WO2016009341A1 - Promédicaments mimétiques de la thiorédoxine et leurs utilisations - Google Patents

Promédicaments mimétiques de la thiorédoxine et leurs utilisations Download PDF

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WO2016009341A1
WO2016009341A1 PCT/IB2015/055307 IB2015055307W WO2016009341A1 WO 2016009341 A1 WO2016009341 A1 WO 2016009341A1 IB 2015055307 W IB2015055307 W IB 2015055307W WO 2016009341 A1 WO2016009341 A1 WO 2016009341A1
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alkyl
compound
alkylene
absent
coch
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PCT/IB2015/055307
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Andrew Lurie Salzman
Prakash Jagtap
Garry John SOUTHAN
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Radikal Therapeutics Inc.
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Publication of WO2016009341A1 publication Critical patent/WO2016009341A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention relates to thioredoxin (TRX) mimetic prodrugs, pharmaceutical compositions comprising them, and uses thereof.
  • ALT alanine transaminase
  • AST aspartate transaminase
  • BAL bronchoalveolar lavage fluid
  • BUN blood urea nitrogen
  • CILI chlorine (Cl 2 ) inhalational lung injury
  • CIN contrast media-induced nephropathy
  • Fi0 2 fraction of inspired oxygen
  • IMV intermittent mandatory ventilation
  • IP intraperitoneally
  • I/R ischemia-reperfusion
  • IV intravenously
  • LAD left anterior descending
  • LCMS Liquid chromatography-mass spectrometry
  • LPS lipopolysaccharide
  • MDA malondialdehyde
  • MIRI myocardial ischemia-reperfusion injury
  • MPO myeloperoxidase
  • NAC N-acetylcysteine
  • Pa0 2 partial pressure of oxygen
  • PBS phosphate-buffered saline
  • PC0 2 partial pressure of oxygen
  • PBS phosphate-buffered
  • l-(2-acetamido-3-mercaptopropanoyl)-N-(l-amino-3-mercapto-l-oxopropan-2- yl)pyrrolidine-2-carboxamide, 2-(l-(2-amino-3-mercaptopropanoyl) pyrrolidine-2- carboxamido)-3-mercaptopropanoic acid, and analogues thereof are TRX mimetics, thiol- rich tripeptide containing cysteine -proline-cysteine (Cys-Pro-Cys) or analogues, which are closely analogous to the native TRX motif.
  • TRX is a multifunctional redox-active protein that scavenges ROS by itself or together with TRX-dependent peroxiredoxin, and is a critical element in the defense against redox stress. TRX also has chemotaxis-modulating functions and suppresses PMN infiltration into sites of inflammation (Hoshino et ah, 2003). The redox stress of acute lung injury is initially countered by endogenous reductants, especially TRX, but such thiol-rich reductant defenses are readily overwhelmed by massive oxidant insults. The subsequent depletion of TRX increases susceptibility to acute lung injury, as noted in models of hyperoxic lung injury (Tipple et ah, 2007).
  • US 8,530,407 discloses antioxidant compounds of the general formula A-Yi-Cys- Y2-Cys-Y 3 -B, wherein Cys is a cysteine residue, A and B are each individually a hydrophobic or non-charged moiety; and Yi, Y 2 and Y 3 are each individually one or more amino acid residues in the range of 0-30 residues, with the provision that Yi, Y 2 and Y 3 collectively provide for at least two amino acid residues in the peptide.
  • the present invention provides a compound of the formula I:
  • Xi is absent or an amino acid residue forming a peptide bond with the -NH- group adjacent thereto;
  • X 2 is absent or an amino acid residue forming peptide bonds with the carbonyl group and nitrogen atom adjacent thereto;
  • X 3 is absent or an amino acid residue forming peptide bonds with the carbonyl and -NH- groups adjacent thereto;
  • X 4 is absent or an amino acid residue forming a peptide bond with the carbonyl group adjacent thereto;
  • Ri is H, -CO(Ci-C 8 )alkyl, -CO(Ci-C 8 )alkylene-NH((Ci-C 8 )alkyl, -CO(C C 8 )alkylene-N((Ci-C 8 )alkyl) 2 , or -CO(Ci-C 8 )alkylene-N + ((Ci-C 8 )alkyl) 3 , covalently linked to the N atom of the amino moiety of Xi, when present;
  • R 2 is H or (Ci-Cs)alkyl, covalently linked to the carbonyl moiety of X 4 , when present; and
  • R 3 and R 3 ' each independently is -SH, -S-CO(d-C 8 )alkyl, -S-CO(Ci-C 8 )alkylene- N((Ci-C 8 )alkyl) 2 , or -S-CO(Ci-C 8 )alkylene-N + ((Ci-C 8 )alkyl) 3 ; or R 3 and R 3 ' are both -S- and together form a disulfide bond; or one of R 3 and R 3 ' is -S- forming a disulfide bond with one of R 3 and R 3 ' of another identical or different compound of the formula I, and another of R 3 and R 3 ' is -SH, -S-CO(Ci-C 8 )alkyl, -S-CO(Ci-C 8 )alkylene-N((Ci-C 8 )alkyl) 2 , or -S-CO(Ci-C 8 )alkylene-
  • Ri and R 2 is not H, or at least one of R 3 and R 3 ' is not -SH, but excluding the compounds wherein R 3 and R 3 ' are each -SH, Ri is -CO(Ci- C 8 )alkyl, R 2 is H, and Xi, X 2 , X 3 and X 4 are absent.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.
  • the compounds and pharmaceutical compositions of the invention are useful for prevention, treatment or management of a disease, disorder or condition mediated by redox stress.
  • the present invention thus relates to a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof, for use in prevention, treatment or management of a disease, disorder or condition mediated by redox stress.
  • the present invention relates to use of a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof, for the preparation of a pharmaceutical composition for prevention, treatment or management of a disease, disorder or condition mediated by redox stress.
  • the present invention relates to a method for prevention, treatment or management of a disease, disorder or condition mediated by redox stress in an individual in need thereof, comprising administering to said individual an effective amount of a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof.
  • Fig. 1 shows the number of living mice at each time post-NaSH injection. Mice were injected IP with 30 mg/kg NaSH (LD 7 o). 1 hour post NaSH injection, the treated group was injected with R-911 (130 mg/kg), whereas the control group was injected with vehicle.
  • P value for difference between vehicle and R-911 groups was found to be highly significant (p ⁇ 10 ⁇ ).
  • Fig 3 shows the effect of R-911 in a lung radiation (RAD)-induced injury model.
  • C57BL/6 male mice were exposed to 14 Gy radiation to the thorax.
  • R-911 125 mg/kg or vehicle (RAD only group) was delivered IP.
  • a second delivery of R-911 or vehicle was given 4 hours later on the day of radiation, and treatments continued twice daily for the next 6 days.
  • Apoptosis was assessed by TUNEL staining on Day 7 post-radiation and the quantitative assessment of percent of apoptotic cells per lung section is shown.
  • Un-irradiated mice (No RAD group) are shown as a negative control. * indicates significant difference by one-way ANOVA followed by Tukey' s multiple comparison test comparing the indicated columns; overall ANOVA /? ⁇ 0.0001.
  • Fig. 4 shows the prophylactic vs. therapeutic effect of R-911 in a lung radiation (RAD)-induced injury model.
  • C57BL/6 male mice were exposed to 14 Gy radiation to the thorax. Either five minutes before exposure (pre) or 24 hours after exposure (post), R-911 (125 mg/kg) or vehicle (RAD only group) was delivered IP. A second delivery of R-911 or vehicle was given 4 hours later, and treatments continued twice daily for the next 5-6 days. Apoptosis was assessed by TUNEL staining on Day 7 post-radiation and the quantitative assessment of percent of apoptotic cells per lung section is shown. Un-irradiated mice (No RAD group) are shown as a negative control. * indicates significant difference by one-way ANOVA followed by Tukey' s multiple comparison test comparing the indicated columns; overall ANOVA /? ⁇ 0.0003.
  • Fig. 5 shows that the reduction in lung oxygenation is ameliorated in R-911 treated group.
  • the significant elevation of Pa0 2 in R-911 treated group indicates enhanced lung oxygenation.
  • Figs. 6A-6B show that R-911 resuscitated lung I/R- induced histological injury. Histlogical injury score (6A) and H&E stained histological images (6B) demonstrate the protective effect of R-911 in restoring lung tissue architecture.
  • Figs. 7A-7B show that R-911 significantly lessens inflammation induced by I/R injury.
  • MPO (7A) and MDA (7B) were determined in wet lung tissue. Both markers were increased in treatment groups, and significantly reduced upon R-911 administration.
  • Fig. 8 shows that R-911 significantly reduces protein exacerbation.
  • BAL fluid content revealed an augmentation of proteins in treatment group, a sign of alveolar epithelial injury, in contrast to significant decrease of proteins in the R-911 group.
  • Fig. 9 shows that R-911 reduces edema induced by lung I/R injury. Wet/dry ratio was determined in wet lung tissue, portraying a significant decrease in pulmonary edema when treated with R-911.
  • Figs. 10A-10B show that R-911 significantly attenuated CIN-induced BUN (10A) and creatinine (10B) elevations in CIN model in rats treated with R-911 or NAC (equimolar dose to 100 mg/kg/day of R-911). Sham rats are compared to CIN rats treated with a vehicle control or with 3 dose levels of R-911 (10, 30 and 100 mg/kg/day). P values: * ⁇ 0.05, ** ⁇ 0.0002, *** ⁇ 0.005, **** ⁇ 10 ⁇ 5 .
  • FIGs. 11A-11B show that R-911 significantly restored tissue architecture in the kidney of contrast media-treated rats.
  • 11A shows representative H&E staining (x20) of histological samples that were used to generate the injury scoring data.
  • R-911 treatment dramatically decreased the histological injury score and restored normal kidney tissue architecture.
  • 11B shows the histological injury scoring of kidneys of different groups post contrast media injection. P value for difference between vehicle and 100 mg/kg/day R-911 group, as well as the NAC group was found to be highly significant (p ⁇ 0.0008).
  • Figs. 12A-12B show that R-911 significantly attenuated CIN-induced histological injury in a diabetic model.
  • Representative histological kidney sections (12A, all slides are xlO) indicate that CIN caused severe damage to the kidney tissue.
  • R-911 300 mg/kg/day
  • An equimolar dose of NAC (94.5 mg/kg/day) was shown to be less effective when compared to contrast media vehicle groups (p ⁇ 0.05).
  • Figs. 13A-13B show that R-911 significantly attenuated CIN-induced elevations in creatinine and BUN.
  • CIN caused severe damage to the kidney tissue as BUN (13A) and creatinine (13B).
  • Increasing volume did not cause any significant advantage when administered with 300 mg/kg/day of R-911.
  • FIGs. 14A-14B show that R-911 significantly restored tissue architecture in the lung in mouse lungs exposed to chlorine.
  • 14A shows representative H&E staining (x20) of histological samples that were used to generate the injury scoring data (14B). While Cl 2 injury elevated the histology score, R-911 treatment dramatically decreased this impacted histological injury score and restored normal lung tissue architecture.
  • Fig. 15 shows that R-911 significantly restored VILI-induced decrease in Pa0 2 .
  • Figs. 18A-18B show that R-911 significantly attenuated RIRI- induced creatinine elevations.
  • R-911 treatment groups dramatically reduced, in a dose dependent manner, creatinine elevations seen at 6 (18A) and 24 (18B) hours post reperfusion.
  • FIGs. 19A-19B show that R-911 significantly attenuated RIRI-induced histological damage and restored histological tissue architecture in a dose dependent fashion. Representative slides are shown in 19A, and the SEM values of histological injury scoring of all kidneys from the indicated treatment groups are shown in 19B.
  • Figs. 20A-20C show that R-911 significantly decreases MIRI-induced augmentation of infarct size (20A), neutrophil infiltration (20B, /? ⁇ 0.01), and lipid peroxidation (20C, /? ⁇ 0.0001).
  • Figs. 21A-21B show that R-911 significantly reduces histological injury and restores cardiac tissue architecture as can be visualized in the representative slides (21A) and in 21B, presenting the SEM histological injury scores indicating a dramatic attenuation of injury by R-911 (40%, /? ⁇ 0.0003 in a 2-tailed t-test).
  • SEM histological injury scores indicating a dramatic attenuation of injury by R-911 (40%, /? ⁇ 0.0003 in a 2-tailed t-test).
  • the present invention provides a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof.
  • TRX mimetic prodrugs i.e., derivatives or analogues of the amino acid sequence Cys-Pro-Cys or Ai-Cys-A 2 -Pro-A 3 -Cys-A 4 wherein Ai to A 4 each independently is either absent or an amino acid residue, which upon hydrolysis under physiological conditions are converted into the native Cys-Pro-Cys or Ai-Cys-A 2 -Pro-A 3 -Cys-A 4 sequence, and are thus expected to be effective in all those clinical indications wherein administration of the native TRX motif is beneficial.
  • the TRX mimetic prodrug of the present invention is a derivative or analogue of the amino acid sequence Cys-Pro-Cys or Ai-Cys-A 2 -Pro-A 3 - Cys-A 4 in which the thiol group of at least one of the Cys residues is protected by a group independently selected from -CO(Ci-C 8 )alkyl, -CO(Ci-C 8 )alkylene-N((Ci-C 8 )alkyl) 2 , or - CO(Ci-C 8 )alkylene-N + ((Ci-C 8 )alkyl)3; and/or the terminal amino group is protected by a group selected from -CO(Ci-C 8 )alkyl, -CO(Ci-C 8 )alkylene-NH((Ci-C 8 )alkyl, -CO(C C 8 )alkylene-N((Ci-C 8 )
  • the TRX mimetic prodrug of the invention is a derivative or analogue of the amino acid sequence Cys-Pro-Cys or Ai-Cys-A 2 -Pro-A 3 -Cys- A 4 in which at least one of the terminal amino group and terminal carboxyl group is protected as defined above, and/or the thiol group of each one of the Cys residues is deprotonated and the two sulfur atoms together form a disulfide bond.
  • the TRX mimetic prodrug of the invention is a derivative or analogue of the amino acid sequence Cys-Pro-Cys or Ai-Cys-A 2 -Pro-A 3 -Cys- A 4 in which at least one of the terminal amino group and terminal carboxyl group is protected as defined above, and/or the thiol group of at least one of the Cys residues is deprotonated wherein the sulfur atom forms a disulfide bond with the sulfur atom of a deprotonated thiol group of another identical or different compound of the formula I, and the thiol group of the other Cys residue is optionally protected as defined above.
  • a compound according to this particular aspect is thus a dimer- or higher multimer-like compound, in which two or more identical or different entities, each independently according to the formula I, are linked via disulfide bonds, wherein each disulfide bond links two entities.
  • the dimer-like compound of the present invention results from a linkage between two compounds of the formula I, wherein only one of the thiol groups of each one of the compounds is deprotonated and the sulfur atom of the deprotonated thiol group of one of the compounds forms a disulfide bond with the sulfur atom of the deprotonated thiol group of the other compound.
  • the dimer-like compound of the present invention is a cyclic dimer resulting from a linkage between two compounds of the formula I, wherein the thiol group of each one of the Cys residues in each one of the compounds is deprotonated and each one of the sulfur atoms of the deprotonated thiol groups of one of the compounds forms a disulfide bond with a different one of the sulfur atoms of the deprotonated thiol groups of the other compound.
  • the compound of the present invention is a higher multimer-like compound resulting from a linkage between more than two compounds of the formula I, wherein the thiol group of each one of the Cys residues in at least one of those compounds is deprotonated, and the sulfur atom of each one of the deprotonated thiol groups forms a disulfide bond with the sulfur atom of a deprotonated thiol group of a different compound.
  • cysteine and proline residues or residue derivatives constituting the TRX mimetic prodrug of the present invention may independently be of either L or D configuration, indicating the optical activity of the isomer of glyceraldehyde from which that amino acid can, in theory, be synthesized.
  • the (S) and (R) designators may be used to indicate the absolute stereochemistry of the compound, wherein proline naturally being (S) at the a carbon, and cysteine, having a sulfur atom at the second position in its side chain, naturally being (R).
  • alkyl typically means a linear or branched saturated hydrocarbon radical having 1-8 carbon atoms and includes, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, n- heptyl, n-octyl, and the like.
  • Preferred are (Ci-C6)alkyl groups, more preferably (Ci- C 4 )alkyl groups, most preferably methyl and ethyl.
  • alkylene typically means a divalent linear or branched hydrocarbon radical having 1-8 carbon atoms and includes, e.g., methylene, ethylene, propylene, butylene, 2-methylpropylene, pentylene, 2-methylbutylene, hexylene, 2-methylpentylene, 3-methylpentylene, 2,3-dimethylbutylene, heptylene, octylene, and the like.
  • amino acid refers to an organic compound comprising both amine and carboxylic acid functional groups, which may be either a natural or non- natural amino acid and may be of either L or D configuration.
  • the twenty two natural amino acids are aspartic acid (Asp), tyrosine (Tyr), leucine (Leu), tryptophan (Trp), arginine (Arg), valine (Val), glutamic acid (Glu), methionine (Met), phenylalanine (Phe), serine (Ser), alanine (Ala), glutamine (Gin), glycine (Gly), proline (Pro), threonine (Thr), asparagine (Asn), lysine (Lys), histidine (His), isoleucine (lie), cysteine (Cys), selenocysteine (Sec), and pyrrolysine (Pyl).
  • Non-limiting examples of non-natural amino acids include diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine (Orn), aminoadipic acid, ⁇ -alanine, 1-naphthylalanine, 3-(l-naphthyl)alanine, 3-(2- naphthyl)alanine, ⁇ -aminobutiric acid (GABA), 3-(aminomethyl) benzoic acid, p-ethynyl- phenylalanine, p-propargly-oxy-phenylalanine, m-ethynyl-phenylalanine, p- bromophenylalanine, p-iodophenylalanine, p-azidophenylalanine, p-acetylphenylalanine, azidonorleucine, 6-ethynyl-tryptophan, 5-ethynyl-tryp
  • amino acid residue refers to a residue of an amino acid after removal of hydrogen atom from an amino group thereof, e.g., its a-amino group or side chain amino group if present, and -OH group from a carboxyl group thereof, e.g., its a-carboxyl group or side chain carboxyl group if present.
  • peptide bond or "amide bond” as used herein refers to the covalent bond -C(0)NH- formed between two molecules, e.g., two amino acids, when a carboxyl group of one of the molecules reacts with an amino group of the other molecule, causing the release of a molecule of water.
  • the compound of the present invention is a compound of the formula I, wherein Ri is H, -CO(Ci-C 4 )alkyl, preferably -CO(Ci-C 2 )alkyl, -CO(Ci- C 4 )alkylene-NH((Ci-C 4 )alkyl, preferably -CO(Ci-C 2 )alkylene-NH((Ci-C 2 )alkyl, -CO(d- C 4 )alkylene-N((Ci-C 4 )alkyl) 2 , preferably -CO(Ci-C 2 )alkylene-N((Ci-C 2 )alkyl) 2 , or - CO(Ci-C 4 )alkylene-N + ((Ci-C 4 )alkyl) 3 , preferably -CO(Ci-C 2 )alkylene-N + ((Ci-C 2 )alkyl) 3
  • the compound of the present invention is a compound of the formula I, wherein R 2 is (Ci-C 4 )alkyl, preferably (Ci-C 2 )alkyl, or H. Particular such embodiments are those wherein R 2 is methyl, ethyl or H.
  • the compound of the present invention is a compound of the formula I, wherein R 3 and R 3 ' each independently is -SH, -S-CO(Ci-C 4 )alkyl, preferably -S-CO(Ci-C 2 )alkyl, -S-CO(Ci-C 4 )alkylene-N((Ci-C 4 )alkyl) 2 , preferably -S- CO(Ci-C 2 )alkylene-N((Ci-C 2 )alkyl) 2 , or -S-CO(Ci-C 4 )alkylene-N + ((Ci-C 4 )alkyl) 3 , preferably -S-CO(Ci-C 2 )alkylene-N + ((Ci-C 2 )alkyl) 3 ; or R 3 and R 3 ' are both -S- and together form a disulfide bond.
  • R 3 and R 3 ' each independently is -SH, -S-COCH 3 , -S-CO-CH 2 -N(CH 3 ) 2 , or -S-CO-CH 2 -N + (CH 3 ) 3 ; or R 3 and R 3 ' are both -S- and together form a disulfide bond.
  • the compound of the present invention is a dimer- or higher multimer-like compound as defined above, i.e., a compound of the formula I, wherein one of R 3 and R 3 ' is -S- forming a disulfide bond with one of R 3 and R 3 ' of another identical or different compound of the formula I, and another of R 3 and R 3 ' is -SH, -S- CO(Ci-C 4 )alkyl, preferably -S-CO(Ci-C 2 )alkyl, -S-CO(Ci-C 4 )alkylene-N((Ci-C 4 )alkyl) 2 , preferably -S-CO(Ci-C 2 )alkylene-N((Ci-C 2 )alkyl) 2 , or -S-CO(Ci-C 4 )alkylene-N + ((Ci- C 4 )alkyl) 3 , preferably -S-S-
  • one of R 3 and R 3 ' is -S-, and another of R 3 and R 3 ' is -SH, -S-COCH 3 , -S-CO-CH 2 -N(CH 3 ) 2 , or -S- CO-CH 2 -N + (CH 3 ) 3 ; or R 3 and R 3 ' are both -S-.
  • the compound of the present invention is a compound of the formula I, wherein Xi, X 2 , X 3 and X 4 are absent, or at least one of Xi, X 2 , X 3 and X 4 is present.
  • Particular such embodiments are those wherein (i) one of Xi, X 2 , X 3 and X 4 is an amino acid residue as defined above, and the other three of Xi, X 2 , X 3 and X 4 are absent, e.g., wherein Xi, X 2 and X 3 are absent, and X 4 is a glycine residue; Xi, X 2 and X 4 are absent, and X 3 is a glycine residue; Xi, X 3 and X 4 are absent, and X 2 is a glycine residue; or X 2 , X 3 and X 4 are absent, and Xi is a glycine residue; (ii) two of Xi, X 2 , X 3 and X 4 are amino acid residues each independently as defined above, and the other two of Xi, X 2 , X 3 and X 4 are absent, e.g., wherein Xi and X 2 are absent
  • the compound of the present invention is a compound of the formula I according to some of the embodiments defined above, wherein Ri is H, -CO(Ci-C 4 )alkyl, preferably -CO(d-C 2 )alkyl, -CO(Ci-C 4 )alkylene-NH((Ci- C 4 )alkyl, preferably -CO(Ci-C 2 )alkylene-NH((Ci-C 2 )alkyl, -CO(Ci-C 4 )alkylene-N((Ci- C 4 )alkyl) 2 , preferably -CO(Ci-C 2 )alkylene-N((Ci-C 2 )alkyl) 2 , or -CO(Ci-C 4 )alkylene- N + ((Ci-C 4 )alkyl) 3 , preferably -CO(Ci-C 2 )alkylene-N + ((Ci-C-C 4 )alkyl
  • Ri is H, -CO-CH 2 -N(CH 3 ) 2 , or -CO-CH 2 -N + (CH 3 ) 3 ;
  • R 2 is H, methyl, or ethyl; and
  • R 3 and R 3 ' each independently is -SH, - S-COCH 3 , -S-CO-CH 2 -N(CH 3 ) 2 , or -S-CO-CH 2 -N + (CH 3 ) 3 , or R 3 and R 3 ' are both -S- and together form a disulfide bond, wherein each one of the combinations of Ri, R 2 , R 3 and R 3 ' constitutes a separate embodiment.
  • the compound of the present invention is a compound of the formula I, wherein Xi, X 2 , X 3 and X 4 are absent, i.e., a derivative or analogue of the amino acid sequence Cys-Pro-Cys, and: (i) Ri is H, R 2 is ethyl, R 3 is -SH, and R 3 ' is -SH (compound 1; R-951); (ii) Ri is -CO(CH 2 )-N(CH 3 ) 2 , R 2 is H, R 3 is -SH, and R 3 ' is -SH (compound 2; R-956); (iii) Ri is -CO(CH 2 )-N(CH 3 ) 2 , R 2 is ethyl, R 3 is -SH, and R 3 ' is -SH (compound 3; R-952); (iv) Ri is H, R 2 is H, R 3 is -S-COCH 3 , and
  • the compound of the present invention is a compound of the formula I, wherein X 2 is a glycine residue; and Xi, X 3 and X 4 are absent, i.e., a derivative or analogue of the amino acid sequence Cys-Gly-Pro-Cys, and wherein: (i) Ri is H, R 2 is ethyl, R 3 is -SH, and R 3 ' is -SH (compound 10); (ii) Ri is -CO(CH 2 )- N(CH 3 ) 2 , R 2 is H, R 3 is -SH, and R 3 ' is -SH (compound 11); (iii) Ri is -CO(CH 2 )-N(CH 3 ) 2 , R 2 is ethyl, R 3 is -SH, and R 3 ' is -SH (compound 12); (iv) Ri is H, R 2 is H, R 3 is -S- COCH 3 ,
  • the compound of the present invention is a dimer- or higher multimer-like compound as defined above, i.e., a compound of the formula I, wherein Ri is H, -CO(Ci-C 4 )alkyl, preferably -CO(Ci-C 2 )alkyl, -CO(d- C 4 )alkylene-NH((Ci-C 4 )alkyl, preferably -CO(Ci-C 2 )alkylene-NH((Ci-C 2 )alkyl, -CO(C C 4 )alkylene-N((Ci-C 4 )alkyl) 2 , preferably -CO(Ci-C 2 )alkylene-N((Ci-C 2 )alkyl) 2 , or - CO(Ci-C 4 )alkylene-N + ((Ci-C 4 )alkyl) 3 , preferably -CO(Ci-C 2 )
  • Ri is H, -CO-CH 2 -N(CH 3 ) 2 , or - CO-CH 2 -N + (CH ) ;
  • R 2 is H, methyl, or ethyl; and either one of R and R ' is -S-, and another of R 3 and R 3 ' is -SH, -S-COCH 3 , -S-CO-CH 2 -N(CH 3 ) 2 , or -S-CO-CH 2 -N + (CH 3 ) 3 , or R 3 and R 3 ' are both -S-, wherein each one of the combinations of Ri, R 2 , R 3 and R 3 ' constitutes a separate embodiment.
  • dimer- or higher multimer- like compounds wherein at least one of Xi, X 2 , X 3 and X 4 is a glycine residue, and the other of Xi, X 2 , X 3 and X 4 are absent.
  • the compounds of the formula I may be synthesized according to any technology or procedure known in the art, e.g., as described in Example 1 hereinafter.
  • the compounds of the formula I may have one or more asymmetric centers, and may accordingly exist both as enantiomers, i.e., optical isomers (R, S, or racemate, wherein a certain enantiomer may have an optical purity of 90%, 95%, 99% or more) and as diastereoisomers.
  • those chiral centers may be, e.g., in each one of the carbon atoms located at position alpha to any one of the carbonyl groups in the formula I. It should be understood that the present invention encompasses all such enantiomers, isomers and mixtures thereof, as well as pharmaceutically acceptable salts and solvates thereof.
  • Optically active forms of the compounds of the formula I may be prepared using any method known in the art, e.g., by resolution of the racemic form by recrystallization techniques; by chiral synthesis; by extraction with chiral solvents; or by chromatographic separation using a chiral stationary phase.
  • a non-limiting example of a method for obtaining optically active materials is transport across chiral membranes, i.e., a technique whereby a racemate is placed in contact with a thin membrane barrier, the concentration or pressure differential causes preferential transport across the membrane barrier, and separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through.
  • Chiral chromatography including simulated moving bed chromatography, can also be used.
  • a wide variety of chiral stationary phases are commercially available. Table 2: Compounds of the formula I, herein identified 1-9
  • TRX superfamily proteins Reduction of the disulfide bond of TRX superfamily proteins, a critical step in providing intracellular redox defense, results from the donation of NADPH-derived electrons to TRX, followed by their transfer to recipient TRX superfamily proteins via a dithiolate exchange mechanism (R0hr et al. , 2013).
  • the compounds of the present invention e.g., compound 2, are TRX mimetics with vicinal thiol groups that may substitute for deficient TRX in mediating dithiolate exchange.
  • TRX mimetics and compounds providing reducing thiol equivalents are useful in the treatment of indications where oxidative stress occurs and ROS are generated. These compounds may scavenge ROS directly or via TRX-mimetic activity.
  • compounds containing thiols and especially those with multiple thiols in close proximity are subject to facile oxidation to various disulfides and other products. This can occur on standing, as a solid, in solution and in biological matrixes.
  • Prodrugs that mask the thiols prevent their instability, but allow the thiol equivalents to be released when administered to subjects.
  • the compounds of the present invention which are TRX mimetic prodrugs, are thus expected to be effective in all those clinical indications wherein administration of the native TRX motif is beneficial, i.e., in the prevention, treatment or management of any disease, disorder, or condition mediated by redox stress.
  • Such diseases, disorders or conditions include, without being limited to, chlorine inhalational lung injury, phosgene inhalational lung injury, hydrogen sulfide inhalational lung injury, ventilator-induced lung injury, lung ischemia reperfusion injury, chronic obstructive pulmonary disease (COPD), bronchopulmonary dysplasia, adult respiratory distress syndrome (ARDS), radiation induced lung fibrosis, congestive heart failure (CHF), myocardial infarction, myocardial ischemia-reperfusion injury, stroke, cardiopulmonary bypass surgery, doxorubicin-induced cardiomyopathy, limb ischemia reperfusion injury, renal ischemia repercussion injury, contrast media-induced nephropathy, hemorrhagic shock, endotoxic shock, septic shock, hepatic reperfusion injury, primary lung graft dysfunction, lung transplantation, renal transplantation, burn injury, angioplasty, traumatic brain injury, Parkinson's disease, ischemic bowel disease, mesenteric ischemia,
  • the present invention thus provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an active agent, i.e., a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof.
  • the active agent is compound selected from compounds 1-18, or a pharmaceutically acceptable salt or solvate thereof.
  • compositions of the present invention can be provided in a variety of formulations, e.g., in a pharmaceutically acceptable form and/or in a salt form, as well as in a variety of dosages.
  • the pharmaceutical composition of the present invention comprises a non-toxic pharmaceutically acceptable salt of the active agent as defined above.
  • suitable pharmaceutically acceptable salts include acid addition salts such as, without being limited to, the mesylate salt, the maleate salt, the fumarate salt, the tartrate salt, the hydrochloride salt, the hydrobromide salt, the esylate salt, the /?-toluenesulfonate salt, the benzenesulfonate salt, the benzoate salt, the acetate salt, the phosphate salt, the sulfate salt, the citrate salt, the carbonate salt, and the succinate salt.
  • Additional pharmaceutically acceptable salts include salts of ammonium (NH 4 + ) or an organic cation derived from an amine of the formula R 4 N + , wherein each one of the Rs independently is selected from H, Q-C 22 , preferably Ci-C 6 alkyl, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, and the like, phenyl, or heteroaryl such as pyridyl, imidazolyl, pyrimidinyl, and the like, or two of the Rs together with the nitrogen atom to which they are attached form a 3-7 membered ring optionally containing a further heteroatom selected from N, S and O, such as pyrrolydine, piperidine and morpholine.
  • N, S and O such as pyrrol
  • suitable pharmaceutically acceptable salts thereof may include metal salts such as alkali metal salts, e.g., lithium, sodium or potassium salts, and alkaline earth metal salts, e.g., calcium or magnesium salts.
  • Further pharmaceutically acceptable salts include salts of a cationic lipid or a mixture of cationic lipids.
  • Cationic lipids are often mixed with neutral lipids prior to use as delivery agents.
  • Neutral lipids include, but are not limited to, lecithins; phosphatidylethanolamine; diacyl phosphatidylethanolamines such as dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, palmitoyloleoyl phosphatidylethanolamine and distearoyl phosphatidylethanolamine; phosphatidylcholine; diacyl phosphatidylcholines such as dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine and distearoyl phosphatidylcholine; phosphatidylglycerol
  • Examples of cationic lipid compounds include, without being limited to, Lipofectin ® (Life Technologies, Burlington, Ontario) (1 : 1 (w/w) formulation of the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoylphosphatidyl-ethanolamine); LipofectamineTM (Life Technologies, Burlington, Ontario) (3: 1 (w/w) formulation of polycationic lipid 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl] - ⁇ , ⁇ -dimethyl- 1 -propanamin-iumtrifluoroacetate and dioleoylphosphatidyl-ethanolamine), Lipofectamine Plus (Life Technologies, Burlington, Ontario) (Lipofectamine and Plus reagent), Lipofectamine 2000 (Life Technologies, Burlington, Ontario) (Cationic lipid), Effectene
  • the pharmaceutically acceptable salts of the present invention may be formed by conventional means, e.g., by reacting the free base form of the active agent with one or more equivalents of the appropriate acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is removed in vacuo or by freeze drying, or by exchanging the anions of an existing salt for another anion on a suitable ion exchange resin.
  • the present invention encompasses solvates of the various active agents defined above as well as salts thereof, e.g., hydrates.
  • compositions provided by the present invention may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19 th Ed., 1995.
  • the compositions can be prepared, e.g., by uniformly and intimately bringing the active agent, i.e., the compound of the formula I, into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.
  • the compositions may be in liquid, solid or semisolid form and may further include pharmaceutically acceptable fillers, carriers, diluents or adjuvants, and other inert ingredients and excipients.
  • the pharmaceutical composition of the present invention is formulated as nanoparticles.
  • compositions can be formulated for any suitable route of administration, but they are preferably formulated for parenteral administration, e.g., intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, intrapleural, intratracheal or subcutaneous administration, as well as for inhalation.
  • parenteral administration e.g., intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, intrapleural, intratracheal or subcutaneous administration, as well as for inhalation.
  • the dosage will depend on the state of the patient, and will be determined as deemed appropriate by the practitioner.
  • the pharmaceutical composition of the invention may be in the form of a sterile injectable aqueous or oleagenous suspension, which may be formulated according to the known art using suitable dispersing, wetting or suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
  • Acceptable vehicles and solvents include, without limiting, water, Ringer's solution and isotonic sodium chloride solution.
  • compositions according to the present invention when formulated for inhalation, may be administered utilizing any suitable device known in the art, such as metered dose inhalers, liquid nebulizers, dry powder inhalers, sprayers, thermal vaporizers, electrohydrodynamic aerosolizers, and the like.
  • compositions according to the present invention when formulated for administration route other than parenteral administration, may be in a form suitable for oral use, e.g., as tablets, troches, lozenges, aqueous, or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active agent(s) in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets.
  • excipients may be, e.g., inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, e.g., corn starch or alginic acid; binding agents, e.g., starch, gelatin or acacia; and lubricating agents, e.g., magnesium stearate, stearic acid, or talc.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate
  • granulating and disintegrating agents e.g., corn starch or alginic acid
  • binding agents e.g., starch, gelatin or acacia
  • lubricating agents e.g.,
  • the tablets may be either uncoated or coated utilizing known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated using the techniques described in the US Patent Nos. 4,256, 108, 4, 166,452 and 4,265,874 to form osmotic therapeutic tablets for control release.
  • the pharmaceutical composition of the invention may also be in the form of oil-in-water emulsion.
  • compositions of the invention may be formulated for controlled release of the active agent.
  • Such compositions may be formulated as controlled- release matrix, e.g., as controlled-release matrix tablets in which the release of a soluble active agent is controlled by having the active diffuse through a gel formed after the swelling of a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastro-intestinal fluid (in vivo).
  • Many polymers have been described as capable of forming such gel, e.g., derivatives of cellulose, in particular the cellulose ethers such as hydroxypropyl cellulose, hydroxymethyl cellulose, methylcellulose or methyl hydroxypropyl cellulose, and among the different commercial grades of these ethers are those showing fairly high viscosity.
  • compositions comprise the active agent formulated for controlled release in microencapsulated dosage form, in which small droplets of the active agent are surrounded by a coating or a membrane to form particles in the range of a few micrometers to a few millimeters.
  • Another contemplated formulation is depot systems, based on biodegradable polymers, wherein as the polymer degrades, the active agent is slowly released.
  • the most common class of biodegradable polymers is the hydrolytically labile polyesters prepared from lactic acid, glycolic acid, or combinations of these two molecules.
  • Polymers prepared from these individual monomers include poly (D,L-lactide) (PLA), poly (glycolide) (PGA), and the copolymer poly (D,L-lactide-co-glycolide) (PLG).
  • compositions of the present invention are useful for prevention, treatment or management of a disease, disorder or condition mediated by redox stress as defined above.
  • treatment refers to administration of a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof, after the onset of symptoms of said disease, disorder or condition, regardless of the cause for that medical condition.
  • prevention refers to administration of a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof, prior to the onset of symptoms of said disease, disorder or condition, particularly to patients at risk for such disorder or condition; and the term “management” as used herein refers to prevention of recurrence of said disease, disorder or condition in a patient previously suffered from that medical condition.
  • the present invention thus relates to a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof, for use in prevention, treatment or management of a disease, disorder or condition mediated by redox stress.
  • Particular such compounds are those herein identified as compounds 1-18, e.g., R-911, or pharmaceutically acceptable salts or solvates thereof.
  • the present invention relates to use of a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof, for the preparation of a pharmaceutical composition for prevention, treatment or management of a disease, disorder or condition mediated by redox stress.
  • Particular such compounds are those herein identified as compounds 1-18, e.g., R-911, or pharmaceutically acceptable salts or solvates thereof.
  • the present invention relates to a method for prevention, treatment or management of a disease, disorder or condition mediated by redox stress in a subject, e.g., an individual, in need thereof, comprising administering to said subject an effective amount of a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof.
  • a compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof.
  • Particular such compounds are those herein identified compounds 1-18, e.g., R-911, or pharmaceutically acceptable salts or solvates thereof.
  • an effective amount refers to the quantity of the compound of the formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof, that is useful to prevent, treat, or manage the particular disease, disorder or condition that is to be prevented, treated or managed in said individual, and may thus, in fact, be a “prophylactically effective amount” or "therapeutically effective amount”.
  • tetra-peptide prodrug R-911 was assembled as described below, following the linear synthesis shown in Scheme 1 (see Appendix) performed from the commercially available staring materials Boc-L-Cys(trt)-OH, Boc-L-Pro-OH, Fmoc-L-Cys(trt)-OH and Fmoc-Pro-OH.
  • step 1 N-Boc-(S-trityl)-Cys(OH) (a; 7.6 gm, 0.016 mol) was suspended in dry acetonitrile (50 ml). Diisopropylethylamine (4 ml, 0.025 mol) and iodoethane (2.6 ml, 0.032 mol) was added. After the addition, the reaction mixture became clear. It was stirred at RT for 2 days. Additional, 2.6 ml of iodoethane was added and stirred further for 6 hours. The reaction mixture was concentrated. Water (200 ml) and EtOAC (200 ml) was added. N-Boc-(S-trityl)-Cys(OEt) (b) was collected, dried on Na 2 S0 4 and concentrated (7.7 gm).
  • step 2 N-Boc-(S-trityl)-Cys(OEt) (7.5 gm) was dissolved in dry EtOAc (40 ml). Solution of HCl in dioxane (4M, 5 eq) was added, and the reaction was stirred at RT until the starting material disappeared. The reaction mixture was concentrated on rotary evaporator at 40°C. Residue was washed with hexane (50 ml) to remove the trityl chloride impurity, and was then dried under vacuum (-100% yield of crude residue); and the material was used as such for the next reaction.
  • step 3 N-Boc-Pro(OH) (3.5 gm, 0.016 mol) was suspended in dry CH 2 C1 2 (50 ml). ⁇ , ⁇ '-dicyclohexylcarbodiimide (DCC; 3.350 gm) and N-methyl morpholine (NMM, 3 eq.) was added. The mixture was stirred at RT for 30 min. To the above mixture, a solution of NH 2 -(S-trityl)-Cys(OEt) HCl salt (0.016 mol) in dichloromethane (25 ml) was added, and the reaction was stirred at RT until the starting material disappeared ( ⁇ 4 hours).
  • step 4 d (7.3 gm) was dissolved in dry EtOAc (50 ml) and 4M solution of HCl in dioxane (15.5 ml, 0.062 mol) was added. The reaction was stirred at RT until the starting material disappeared. The reaction mixture was concentrated on rotary evaporator at 50°C. Residue was washed with hexane (50 ml) to remove the trivial amount of trityl chloride impurity and HCl, and was then dried under vacuum to remove the excess HCl from product (e).
  • step 5 a suspension of Fmoc-Cys(OH)-S-Trt (730 mg, 1 eq), DCC (260 mg), NMM (3 eq) in CH 2 C1 2 (20 ml) was stirred at RT for 30 min. Intermediate e HCl salt (650 mg, 0.0012 mol) was added and stirred at RT for overnight. The reaction mixture was concentrated. EtOAC (20 ml) was added and the DCC urea was filtered. The filtrate was washed with water, NaHC0 3 and brine (to reduce the emulsion), and was then dried on sodium sulfate and concentrated. The residue was purified on silica gel column using 50% EA-hexane (820 mg).
  • step 6 intermediate f (820 mg) was dissolved in acetonitrile (15 ml) and diethylamine (5 ml) was added. The mixture was stirred at RT for 2 hours, and concentrated on rotary evaporator. The residue was purified on silica gel column 5% MeOH-CH 2 Cl 2 , which provided the amino intermediate g (425 mg).
  • step 7 intermediate g HCl salt (27 gm mixture of two isomers) was dissolved in EtOAC (200 ml) and NaHC0 3 (200 ml), and the solution was stirred for 5-10 min. Chloroacetyl chloride (3 eq) was slowly added. The reaction mixture was stirred for 1 hour. EtOAC layer was separated, and washed with water. It was dried on Na 2 S0 4 and concentrated. The residue was purified on silica gel column in 3-4% MeOH-CH 2 Cl 2 to provide h as a mixture of isomer 1 (18 gm) and isomer 2 (4 gm).
  • step 8 intermediate h (mixture of isomers, 8 gm) was dissolved in tetrahydrofuran (THF), and a 2M solution of dimethylamine in THF (70 ml) was added. The mixture was stirred at RT for overnight, and was then concentrated. The residue was purified on silica gel column using 3-5% methanol-CH 2 Cl 2 to provide intermediate i (6 gm).
  • step 9 intermediate i (2.2 gm) was dissolved in methylene chloride (25 ml) and triethyl silane (4 eq) was added. The mixture was treated with trifluoroacetic acid (TFA; 4 eq) at RT, and was then stirred for 6 hours. It was concentrated on rotary evaporator. The residue was suspended in hexane (20 ml). Hexane was removed. The residue was washed again with hexane (20 ml) and dried under vacuum to give the TFA salt of R-952 (compound 3), which was used as such for the next reaction.
  • TFA trifluoroacetic acid
  • step 10 triethylamine (4 eq) and acetic anhydride (4 eq) was added to a solution of R-952 (1.2 gm) in methylene chloride (25 ml). The reaction mixture was stirred at RT for 16 hours. Excess triethylamine and acetic anhydride was removed on rotary evaporator. The corresponding crude acetate salt was dissolved in water and purified on C- 18 column using 0.1% solution of acetic acid in water and acetonitrile.
  • Example 2 R-911 is converted under physiological conditions to its corresponding active form
  • Example 4 has prophylactic and therapeutic anti-apoptotic effects in a lung radiation-induced injury model
  • R-911 was delivered IP at a dose of 250 mg/kg/day for 7 days.
  • C57BL/6 male mice were exposed to 14 Gy radiation to the thorax.
  • R-911 125 mg/kg or vehicle (radiation only group) was delivered.
  • a second delivery of R-911 or vehicle was given 4 hours later on the day of radiation and treatments continued twice daily for the next 6 days.
  • Mice were necropsied on Day 7 post-radiation and their lungs examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to assess the percent apoptotic cells.
  • TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
  • R-911 was delivered IP at a dose of 250 mg/kg/day for 6-7 days.
  • C57BL/6 male mice were exposed to 14 Gy radiation to the thorax.
  • group A pre- radiation
  • group B post-radiation
  • treatment was initiated 24 hours after radiation was delivered, and continued 4 hours later and twice daily for the next 5 consecutive days.
  • mice were necropsied on Day 7 post-radiation and their lungs examined by TUNEL assay to assess the percent apoptotic cells.
  • R-911 treatment significantly decreased radiation- induced apoptosis when delivered either pre- or post-radiation.
  • LPS (0.25 mg/25 g mouse) was IP injected to mice in Vehicle and R-911 groups. At 1, 6 and 11 hours post LPS injection, R-911 (100 mg/kg per each dose) was IP administered. Mice in all groups were sacrificed 18 hours post LPS injection. Serum was collected as well as liver, kidney, lung and ileum tissues for further analysis.
  • R-911 protected mice from LPS-induced injury as indicated by multiple biomarkers of LPS pathology. As shown in Table 5, creatinine and BUN levels, important markers of renal health, were reduced, determining a protective effect of R-911 on kidney function.
  • ALT and AST levels which increase during acute liver failure, were also markedly reduced, demonstrating efficacy of R-911 in preventing liver damage. Histological injury was evaluated in lung, kidney and liver, and the histological score was based on a scale between 0 (no damage) and 4 (severe injury). All three yielded scores portraying significant reduction of histological destruction and restoration of tissue architecture. The / ⁇ -values in the chart are the difference between the vehicle control group and R-911 treatment group, all of which suggest data are exceedingly significant (+ value is Standard Error).
  • Example 6 is effective in lung ischemia-reperfusion injury model
  • R-911 protected the lung from VR injury as indicated by multiple biomarkers of the disease.
  • Evidence of improved lung oxygenation was demonstrated by elevation of Pa0 2 (222 mmHg in control group vs. 351 mmHg in R-911 group, /? ⁇ 0.001, Fig. 5).
  • a histological examination of lung tissue revealed a dramatic reduction in injury to the treatment group (2.33 in the treatment group vs. 4.17 in the control group, /? ⁇ 0.001, Fig. 6).
  • MPO concentration, an index of neutrophil infiltration in lung tissue, and MDA concentration, an index of lipid peroxidation are commonly utilized as markers of inflammation in VR injury model. Both significantly displayed the protective effect of R- 911.
  • MPO was 0.83 U/gr in the treatment group, compared to 1.22 U/gr in the control group (p ⁇ 0.001, Fig. 7A).
  • MDA was 283.83 ⁇ /100 mg of wet tissue in treatment group, compared to 450.67 ⁇ /100 mg of wet tissue in the control (p ⁇ 0.001, Fig. 7B).
  • Protein concentration was quantified in BAL, a method used to determine the integrity of the lung epithelium. A significant reduction was found in the protein concentration in the BAL of R- 911 treated group (691.33 mg/ml in control group to 409.5 mg/ml in R-911 treated group, /? ⁇ 0.001, Fig. 8).
  • Wet/dry ratio a method that measures the increase of water in the lungs, is utilized to determine the presence of edema induced by pulmonary injury.
  • the data collected establish R-911 as a potent therapy in improving the symptoms of lung I/R injury and modifying the course of injury by restoring lung function.
  • Example 7 R-911 prevents kidney damage in models of contrast media-induced nephropathy
  • the rats were divided into the following treatment groups: (i) Vehicle (saline); (ii) R-911 10 mg/kg/day; (iii) R-911 30 mg/kg/day; (iv) R-911 100 mg/kg/day; (v) NAC (equimolar to 100 mg/kg/day of R-911); and (vi) Sham operation.
  • a full treatment was divided into three equal portions, which were administered by IP injection during the course of the study.
  • Kidney tissue samples were fixed for 1 week in 10% (w/v) PBS-buffered formaldehyde solution at RT, dehydrated using graded ethanol, and embedded in Paraplast (Sherwood Medicab 1, Mahwah, NJ, USA). Sections were then deparaffinized with xylene, and stained with hematoxylin and eosin. All sections were studied using Axiovision Zeiss (Milan, Italy) microscope. The following morphological criteria were used for scoring: 0, normal kidney; grade 1, minimal edema or infiltration; grade 3, moderate edema and inflammatory cell infiltration without obvious damage to kidney architecture; grade 4, severe inflammatory cell infiltration with obvious damage to kidney architecture. All the histological studies were performed in a blinded fashion.
  • the study included the following treatment groups: (i) Sham (n 3) no contrast media, saline injections 0.5 ml; (ii) Vehicle (saline) injections 0.5 ml; (iii) R-911 300 mg/kg/day (100 mg/kg/dose x3 doses) - each injection in 0.5 ml saline; (iv) NAC 94.5 mg/kg/day (31.5 mg/kg/dose x3 doses) in 0.5 ml saline; (v) R-911 300 mg/kg/day (100 mg/kg/dose x3 doses) in increased volume 10 ml/kg - each injection 2.5 ml saline for a 250g rat; and (vi) increased volume alone 10 ml/kg - each injection 2.5 ml saline for a 250g rat.
  • Example 8 is effective as a rescue therapy in a murine Cl 2 exposure model
  • mice were exposed in a cylindrical glass chamber (4 mice per exposure) that is flushed continuously for 30 minutes at a rate of 2 liters/minute with humidified gas obtained from a calibrated cylinder containing air and 400 ppm Cl 2 . After the end of the 30-minute exposure, the chamber was opened and mice were removed and placed immediately in cages in room air. Thirty minutes after the conclusion of Cl 2 exposure, mice were administered IP with 300 mg/kg/dose of R-911 in 200 ⁇ . At 24 hours post-exposure to the Cl 2 -containing air, a midline incision from the neck to the pubis was created for access to the chest and abdominal cavities.
  • the pulmonary circulation was flushed through the main pulmonary artery with 20 ml of normal saline.
  • the lungs were separated from the mediastinal tissues and were taken for histological examination (H&E staining).
  • H&E staining The following morphological criteria were used for scoring: 0, normal lung; grade 1, minimal edema or infiltration of alveolar or bronchiolar walls; grade 3, moderate edema and inflammatory cell infiltration without obvious damage to lung architecture; grade 4, severe inflammatory cell infiltration with obvious damage to lung architecture. All the histological studies were performed in a blinded fashion.
  • Example 9 is effective as a rescue therapy in a ventilator- induced lung injury model
  • MPO was measured and lung histology was assessed.
  • lung tissue samples were fixed for 1 week in 10% (w/v) PBS-buffered formaldehyde solution at room temperature, dehydrated using graded ethanol, and embedded in Paraplast (Sherwood Medicab 1, Mahwah, NJ, USA). Sections were then deparaffinized with xylene, and stained with hematoxylin and eosin. All sections were studied using Axiovision Zeiss (Milan, Italy) microscope.
  • Example 10 is effective as a therapy in a renal ischemia reperfusion injury model
  • the rats were divided into the following treatment groups: (i) Vehicle (saline); (ii) R-911 30 mg/kg loading dose, 10 mg/kg/hr infusion rate over; (iii) R-911 100 mg/kg loading dose, 33 mg/kg/hr infusion rate; (iv) R-911 300 mg/kg loading dose, 100 mg/kg/hr infusion rate; and (v) Sham operation.
  • R-911 (or vehicle) dose was administered 5 minutes prior to reperfusion, and subsequent infusions immediately followed reperfusion. Blood samples were drawn at 6 and at 24 hours, when the animals were terminated, to determine plasma creatinine levels. At termination, kidneys were removed and samples were fixed for histological examination. As shown in Fig. 18, administration of R-911 before reperfusion profoundly attenuated elevations in plasma creatinine in a dose dependent manner (Fig. 18). Histologic studies (Fig. 19) revealed that R-911 provided near total protection against severe renal tubular necrosis. These results indicate that R-911 can be a useful therapy for preventing RIRI.
  • Example 11 reduces infarct size and myocardial damage in a rodent model of myocardial ischemia-reperfusion injury
  • the LAD was re-occluded, and 1 ml of Evans blue dye (2% wt/vol) was injected to the animal via the jugular vein.
  • the area at risk i.e., the non-perfused and thus non-stained myocardium, was separated from the non-ischemic tissue and expressed as a percentage of the left ventricle.
  • the tissue from the are at risk staining with p-nitroblue tetrazolium was separated from the infarcted tissue and weighed, and the infarct size was expressed as a percentage of the are at risk.
  • Fig. 20 shows that R-911 significantly reduced tissue infarction, myocardial MPO, and lipid peroxidation.
  • Fig. 21A shows that MIRI induced severe myocyte necrosis, edema and neutrophil infiltration, compared to a sham control, and administration of R-911 prior to reperfusion profoundly attenuated virtually all histologic features of injury.
  • Fig. 21B presenting histological injury scores, indicates a dramatic attenuation of injury by R- 911 (40%, /? ⁇ 0.0003 in a 2-tailed t-test). These results indicate that R-911 can be a useful therapy for preventing MIRI.

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Abstract

La présente invention concerne des promédicaments mimétiques de la thiorédoxine (TRX), plus particulièrement, des dérivés ou des analogues de la séquence d'acides aminés Cys-Pro-Cys ou A1-Cys-A2-Pro-A3-Cys-A4 dans laquelle chaque A1 à A4 est indépendamment absent ou représente un résidu d'acide aminé, ainsi que des compositions pharmaceutiques les comprenant. Ces composés, après hydrolyse en conditions physiologiques, sont convertis en séquence Cys-Pro-Cys ou A1-Cys-A2-Pro-A3-Cys-A4 native, et sont donc utiles pour la prévention, le traitement ou la prise en charge de maladies, troubles ou affections médiées par un stress oxydatif.
PCT/IB2015/055307 2014-07-14 2015-07-14 Promédicaments mimétiques de la thiorédoxine et leurs utilisations WO2016009341A1 (fr)

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Publication number Priority date Publication date Assignee Title
US11333384B1 (en) 2020-03-05 2022-05-17 Trane International Inc. Systems and methods for adjusting detected temperature for a climate control system

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11333384B1 (en) 2020-03-05 2022-05-17 Trane International Inc. Systems and methods for adjusting detected temperature for a climate control system

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