Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention features administering to a patient a composition comprising alpha-ketoalkanoic acid and/or one of its derivatives, which specifically includes a physiologically-acceptable salt of alpha-ketoalkanoic acid, an ester of alpha-ketoalkanoic acid, or an amide of alpha- ketoalkanoic acid for the treatment of a cytokine-mediated inflammatory condition.
• Suitable alpha-ketoalkanoic acids include C 3 -Cs straight chained or branched alpha- ketoalkanoic acids, for example, pyruvic acid.
• Physiologically acceptable salts, of ⁇ -ketoalkanoic acids include Na + , K + , Ca ++ , Mg + , NH 4 + and the like.
• the pharmaceutically acceptable inert carrier in the composition of the invention can be any carrier substance generally recognized as safe for administering a therapeutic agent to a mammal, e.g., a buffer solution for infusion, a tablet for oral administration or in gel, micelle or liposome form for on-site delivery.
• delivery solutions include isotonic or hypertonic saline; bicarbonate, phosphate or citrate buffer, plasma extender, or a microcolloid or microcrystalline solution.
• Figs. 1A-B show the structures of the preferred alpha- ketoalkanoic acid esters and amides in the composition of the invention
• Fig. 2 depicts a bar graph showing the effect of ethyl pyruvate on the permeability of Caco-2 monolayers incubated for 48 hours with a cocktail of cytokines ("cytomix")
• Nuclear extract of macrophages stimulated with LPS and incubated with antibody against p65-Rel to induce specific supershift of NF- ⁇ B complex were incubated with 100-fold molar excess of unlabeled (cold) NF- ⁇ B or HIF- 1 probe for competition analysis, (c) RAW264.7 cells were stimulated with LPS in the presence of the indicated concentration of ethyl pyruvate.
• the phosphorylation of p38MAPK was analyzed by western blot using antibodies against phosphorylated (thrl80/tyrl82) p38MAPK (in accordance with the manufacturer (New England Biolabs, Inc Cat.
• compositions comprising alpha-ketoalkanoic acid and/or one of its derivatives can ameliorate a cytokine-mediated inflammatory condition.
• the method of the invention has been tested, e.g., in a standard mouse model of murine sepsis, in which it was shown that ethyl pyruvate rescues mammals from lethal sepsis caused by peritonitis, even when the first doses of a derivative of pyruvate were administered 24 hours after sepsis was established.
• the precise mechanism(s) responsible for the protective actions of pyruvate and/or its derivatives remain to be elucidated.
• Macrophage activation by endotoxin, cytokines, and products of cell injury lead to the nuclear translocation of NF- ⁇ B, a transcription factor that enhances the transcription of TNF and other products of the activated macrophage (Senftleben et al., Crit . Care Med. (2002) 30(1 Suppl) : S18-26) .
• Macrophage activation also leads to phosphorylation of p38 MAP kinase, a pathway that has been implicated in stabilizing TNF mRNA, and increasing TNF translation efficiency (Adams et al., Prog. Med. Chem . (2001) 38:1-60).
• ethyl pyruvate significantly inhibited HMGB-1 release from macrophages, and decreased serum HMGB-1 levels in septic mice. Macrophages contain large quantities of HMGB-1 in the cytosol that provide a pre-formed pool of HMGB-1 that can be released during activation. The mechanism(s) that regulate HMGB-1 release from macrophages is enigmatic, but the present data indicate that ethyl pyruvate inhibits the release of the cytosolic protein from activated macrophages. Inhibition of HMGB-1 release by ethyl pyruvate was specific, because ethyl pyruvate did not affect intracellular HMGB-1 protein expression or stability.
• ethyl pyruvate inhibition of signaling through NF- ⁇ B and p38 MAPK pathway may underlie the ability of ethyl pyruvate to suppress HMGB-1 release from LPA-stimulated cells. Additionally, inhibition of TNF in the local macrophage milieu contributes to inhibition of HMGB-1 release, because TNF is a potent inducer of HMGB-1 release.
• pyruvate and/or its derivatives have the therapeutic potential for disease mediated by an excess of TNF- and HMGB-1.
• ethyl pyruvate is a relatively non-toxic food additive, and the observed effects occur in therapeutically achievable and safe levels.
• Ethyl pyruvate can be administered in Ringer' s- type crystalloid fluid, a calcium- and potassium-containing balanced salt solution that is widely used as resuscitating agent for the treatment of shock. Accordingly, it is a primary object of this invention to provide new method of treating inflammatory conditions, particularly those that are mediated by cytokines, using an improved composition containing alpha-ketoalkanoic acid and/or its derivatives.
• one composition of this invention comprises a alpha-ketoalkanoic acid ester, in accordance with the molecular structures shown in Fig. 1, admixed with a sufficient concentration of biologically safe organic or inorganic cations to induce enolization of the alpha-keto functionality of the ester at physiological pH values.
• the composition comprises an alkyl ester of alpha-ketopropionic acid (pyruvic acid) , the ester is the ethyl analog and the cation is a divalent cation, particularly either calcium or magnesium.
• the ester compound is ethyl pyruvate admixed with calcium ion in a Ringer' s solution at a pH of about 7-8.
• the therapeutic compositions of the invention may be administered orally, topically (e.g., ointment, gel or cream), or parenterally, (e.g., intranasally, subcutaneously, intramuscularly, intravenously, intraluminally, intra-arterially, intravaginally, transurethrally or rectally) by routine methods in pharmaceutically acceptable inert carrier substances.
• the therapeutic compositions of the invention may be administered in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using micelles, gels, liposomes, or a buffer solution.
• Exemplary cytokine-mediated inflammatory conditions include, but are not limited to, local and systemic inflammation, inflammatory bowel disease (Crohn's disease and ulcerative colitis), rheumatoid arthritis, asthma (including status asthmaticus) , endotoxemia, sepsis and septic shock, also including inflammatory skin conditions, for example, psoriasis and eczema.
• Ethyl Pyruvate Solution Ethyl pyruvate was prepared in solution with sodium (130 mM) , potassium (4 mM) , calcium (2.7 mM) , chloride (130 mM) , and ethyl pyruvate (28 mM) ; pH 7.0). For injections in mice solutions were diluted so that each injection volume was 0.4 ml per dose.
• BALB/c murine macrophage-like RAW 264.7 cells obtained from the American Type Culture Collection (ATCC Number TIB-71, ATCC, Rockville, MA) (61), were cultured in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% heat inactivated fetal bovine serum (Gemini, Calabasas, CA) , 2 mM glutamine (Cat. # 25030-149, Gibco BRL, Rockville, MA) and antibiotic-antimycotic mix (Cat. # 15240- 062, Gibco BRL, Rockville, MA) in a humidified incubator with 5% C0 2 . Cells were mechanically removed and resuspended in serum-free Opti-MEM I medium (Life Technology, Grand Island, NY) to perform experiments at 75% confluence.
• Recombinant mouse TNF standards were obtained from R&D Systems (Inc., Minneapolis, MN) and dissolved in 0.1% bovine serum albumin solution (BSA, low endotoxin grade from Sigma Chemical Co., St. Louis, MO).
• BSA bovine serum albumin solution
• Monoclonal antibody to mouse TNF was purchased from Biosource International, Inc., Camarillo, CA. Human TNF monoclonal antibody, human TNF antiserum and mouse TNF antiserum were prepared and contributed by Dr. Christine Metz.
• HMGB-1 Mouse serum IL-6 and IL- l ⁇ levels were measured using ELISA kits (R&D Systems, Inc., Minneapolis, MN) .
• HMGB-1 was analyzed by western blot as ⁇ previously described by Wang et al, 1999. Briefly, serum or cell culture conditioned media was first filtrated through centricon YM-100 (Millipore Corp., Bedford, MA) to clear the samples from cell debris and macromolecular complex formed during clotting. Then, samples were concentrated 15 folds by using centricon YM-30 and subjected to 12% SDS- polyacrylamide gels (SDS-PAGE) .
• SDS-PAGE SDS- polyacrylamide gels
• HMGB- 1 was analyzed using polyclonal 241 antibody and secondary anti-rabbit horseradish peroxidase (Amersham, Pitscataway, NJ) . Standard curves were constructed using r-HMGB-1, and the intensity of the 30kD band analyzed by densitometry.
• RNA extraction and RNase protection assay Total RNA extraction and RNase protection assay.
• Total RNA was extracted from cultured cells by using RNAzol B in ⁇ accordance with the manufacturer's instructions (Tel-Test "B” Inc, Friendswood, TX) . The integrity of the RNA was verified by electrophoresis on 1.2% agarose/17% formaldehyde gels. The levels of TNF and cyclophilin mRNA in RAW cells were measured using an RNase protection assay kit from PharMingen in accordance with the manufacturer' s instructions (San Diego, CA) . The antisense RNA probe was labeled with ⁇ 32 p-uTP (800 Ci/mmol, Amersham, IL) using T7 RNA polymerase.
• ethyl pyruvate on a stabilizing calcium salt solution (REPS) in a cytokine- induced intestinal epithelial hyperpermeability condition
• REPS stabilizing calcium salt solution
• Exemplary materials and methods include the following, while minor modifications can be made: Caco-2 human enterocytes (10 5 cells/well) were plated on permeable filters in 12-well Transwell bicameral chambers (COSTAR, Corning, NY) and fed biweekly. Permeability studies were carried out using confluent monolayers between 21-28 d after seeding.

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• 2002
  • 2002-03-15 EP EP02725226A patent/EP1379230A4/en not_active Withdrawn
  • 2002-03-15 WO PCT/US2002/008283 patent/WO2002074301A1/en not_active Ceased
  • 2002-03-15 CA CA002440480A patent/CA2440480A1/en not_active Abandoned
  • 2002-03-15 AU AU2002255805A patent/AU2002255805B2/en not_active Ceased
  • 2002-03-15 JP JP2002573009A patent/JP2004528307A/ja active Pending
• 2003
  • 2003-09-15 US US10/662,975 patent/US6943190B2/en not_active Expired - Fee Related
• 2005
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