cannot or does not distinguish between exogenous (foreign) substances within the mammal and autologous tissues or substance.
  • the immune system treats autologous tissues and substances (self antigens) as if they were foreign and evokes the proliferative immune defense that is initiated against exogenous (foreign) tissues or invading organisms.
  • the normal immune system begins a proliferative response against autologous tissues.
  • the term "mammal" refer to a life form which has an immunoregulatory system. - 16 -
  • the fatty acids include any fatty acids containing at least 2 carbon atoms, saturated fatty acids, and non-saturated fatty acids.
  • conjugate of (1) and (2) includes products comprising (1) bond to (2) by an effective means for covalent bonding.
  • acyl derivative refers to any of said conjugate.
  • autoantigen refers to any substances normally found within a mammal that (i) is not recognized as part of the mammal itself by the lymphocytes or antibodies of that mammal, (ii) is attacked by the immunoregulatory system of the mammal as though such antigen were a foreign substance and (iii) acts to downregulate the arm of the . immune system that is responsible for causing a specific autoimmune disease.
  • autoantigen also includes antigenic substances which induce conditions having the symptoms of an autoimmune disease when administered to mammals.
  • autoantigen and "autoimmune-suppressive protein” are used interchangeably.
  • autoimmune suppressive peptide or "disease-suppressive peptide” includes any peptides or polypeptides containing partial amino acid sequences or moieties of autoantigens and possessing the ability to suppress or prevent an autoimmune response upon administration. Such fragments need not possess the autoantigenic properties of the entire autoantigen.
  • MBP is administered - 17 - parenterally to mammals in the presence of an adjuvant it induces EAE (experimental allergic encephalomyelitis ) in susceptible mammals.
  • EAE experimental allergic encephalomyelitis
  • analogs of such autoantigens or peptide fragments thereof refers to compounds that are structurally related to autoantigens or their autoimmune-suppressive peptides and which possess the same biological activity, i.e. the ability to eliminate or suppress the autoimmune response, upon administration.
  • the term includes peptides having amino acid sequences which differ from the amino acid sequence of the autoantigen or disease suppressive peptides thereof by one or more amino acid residues (while still retaining the autoimmune-suppressive activity of the autoantigen or fragment) as well as compounds or compositions which mimic the autoimmune- suppressive activity of the autoantigen in its ability to suppress or alleviate the symptoms of the disease.
  • tissue from an organ that is the target of attack by an arm of the immune system in an autoimmune disease e.g. the pancreas in diabetes or the white matter of the central nervous system in MS.
  • autoimmune-disease suppressive agent or autoimmune suppressive agent refers to a compound or composition which can be administered to suppress, prevent or delay the clinical onset or manifestation of a specific autoimmune disease.
  • the term includes antoantigens, autoimmune-suppressive peptide of autoantigen and analogs of said peptide thereof as defined above. - 18 -
  • mucosal immune tissues include mucosal immune tissues present in the intestinal, nasal, tracheal, colonic, rectal, buccal, and vaginal mucosa tissues.
  • non-parentera1 delivery means delivery via the oral, nasal, tracheal, colonic, rectal, buccal, and vaginal mucosa tissues.
  • aerosols for delivery to the trachea and nasal sprays for delivery to the nasal mucosal tissue As employed herein the term "intestine” includes the small intestine, the caecum, the colon, and the rectum.
  • acylating or “acylation” means the introduction of one or more acyl groups covalently bonded to a functional group of autoantigen or autoimmune suppressive peptide of said autoantigen or analogs of said autoantigen or analogs of said peptides.
  • a functional group is present in, or added to, said amino acid residue or amino acid analog of said amino acid residue.
  • the acyl groups are from fatty acids.
  • acylation typically attachment sites for fatty acid chains in peptides, proteins, protein analogs, and peptide analogs re tyrosine (acylation of the phenolic hydroxyl) , serine (acylation of methyl hydroxyl) , cysteine (acylation of the side chain sulfhydryl) , lysine (acylation of the e amine) and the N-terminus (acylation of the ⁇ amine) .
  • acylation is through the free hydroxyl group or the N-terminal ⁇ amine or free e amine of comprising amino acid residues or free sulfhydryl group of autoantigen or autoimmune- suppressive peptides or their analogs.
  • fatty acid means saturated or unsaturated C 2 -C 2 ⁇ fatty acids as well as high molecular weight fatty acids.
  • the saturated fatty acids include (but not limited to) acetic acid. - 19 - proprionic acid, butyric acid, caproic acid, caprylic acid, decanoic acid (capric acid) , lauric acid, myristic acid, palmitic acid and stearic acid.
  • the non-saturated fatty acids generally have the formula of C n Hj ⁇ COOH, and include (but are not limited to) oleic acid, linoleic acid, and arachidonic acid.
  • the fatty acids are saturated fatty acids, unsaturated fatty acids and high molecular weight fatty acids, including, acetic acid, proprionic acid, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, lignoceric acid, cerebronic acid, nervonic acid and oxynervonic acid.
  • acetic acid proprionic acid
  • butyric acid caproic acid
  • caprylic acid capric acid
  • lauric acid myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, lignoceric acid, cerebronic acid, nervonic acid and oxynervonic acid.
  • the ⁇ -amine group of N-terminal amino acid residue can be from any amino acids including alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, arginine, aspartic acid, asparagine, cysteine, glutamic aid, glutamine, glycine, histidine, lysine, serine, threonine, tyrosine, homocysteine, cysteinesulfinic acid, homoserine, ornithine, citrulline, argininosuccinic acid, and organic compounds containing both amino and carboxyl groups including but not limited to ⁇ -aminohexadecanoic acid and e- amino caproic acid.
  • the N-terminal amino acid residue or organic compound used is either present in, or added to, autoantigen or autoimmune-suppressive peptides of said autoantigen or analogs of said autoantigen or analogs of said peptide.
  • the free hydroxyl group can be present in an amino acid residue of, or be introduced to via the N- - 20 - terminus or C-terminus of, an autoantigen or its autoimmune-suppressive peptides or analogs of said autoantigen and said peptide.
  • Amino acid residues containing such free hydroxyl group include but are not limited to tyrosine, serine, threonine, hydroxylysine, 4-hydroxyproline, and homoserine.
  • One or more hydroxyl groups can be introduced by adding the above-mentioned amino acids, glycolic acid or lactic acid to the autoantigen or its autoimmune- suppressive peptide or analogs of said autoantigen and peptide through an amide linkage to an amino acid residue.
  • any compounds which contain a free carboxyl group or amino group for amide bond formation with an amino acid residue as well as a free hydroxyl group for ester linkage, can be added to the autoimmune-suppressive protein or its peptide fragments to introduce a free hydroxyl group.
  • Such compounds include but are not limited to tyrosine, serine, threonine, hydroxylysine, 4-hydroxyproline, homoserine, glycolic acid, lactic acid, gluconic acid, saccharic acid, mevalonic acid and glycuronic acid.
  • Such compounds can be linked to an autoantigen or autoimmune-suppressive peptide of autoantigen or analogs of said autoantigen and said peptide via amide linkage, leaving one or more free hydroxyl group available for ester linkage.
  • acylation to form a conjugate is accomplished by one or more ester linkage between the free hydroxyl group of a tyrosine residue and the carboxyl group of a fatty acid, or by one or more amide linkage between an N-terminal ⁇ or e amine group (either primary or secondary) and the carboxyl group of a fatty acid, or by a mixture of such ester and amide linkage.
  • acyl derivatives are obtained by utilizing the free hydroxyl group on a tyrosine residue or the amine group ( ⁇ or e) on the N-terminal amino acid residue, which is either intrinsically - 21 - present or covalently introduced to the autoantigen or autoimmune-suppressive peptide or analogs of said autoantigen and peptide via the N-terminus or C- terminus.
  • the fatty acid conjugate of autoantigen or autoimmune-suppressive peptide of autoantigen or analogs of said autoantigen and said peptide has at least one fatty acid attached via covalent bond(s) .
  • the reaction typically is diluted with water.
  • the acylated autoantigen or acylated autoimmune-suppressive peptide of said autoantigen or acylated analogs of said autoantigen or acylated analogs of said peptide is placed in a properly buffered aqueous solution for further processing.
  • processing particularly includes purification by standard - 22 - chromatographic methods such as reverse phase or hydrophobic chromatography, concentration by crossflow filtration, solvent exchange by ultrafiltration and the like (Pharm. Res. (1993) 10; 68-74; J. Pharm. Sci. (1995) 84:682-687). All peptides will be purified by semi- preparative HPLC and structures will be confirmed by amino acid analysis and mass spectroscopy and, when appropriate, by *H and 13 C NMR.
  • the aqueous solution of the purified fatty acid-acylated autoantigen or autoimmune-suppressive peptide or analogs of said autoantigen and said peptide can be processed to recover the soluble acyl derivative as a powder.
  • any procedure for recovering the acylated autoantigen or autoimmune-suppressive peptide or analogs of said autoantigen and said peptide as a powder including lyophilization (freeze drying) , crystallization or precipitation techniques, can be used.
  • the present invention is not limited to the way of isolating and recovering the acylated protein and peptide in powder form.
  • the present invention is applicable to administration by injection means, oral (including colonic and rectal) , nasal, tracheal, vaginal and buccal administration, and intrathymic injection, and administration to the tissue targeted by the autoimmune attack.
  • the acyl derivatives can be formulated in any standard pharmaceutical dosage forms, such as solution, pellet, tablet, capsule, spray, aerosol, cream, granule, insert, patches, matrix and mucoadhesive formulations.
  • Intestinal esterases and proteolytic enzymes secreted by the -23- pancreas can act on the acyl derivative of autoantigen or autoimmune-suppressive peptide or analogs of said autoantigen and said peptide, targeting the ester and amide bonds, respectively.
  • These enzymes in general require a pH higher than 7 for optimal activities and lose activities at acidic pH.
  • the distal small intestine has much lower lumenal concentration of digestive enzymes.
  • the lumenal premature degradation of acyl conjugate of autoantigen or autoimmune-suppressive peptide or analogs of said autoantigen and said peptide by esterases or by proteolytic enzymes can be minimized by delivering to the distal ileum and by co-administering any agents which are non-toxic and can reduce intestinal pH or pH at any musoal surface to be lower than 5.5.
  • agents are any organic acids present in food or of pharmaceutical grade or used as food additives.
  • organic acids include, but are not limited to, citric acid, malic acid, poly(acrylic acid) , lactic acid, glycolic acid, Carbopol 971P, Carbopol 974P, Carbopol 934P, and EDTA. These acids can be incorporated using standard pharmaceutical formulation dosage forms and formulating procedures.
  • the acyl derivative of the autoantigen or autoimmune- suppressive peptide or analogs of said autoantigen or analogs of said peptide is delivered by formulation approaches to the human ileum or colon because the concentration of lumenal pancreatic enzymes are much less.
  • formulation approaches are well known in the art.
  • U.S. Patent No. 5,443,841 and U.S. Patent No. 55,78,323 disclose a system of proteinoid microspheres consisting of proteinoids having 2 to 20 amino acid residues.
  • these microspheres are able to deliver proteins or peptides only in the distal intestine when the majority of the comprising - 24 - amino acids are basic amino acids which become ionized at the distal intestinal pH, which is higher than 7.
  • Proteinoid microspheres which are selectively soluble under alkaline pH environments, such as the distal portion of the intestine, are based on base-soluble proteinoids.
  • a base-soluble proteinoid consists of a majority of basic amino acids in its composition.
  • proteinoids thus designed can protect peptides and proteins from gastrointestinal proteolysis and start release only in the ileum.
  • Such exemplified preparation reportedly increases the pharmacological effect of insulin on reducing blood glucose level by protecting insulin from lumenal degradation, thus increasing the amount of insulin reaching the circulation.
  • Eudragit polymers which can be employed to manipulate the release of drugs at different pH.
  • Eudragit L-100 is an acrylic copolymer based on methacrylic acid and ethylacrylate, which dissolves at pH above 5-6
  • Eudragit S is an acrylic copolymer based on methyacrylic acid and mehtylmethacrylate which dissolves at pH higher than 7.
  • the combination of these Eudragit polymers in the coating can manipulate the drug to be released in a desired intestinal environment (Published in "Practical Course in Lacquer Coating, by Rohm Tech Inc., in 1989).
  • Delivery to the colon can be achieved by azo polymer or other type coatings which allows only release in the colon by utilizing colonic bacterial enzymes. Further, mucoadhesive coating can be employed to any pharmaceutical dosage forms to facilitate the anchor of a solid dosage form on the mucosal surface. Delivery to the rectum can be achieved by wax-based or hydrogel-based - 25 - suppositories. Pharmaceutical practice in making suppositories is well developed. Delivery to the vaginal can be achieved by cream, gel and suppositories.
  • the acyl derivative of the autoantigen or ' autoimmune-suppressive peptide or analogs of said autoantigen and said peptide can be administered as a dry powder or in a solution. Dry aerosol in the form of finely divided solid particles that are not dissolved or suspended in a liquid are also useful in the practice of the present invention.
  • the acyl derivative of the autoantigen or autoimmune- suppressive peptides of said autotantigen or analogs of said autoantigen or analogs of said peptide may be in the form of powder and comprises finely divided particles having an average particle size of between about 1 and 5 ⁇ m, preferably between 2 and 3 ⁇ m. Finely divided particles may be prepared by potizatin or screen filtration or lyophilization. The particles may be administered by inhaling a predetermined dose of the finely divided material, which can be in the form of a powder.
  • the pharmaceutical formulation for the present invention may be administered in the form of an aerosol spray using, for example, a nebulizer such as those described in U.S. Pat. Nos. 4,624,251, 3,703,173, 3,561,444, and 4,635,627.
  • the aerosol material is inhaled by the subject to be treated.
  • Other systems of aerosol delivery such as the pressurized metered dose inhaler (MDI) and the dry powder inhaler as disclosed in Newman, S. P. in Aerosols and the Lung (Clarke, S.W. and Davis, D. eds, pp 197-224, Butterworths, London, England, 1984) can be used when practicing the present invention.
  • MDI pressurized metered dose inhaler
  • the dry powder inhaler as disclosed in Newman, S. P. in Aerosols and the Lung (Clarke, S.W. and Davis, D. eds, pp 197-224, Butterworths, London, England, 1984
  • Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources - 26 - including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, N.J.), American Pharmaseal Co., (Valencia, CA) , and Dura Pharmaceutical Inc. (San Diego, CA) .
  • Autoantigen and its suppressive peptide thus employed herein include, but are not limited to, self-antigens and nonself-antigens implicated in autoimmune diseases, and their autoimmune-suppressive fragments, such as insulin, insulin B-chain (9-23) , insulin A-chain (7-21) , glutamic acid decarboxylase, heat shock protein 65, heat shock protein 60, carboxypeptidase H, ICA 512/IA, perpherin, 38 kD antigen, Gm2-1, and ICA-69 for juvenile diabetes, type II collagen and its effective disease- suppressive fragments for rheumatoid arthritis, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein and their effective disease-suppressive peptide fragments for multiple sclerosis, thyroglobulin for autoimmune thyroditis, acetylcholine receptor for myasthenia Gravis
  • autoimmune-suppressive fragments such as insulin, insulin B-chain (9
  • the autoantigens effective for the treatment of individual autoimmune disease models also include, but are not limited to, liver extract for chronic active hepatitis, adrenal gland extract for adrenalitis, muscle extract for polymyositis, hematopoietic cells for autoimmune he olytic anemia, - 27 - heart extract for rheumatic carditis, and skin cell extract for scleroderma.
  • the autoimmune diseases include, but are not limited to, colitis, herpes simplex virus-induced autoimmune diseases (including but not limited to herpes stromal keratitis) , systemic lupus erythematosus, dermatomyositis, Sydenham's chorea, rheumatoid arthritis, rheumatic fever, thrombocytopenic purpura, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, henoch- protocollein purpura, post-streptococcal nephritis, systemic lupus erythematosus, erythema nodosum, Takayasu's arteritis, myasthenia gravis, Addison's disease, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylos
  • Immunoadjuvants are known in literature regarding their enhancing effects on immunological responses and can be included in the formulation. They include, but are not limited to, liposaccharides and lipid A.
  • the dose of fatty-acid acylated autoantigen or autoimmune-suppressive peptide of autoantigen or analogs of said autoantigen and said peptide ranges from 0.01 mg/kg to 30 mg/kg. In the preferred embodiment, the dose - 28 - ranges from 0.1 mg to 25 mg/kg.
  • the dosing frequency ranges from once a day, twice a week, once a week ,once every other week, or once a month depending on the disease.
  • EXAMPLE 1 Preparation of acylated PLP (139-159) fragment
  • the procedures of peptide synthesis is well known in the art using solid phase peptide synthesis with Fmoc protection of the ⁇ amine group and appropriate side chain protecting groups (J. Med. Chem. 20:1435, 1977; Pharm, Res., 10:68-74, 1993; Advances Organic Chemistry, 3 rd , Jerry March, Wiley & Sons, NY) .
  • Automated synthesizers are available from many commercial sources such as Applied Biosystems synthesizers (models ABI 431 and 433) or Rainin Symphony multiple peptide synthesizer, or PerSeptive BioSystems Pioneer.
  • the amino acid sequence of the PLP (139-151) is His-Ser-Leu-Gly-Lys-Trp-Leu-Gly-His-Pro-Asp-Lys and it was prepared using the following procedure.
  • the Fmoc and Boc protected amino acids were obtained Advances ChemTech and Bachem.
  • Fmoc-His (Trt) F oc- Ser(tBu), Fmoc-Leu, Fmoc-Gly, Fmoc-Lys(Boc) , Fmoc- Trp, Fmoc-Leu, Fmoc-Gly, Fmoc-His (Trt) , Fmoc-Pro, Fmoc-Asp(tBu) , Fmoc-Lys(Boc) were sequentially coupled to Phe-PEF-PS resin (PerSeptive Biosystems) on ABI 431 Peptide Synthesizer using ABI FastMoc chemistry.
  • Phe-PEF-PS resin PerSeptive Biosystems
  • the final protected product is a 13 amino acid peptide resin, Fmoc-His (Trt) -Ser(tBu) -Leu- Gly-Lys (Boc) -Trp-Leu-Gly-His (Trt) -Pro-Asp (tBu) - Lys (Boc) -Phe-PEG-PS resin.
  • the resin was dried under vacuum and then incubated at room temperature in 20% v/v piperidine in N-methyl pyrrolidone for 25 min. This procedure removed the N-terminal Fmoc. - 29 -
  • the deprotected resin was washed first with N- methylpyrrolidone, then dichloromethane/methanol , and finally dichloromethane and dried under vacuum.
  • the deprotected resin was then incubated in a mixture of trifluoroacetic acid, water and ethanedithiol (9.5 ml: 0.25 ml: 0.25 ml) for one and half hours.
  • the resin was removed using filtration and washed with trifluoroacetic acid and dichloromethane.
  • the final filtrate was rotovapped to 2 ml.
  • the peptide was precipitated using cold ether and the suspension was left at 4° C overnight.
  • PLP (139-159) peptide is acylated at the N-terminal added tyrosine residues with an n-acetic acid.
  • PLP (139-159) peptide acylated at the N-terminal added tyrosine residues with an n-oleic acid.
  • PLP (139-159) peptide is acylated at the N-terminal added tyrosine residues with an n-butanoic acid.
  • PLP (139-159) peptide is acylated at the N- terminal added tyrosine residues with an n- caproic acid.
  • the portion eluted at 47 min was analyzed using isocratic HPLC using 22% HPLC solvent 2 and demonstrated to have high purity > 99%.
  • the fraction eluted at 47 min was dried, rotovapped and lyophilized. Acetyl-Tyr- His-Ser-Leu-Gly-Lys-Trp- Leu-Gly-His-Pro-Asp-Lys was thus obtained.
  • Boc-Tyr with no side chain protection was coupled to Fmoc-His (Trt) -Ser(tBu) -Leu-Gl;y-Lys (Boc) - Trp-Leu-Gly-Lys (Boc) -Trp-Leu-Gly-His (Trt) -Pro- Asp (tBu) -Lys (Boc) -Phe-PEG-PS-resin as described above.
  • the crude lipopeptide was then put in 5 ml of dimethylformamide/80% acetic acid (9:1 v/v) and diluted with 50 ml HPLC solvent 1, and then purified with a 15-20 micron semi-prep column and a linear gradient system of 10% to 50% HPLC solvent 2 in 50 min.
  • the fraction eluted at 70 min was then analyzed using isocratic HPLC with 40% HPLC solvent 2.
  • the purest fractions were rotovapped and lyophilized to yield lauryl-Tyr-His-Ser-Leu-Gly- Lys-Trp-Leu-Gly-His-Pro-Asp-Lys with purity greater than 98%.
  • Oleyl-Tyr-His-Ser-Leu-Gly-Lys-Trp-Leu-Gly- His-Pro-Asp-Lys , butyry1-Tyr-His-Ser-Leu-Gly-Lys-Trp- Leu-Gly-His-Pro-Asp-Lys, and caproyl-Tyr-His-Ser-Leu- Gly-Lys-Trp-Leu-Gly-His-Pro-Asp-Lys were all - 31 - synthesized using similar procedures described above.
  • Immunizations are done on day 0 (do) and day 7 (d7) in the posterior flank at two sites with 0.15ml/site subcutaneous injection of 100 ⁇ g of PLP (139-151) in an emulsion of Complete Freund' s adjuvant H37Ra(CFA) (Difco Laboratories) and PBS (1:1 v/v). There were five mice in each group. Individual groups received a nasal dose of 50 or 0 (control) ⁇ g of PLP (139-151) and equal molar amount of fatty acid acylated conjugates of PLP (139-151).
  • the nasal solution was prepared in PBS, and the nasal dosing was administered at days 1, 8, 15, or at days 1, 8, 15, 25 or at days 1, 3, 5, 7 through two nostrils of each mouse (10 ⁇ l for each nostril) using a PE-10 tubing connected to a 25 ⁇ l Hamilton syringe. All clinical neurological disorders are monitored daily and the symptom manifestation is scored as the following: 4+, full hind limb paralysis; 3+ partial hind limb paralysis; 2+ ataxia; 1+ flaccid tail and 0, normal. Re-challenge can be done after day 60 to observe better clinical attenuation of EAE in SJL mice. The results would hint at the induction by acylated conjugates of PLP (139-151) of regulatory memory T cells which act to suppress the clinical symptoms of EAE.
  • oleic acid-conjugaed PLP (139-151) -treated mice " had lower clinical score than PBS-treated mice and PLP (139-151) -treated mice and had no sign of relapse between days 20-35, in comparison to severe episodes of relapse in the PBS- treated mice and mild episodes of relapse in the PLP (131-159) -treated mice.
  • acetic acid-conjugated PLP (139-151) -treated mice had much lower clinical scores in the acute phase than PBS-treated and PLP (139-151) -treated mice, and entered remission much earlier than the PLP (139-151) -treated and PBS-treated mice. Further, the acetic acid-conjugated PLP (139- 151) -treated mice had little remission while PBS- treated group had very serious relapse and the PLP (139-151) -treated mice had mild relapse.
  • the lauric acid-conjugated PLP (139-151) -treated mice had lower clinical scores during the acute phase than the PBS-treated and PLP (139-151) -treated mice, entered remission much earlier and had no sign of relapse than the other two groups.
  • the butyric acid- conjugated PLP (139-151) -treated mice had later onset of EAE and lower clinical scores than the PBS-treated mice and the PLP (139-151) -treated mice.
  • the octanoic acid- conjugated PLP (139-151) -treated mice had later onset - 33 - of EAE and lower clinical scores than the PBS-treated mice and the PLP (139-151) -treated mice.
  • mice Female SJL mice, 6-8 weeks of age, were obtained from the National Cancer Institute (Frederick, MD) . Immunizations are done on days 0 and 7 (d7) in the posterior flank at two sites with 0.15ml/site subcutaneous injection of 100 ⁇ g of PLP (139-151) in an emulsion of Complete Freund' s adjuvant' H37Ra (CFA) (Difco Laboratories) and phosphate buffered saline (PBS) in 1:1 ratio. After the mice were challenged with the PLP (139-151) and CFA at day 0 and day 7, draining lymph node (inquinal and popliteal) and spleen cells were harvested from 3 PLP (139-151) primed mice on day 16.
  • CFA Complete Freund' s adjuvant' H37Ra
  • PBS phosphate buffered saline
  • the lymph node cells are washed and suspended in RPMI 1640 medium containing 2% fresh SJL mouse serum, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 25 mM HEPES, and 5 X 10 "5 M 2-ME.
  • Spleen cells were irradiated at 2000 rad.
  • Spleen cells were plated at 105 cells/well in 50 ml complete RPMI.
  • Lymph node cells were plated at 105 cells/well in 100 ml complete RPMI with spleen cells.
  • Peptides were dissolved in 25 ml complete RPMI and added to appropriate wells (Costar Corp., Cambridge, MA).
  • PLP (139-151) was tested at five concentrations: 5 ⁇ g/ml, 10 ⁇ g/ml, 25 ⁇ g/ml. 40 ⁇ g/ml and 50 ⁇ g/ml and acetyl-Tyr-PLP ( 139-151) was tested at the same concentration.
  • the culture condition was 37°C in 5% C02. After 3 days culture, [ 3 H]thymidine (0.25 ⁇ Ci in 25 ⁇ l) was added and the culture was harvested 18h later and counted using a beta-plate reader. The average of [ 3 H]thymidine uptake was obtained from triplicate wells. The results are shown in Figure 7.
  • Acetyl-Tyr-PLP (139-151) showed better mean T cell proliferative responses than the unmodified peptide, PLP(139-151) at several 34 concentrations. Higher T cell proliferative responses will enable more precise identification of a subject whose T cells are reactive to self proteins in an vital organ.
  • T cells prepared from the draining lvmph nodes of PLP(139-151) - challenged mice that received fatty acid-coniugated PLP (139-
  • Table 2 Organization of animal groups for ELISPOT analysis .
  • mice On day 62 after the mice were immunized with PLP (139- 151) , the mice were sacrificed and the draining lymph nodes (poplineal and inguinal) were obtained.
  • the T cell suspensions from individual groups were prepared and mixed with irradiated spleen cells that were prepared from the PBS-treated mice in a ratio of 1:2 with PLP (139-151) (50 ⁇ g/ml) or PLP (139-151) (25 ⁇ g/ml) or Con A.
  • the cell mixture was incubated for 24 hr in a humidified atmosphere containing 5% C0 2 .
  • mice interferon gamma 10 ⁇ g/ml
  • TGF-alpha mouse tumor necrosis factor-alpha
  • TGF-beta mouse transforming growth factor beta
  • the cells were then removed from the wells by washing several times with 0.25% Tween 20 (v/v) in PBS.
  • the secondary antibodies to individual cytokines were then added to the 96- - 35 well microtiter plate at the concentrations of 4 ⁇ g/ml for interferon gamma, 1 ⁇ g/ml for TGF-beta and 1 ⁇ g/ml for TNF- alpha.
  • the the cytokine-secreting spots in the wells were visualized using the alkaline phosphatase substrate, 5-bromo-4- chloro-3-indolyl phosphate (Sigma) .
  • the Con A-treated wells had very high density of cytokine-secreting spots, serving as an indicator of a successful experiment.
  • ELISPOT analysis was performed to determine PLP (139-151) -responding T cells that secrete interleukin-4 , TGF-beta, and TNF-alpha.
  • concentrations of primary and secondary antibodies for interleukin-4 were 10 ⁇ g/ml and 0.6 ⁇ g/ml. respectively.
  • the organization of animal groups are summarized in Table 3.
  • Table 3 Organization of animal groups for ELISPOT analysis .
  • mice and lauryl-Tyr-PLP (139-151) -treated mice had significantly lower frequency of PLP (139-151) responding T cells secreting interferon gamma than the PBS treated mice ( Figure 9) .
  • PLP 139-151 responding T cells secreting interferon gamma
  • the lauryl-Tyr-PLP (139-151) -treated mice had significantly lower frequency of PLP (139-151) responding T cells secreting TNF-alpha as compared to the PBS-treated and PLP (139-151) -treated mice.
  • the oleyl-Tyr-PLP( 139-151) -treated mice had slightly lower frequency of PLP (139-151) responding T cells secreting TNF-alpha as compared to the PBS-treated and PLP (139-151) -treated mice ( Figure 11).
  • mice The acetyl-Tyr-PLP (139-151) -treated mice, lauryl-Tyr-PLP (139-151) -treated mice, oleyl -Tyr-PLP (139-151) -treated mice all had higher frequency of PLP (139-151) responding T cells secreting TGF-beta ( Figure 10) .
  • the interferon gamma and TNF-alpha are inflammatory cytokines while interleukin-4 and TGF-beta are both suppressive cytokines.
  • EXAMPLE 5 Preparation of acylated insulin B-chain Several acyl derivatives of insulin B-chain (9-26) are prepared using FMOC chemistry and solid phase peptide synthesizers as described in EXAMPLE 1, and they are listed in Table 4.
  • the amino acid sequence of insulin B- chain is Glu-Val-Lys-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-Met-Ser (see, for example, U.S. Patent No. 5,594,100) .
  • Insulin B-chain (9-26) is acylated at the 26 th tyrosine residue with an n-caprylic acid.
  • A. Insulin B-chain (9-26) is acylated at the 26 th tyrosine residue with an n-lauric acid.
  • NOD non-obese diabetic mice
  • Jackson lab Bar Harbor, Main
  • mice are given orally compound E.
  • Each mouse received two doses a week until 35 weeks old.
  • the mice were fed either phosphate-buffered saline (control) or 10 microgram, 100 microgram or 1 gram of compound E in phosphate-buffered saline.
  • the mice were gavaged with an 18 gauge ball-point needle.
  • the urine is collected and tested weekly for the presence of glucose using Glucosuria test tape (Eli Lilly, Indianapolis, IN).
  • mice had more than two positive (the presence of sugar in the urine) on the glucose urine test, a serum sample is taken and tested for blood glucose level (Beckman glucose analyzer) . The mice were declared diabetic and sacrificed if the blood glucose was above 220 or higher.
  • EXAMPLE 7 Inhalation of acylated insulin B-chain (9-26) for the suppression of insulin-dependent diabetes mellitus
  • the NOD mice are given compound E in aerosol using a nebulizer (American Pharmosel Co., Valencia, CA.). All the procedures and experimental details are as described in example 6 except the dosing route and dose. The doses are 0 and 1 mg.
  • the animals are retained in airtight cages, into which the aerosol is dispensed. The amount of material per unit of area can thus be determined and the results quantified in terms of unit of aerosol material per unit volume of cage area.
  • the nebulizer is attached to an air pressure outlet delivering the equivalent of 7.4 liters of oxygen (the amount of oxygen used in the hospital for nebulization) .
  • the nebulizer produces droplets of spray having a diameter of between 0.3 micrometers and about 0.5 micrometers in diameter.
  • One mg of compound E is dispersed in 5 ml phosphate-buffered - 38 - saline and nebulized over a 15 min period per cage. During nebulization, a fine mist is created in the cage and the mice move about freely. All other procedures are as described in Example 6.
  • T cell proliferation responses to acylated insulin B- chain (9-26) Groups of NOD mice are immunized in the hind foot pads by a subcutaneous inoculation of compound E (50 ⁇ g) in complete Freund 's adjuvant. Ten days later, the lymph nodes draining the sites of foot pad injection (popliteal, inguinal and periaoritic) are collected and tested for the proliferative response to derivatized compound E as compared to the control peptide (Con A) or insulin B-chain (9-26).
  • Lymphocytes are seeded in 96-well microtiter plates (Costar Corp., Cambridge, MA), 2 X 10 5 cells in 0.2 ml of HL-1 medium (Ventrex) with 2% autologous serum for 72 hr.
  • the culture medium also contains 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin.
  • EXAMPLE 9 Preparation of acylated Tyr-GP(66-88) fragment Several acyl derivatives of guinea pig myelin basic protein 66-88 fragment with a tyrosine added to the N- terminus, GP (66-88) -Tyr, are prepared using the FMOC chemistry and solid phase peptide synthesizers as described in EXAMPLE 1.
  • the amino acid sequence of guinea pig myelin basic protein 68-88 fragment is Tyr-Gly-Ser-Leu-Pro-Gln-Lys-Ser-Gln-Arg-Ser-Gln-Asp-Glu- Asn-Pro-Val-Val-His-Phe-Phe (J. Immunol . , 155, 1599-1605 (1995)) .
  • Tyr-GP-MBP (66-88) is ' acylated at the N-terminal added
  • Tyr-GP-MBP (66-88) is acylated at the N-terminal added Tyr with two n-capric acids.
  • GP-MBP Experimental autoimmune encephalomyelitis is induced in Lewis rats (Charles River, Wilmington, Mass), aged 6-8 weeks, and experimental procedures are as described previously (J. Immunol . (1995) 155: 1599-1605). There are six rats in each group. Individual groups received a dose of 5, 1 or 0 (control) mg/ml of compound I suspended in phosphate-buffered saline. There are four feedings over an 8-day period and three days after the 4 th feeding, rats received an intradermal injection in both hind foot pads of guinea pig myelin basic protein ( GP- MBP) (25 ⁇ g) combined with Mycobacterium tuberculosis Jamaica strain (4 mg/ml) . All clinical neurological disorders are monitored daily and the symptom manifestation is scored as the following: 4+, full nind lumb paralysis; 3+ partial hind limb paralysis; 2+ ataxia; - 40 -
  • EXAMPLE 11 All procedures are as described in EXAMPLE 10 except that compound I is given using a nebulizer.
  • Chick type II collagen 190-200 fragment is extended by adding a tyrosine at the C-terminus of the 190-200 fragement and is also acylated with various fatty acids using the FMOC chemistry and solid phase peptide synthesizers as described in EXAMPLE 1. These conjugates are listed in Table 6.
  • the amino acid sequence of the 190-200 fragment is GPRGESGTBGS.
  • Collagen (190-200) -Tyr is acylated at the 12 th tyrosine residue with an n-lauric acid.
  • Collagen (190-200) -Tyr is acylated at the 12 th tyrosine residue with an n-palmityl acid.
  • A. Collagen (190-200) -Tyr is acylated at the 12 th tyrosine residue with an n-stearic acid.
  • EXAMPLE 13 Compound K are given to rats at doses of 0.1, 1 and 5 mg per animal.
  • Adjuvant arthritis which is used as the disease model for rheumatoid arthritis, is induced by injecting Freund' s complete adjuvant in the base of the tail.
  • the clinical arthritis score is described on a 0-4 scale for each of four paws of the rats according to J. Exp. Med. 146: 857, 1997 as follows: 4, joint deformity; 3, severe swelling; 2, redness plus mild swelling; 1, redness only; 0, normal. Each day, the rats are evaluated for their clinical arthritis score. . Individual scores of four paws are added together for the score of each animal. The mean clinical score of each treated group is determined by summing the total score of all animals in the group and dividing the total score by the number of rats . in the group . - 42 -
  • EXAMPLE 14 All procedures are as described in EXAMPLE 13 except that the doses are 0.01-, 0.1, and 1 mg and that compound K is given using a nebulizer.
  • EXAMPLE 16 Nasal delivery of acylated GAD peptide for suppressing the onset of insulin-dependent diabetes mellitus
  • NOD non-obese diabetic mice
  • mice are given nasally compound N. Each mouse received two doses a week until 35 weeks old.
  • the mice are fed either PBS (control) or 10 microgram, 100 microgram or 1 gram of compound N in PBS.
  • the mice are gavaged with an 18 gauge ball-point needle. Beginning at 12 weeks the urine is collected and tested weekly for the presence of glucose using Glucosuria test tape (Eli Lilly, Indianapolis, IN) .
  • mice has more than two positive (the presence of sugar in the urine) on the glucose urine test, a serum sample is taken and tested for blood glucose - 43 - level (Beckman glucose analyzer) . The mice are declared diabetic and sacrificed if the blood glucose is above 220 or higher.
  • EXAMPLE 17 Inhalation of acylated GAD peptide for the suppression of insulin-dependent diabetes mellitus
  • the NOD mice are given compound N in aerosol using a nebulizer (American Pharmosel Co., Valencia, CA.). All the procedures and experimental details are as described in EXAMPLE 16 except the dosing route and dose. The doses are 0 and 250 ⁇ g.
  • the animals are retained in airtight cages, into which the aerosol is dispensed. The amount of material per unit of area can thus be determined and the results quantified in terms of unit of aerosol material per unit volume of cage area.
  • the nebulizer is attached to an air pressure outlet delivering the equivalent of 7.4 liters of oxygen (the amount of oxygen used in the hospital for nebulization) .
  • the nebulizer produces droplets of spray having a diameter of between 0.3 micrometers and about 0.5 micrometers in diameter .
  • One mg of compound N is dispersed in 5 ml phosphate-buffered saline and nebulized over a 15 min period per cage. During nebulization, a fine mist is created in the cage and the mice move about freely. All other procedures are as described in EXAMPLE 6.
  • EXAMPLE 18 T cell proliferaton responses to acylated GAD peptide Groups of NOD mice are immunized in the hind foot pads by a subcutaneous inoculation of compound N (50 ⁇ g) in complete Freund 's adjuvant. Ten days later, the lymph nodes draining the sites of foot pad injection (popliteal, inguinal and periaoritic) are collected and tested for the proliferative response to derivatized compound N as compared to the control peptide (Con A) or nonmodified GAD peptide. Lymphocytes are seeded in 96-well microtiter plates (Costar Corp., Cambridge, MA), 2 X 10 5 cells in 0.2 ml of HL-1 medium (Ventrex) with 2% autologous serum for - 44 -
  • the culture medium also contains 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin.
  • the compound N (experimental) or non-modified GAD peptide (positive control) or ConA (negative control) is added at a concentration of 5 mM/ml in triplicate.
  • the culture condition is at 37° C in 5% C02 , and the culture period is 72 hr.
  • 1 mCi [ 3 H] thymidine is added per well.
  • the incorporation of radio-labeled thymidine is measured by liquid scintillation counting.
  • the ratio of the antigen-driven thymidine incorporation to the background incorporation in the absence of antigen is used to determine the degree of stimulation in T cell proliferation.

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Abstract

L'invention porte sur des composés comportant des autoantigènes, leurs analogues, des fragments de leurs peptides et des analogues desdits fragments, sont chimiquement conjugués à des acides gras sous différentes formes. Ces dérivés modulent efficacement la réponse immunitaire selon un mécanisme spécifique aux autoantigènes et jouent de ce fait un rôle dans le traitement de maladies auto-immunes telles que le diabète de type II, la sclérose en plaques, la polyarthrite rhumatoïde et plusieurs autres.
PCT/US1999/001884 1998-01-27 1999-01-27 Derives chimiques d'autoantigenes et de peptides suppresseurs d'auto-immunisation, et preparations pharmaceutiques les contenant WO1999037315A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001012222A1 (fr) * 1999-08-18 2001-02-22 Kim Ho Youn Agent d'induction d'une tolerance immunologique
WO2002020047A3 (fr) * 2000-09-06 2003-01-16 Univ Wayne State Peptides de chlamydiose et leurs mimiques dans une affection demyelinisante
WO2009033669A2 (fr) 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2018182495A1 (fr) * 2017-03-29 2018-10-04 Tcer Ab Auto-antigènes associés à la sclérose en plaques, et utilisation de ces derniers en thérapie et dans un diagnostic

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001012222A1 (fr) * 1999-08-18 2001-02-22 Kim Ho Youn Agent d'induction d'une tolerance immunologique
WO2002020047A3 (fr) * 2000-09-06 2003-01-16 Univ Wayne State Peptides de chlamydiose et leurs mimiques dans une affection demyelinisante
WO2009033669A2 (fr) 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2009039960A1 (fr) * 2007-09-11 2009-04-02 Mondobiotech Laboratories Ag Utilisation du peptide his-ser-leu-gly-lys-trp-leu-gly-his-pro-asp-lys-phe seul ou combiné au peptide gly-ard-gly-asp-asn-pro-oh en tant qu'agent thérapeutique
WO2009033669A3 (fr) * 2007-09-11 2009-05-22 Mondobiotech Lab Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2018182495A1 (fr) * 2017-03-29 2018-10-04 Tcer Ab Auto-antigènes associés à la sclérose en plaques, et utilisation de ces derniers en thérapie et dans un diagnostic
CN110475568A (zh) * 2017-03-29 2019-11-19 Tcer公司 多发性硬化症相关的自身抗原及其在疗法和诊断中的用途
EP3600390A1 (fr) * 2017-03-29 2020-02-05 Tcer Ab Auto-antigènes associés à la sclérose en plaques, et utilisation de ces derniers en thérapie et dans un diagnostic
CN110475568B (zh) * 2017-03-29 2024-08-20 Neogap治疗学公司 多发性硬化症相关的自身抗原及其在疗法和诊断中的用途

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