WO2011150493A1 - Peptides pénétrant dans les mitochondries comme vecteurs d'antimicrobiens - Google Patents

Peptides pénétrant dans les mitochondries comme vecteurs d'antimicrobiens Download PDF

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WO2011150493A1
WO2011150493A1 PCT/CA2011/000609 CA2011000609W WO2011150493A1 WO 2011150493 A1 WO2011150493 A1 WO 2011150493A1 CA 2011000609 W CA2011000609 W CA 2011000609W WO 2011150493 A1 WO2011150493 A1 WO 2011150493A1
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compound
antimicrobial
mpp
mtx
fxr3
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PCT/CA2011/000609
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English (en)
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Shana Kelley
Mark Pereira
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The Governing Council Of The University Of Toronto
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Priority to US13/700,988 priority Critical patent/US9173952B2/en
Priority to CA2800730A priority patent/CA2800730C/fr
Priority to EP11789002.0A priority patent/EP2576594B1/fr
Publication of WO2011150493A1 publication Critical patent/WO2011150493A1/fr

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    • 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
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to cell-permeable peptides that localize to the mitochondria and their use as carriers for antimicrobials.
  • Resistance of a bacterium to antibiotics can arise through various mechanisms including modulation of intracellular concentration of the drug through efflux pumps, hindrance of drug influx (e.g. through biofilm formation), enzymatic inactivation, or through modification of the target of the drug.
  • 1 ' 2 Very few classes of antimicrobials have been marketed in the past 46 years (oxazolidinones 3 and lipopeptides 4 ). At this pace drug research and discovery may not be able to maintain the tenuous hold that we have on infectious disease. This is particularly evident in the emergence of multi-drug resistant pathogens "superbugs" where health care workers are left with few options for treatment. 5 The Gram-positive Staphylococcal, Streptococcocal, Enterococcal, and now Clostridium pathogens have proven to be a particular challenge in this respect. 6 ' 7
  • a compound comprising a mitochondrial penetrating peptide (MPP) conjugated to an antimicrobial, preferably an antibacterial.
  • MPP mitochondrial penetrating peptide
  • the compound described herein for treating infection by a microbe preferably bacteria, further preferably Gram-positive bacteria.
  • a pharmaceutical composition comprising the compound described herein and a pharmaceutically acceptable carrier.
  • a library of compounds comprising a plurality of the compounds described herein.
  • a method of treating an infection by a microbe, preferably bacteria, further preferably Gram-positive bacteria, in a subject comprising administering to the subject a therapeutically effect amount of the composition described herein.
  • composition described herein for the treatment of infection by a microbe, preferably bacteria, further preferably Gram- positive bacteria.
  • a method of optimizing delivery of an antimicrobial to a microbe, preferably bacteria, further preferably Gram-positive bacteria, comprising conjugating the antimicrobial with a MPP.
  • Figure 1 is a schematic representation of the mechanism of antimicrobial delivery using a mitochondrial targeted peptide-drug conjugate (mt-Mtx) to deliver bacteria- specific toxicity. Conjugation to a mitochondria penetrating peptide increases uptake of small molecule inhibitors into bacteria to reach a protein target. In human cells, however, these molecules are effectively sequestered within mitochondria away from human target reducing toxicity.
  • mt-Mtx mitochondrial targeted peptide-drug conjugate
  • FIG. 2 shows the chemical structure of mitochondrial targeted methotrexate compound.
  • A Structure represents direct conjugation of a mitochondria-penetrating peptide (MPP) to methotrexate (Mtx-Fxr3).
  • B Structure represents conjugation of an MPP to a version of methotrexate modified to have higher levels of cellular uptake (MtxA-Fxr3).
  • Mtx Methotrexate
  • Fxr3 Mitochondrial Targeting peptide
  • Mtx-Fxr3 Mitochondrial targeted methotrexate
  • MtxA-Fxr3 Mitochondrial targeted methotrexate analogue.
  • Figure 5 shows a comparison of antimicrobial prospects of methotrexate and methotrexate-peptide conjugates.
  • the ratio of antimicrobial activity to host cell toxicity indicates that Mtx-Fxr3 is approximately 180,000 fold more selectively toxic to bacteria than to human cells compared to methotrexate alone.
  • MtxA-Fxr3 is approximately 20,000 fold more selectively toxic to bacteria than human cells compared to methotrexate alone.
  • Figure 6 shows that fluorescently labelled Mtx-Fxr3 localizes to the mitochondria of HeLa cells.
  • Mitochondrial staining with commercially available Mitotracker Deep Red 633 shows a similar staining pattern.
  • Mtx-Fxr3-to accumulates within the cytoplasm of E. coli JW5503 cells.
  • DIC differential interference contrast; to, thiazole orange.
  • Figure 7 shows: (A) Relative intracellular concentration of Mtx-Fxr3 in E. coli JW5503 and dose-dependent increases in intracellular drug concentrations. With Mtx treatment alone, observable accumulation of drug was not detectable. (B) Mtx-Fxr3 accumulates at significantly higher levels within HeLa cells compared to unfunctionalized Mtx.
  • Figure 8 shows toxicities and therapeutic index calculations for Mtx-Fxr3 and Mtx for Gram-positive pathogens.
  • C Activity of Mtx-Fxr3 and Mtx against S. pneumoniae (ATCC 49619).
  • D Activity of Mtx-Fxr3 and Mtx against S. aureus (ATCC 29213).
  • Peptide MIC > 32 ⁇ , Mtx-Fxr3 MIC 16 ⁇ , Mtx MIC > 32 ⁇
  • E Activity of Mtx-Fxr3 and Mtx against MRSA (ATCC BAA- 1720).
  • Peptide MIC > 32 ⁇ , Mtx-Fxr3 MIC 8 ⁇ , Mtx MIC > 32 ⁇ .
  • the therapeutic index (ratio of bacterial to human cell toxicity) of Mtx and Mtx-Fxr3 were calculated for each organism. The higher the therapeutic index, the better the compound is as an antimicrobial. The fold differences in the calculated therapeutic indices are indicated.
  • Figure 9 shows analysis of mitochondrial toxicity of Mtx-Fxr3.
  • A Mitochondrial membrane potential was monitored using the mitochondria potential sensitive dye JC-1. Treatment of HeLa cells with Mtx-Fxr3 was found to not perturb the mitochondrial membrane potential. The mitochondrial membrane depolarizer FCCP resulted in a significant decrease in membrane potential.
  • B ATP levels were also monitored to assess mitochondrial function. Treatment with Mtx-Fxr3 did not result in a decrease in cellular ATP levels indicating that mitochondrial function was not perturbed. Rotenone, an inhibitor of the electron transport chain, caused a significant decrease in cellular ATP levels.
  • FIG. 10 shows relative intracellular concentration of Mtx-Fxr3 and Mtx in: (A) S. aureus and (B) E. faecalis. Mtx-Fxr3 accumulated within cells in a dose dependent manner with large increases in uptake at the measured MIC of the compound for that organism. In addition, Mtx-Fxr3 accumulated to a greater extent than Mtx alone.
  • a common peptide transporter can be used for selective uptake within bacteria as well as mitochondria.
  • a compound comprising a mitochondrial penetrating peptide (MPP) conjugated to an antimicrobial, preferably an antibacterial.
  • MPP mitochondrial penetrating peptide
  • antimicrobial means a substance that kills or inhibits the growth of microorganisms, such as bacteria ("antibacteriaF), fungi, or protozoans.
  • the antibacterials disclosed herein preferably inhibit or kill Gram-positive bacteria.
  • Antimicrobial drugs can kill microbes (microbicidal) or prevent the growth of microbes (microbistatic).
  • Antimicrobial includes, but is not limited to, the following: Table 1. List of anti-microbial drugs
  • the antimicrobial has a target in human cells that resides in the cytoplasm. In some embodiments, the antimicrobial is conjugated to the N-terminus of the MPP. In one embodiment, the compound is Mtx-Fxr3 or MtxA-Fxr3.
  • MPPs preferably possess both positive charge and lipophilic character, properties determined herein to be important for passage across both the plasma and mitochondrial membranes.
  • MPPs contain cationic and hydrophobic residues to provide a positively charged lipophilic character that facilitates passage through both the plasma and mitochondrial membranes.
  • Cationic amino acids such as lysine (K), arginine (R), aminophenylalanine, and ornithine may be incorporated within the MPPs to provide positive charge, while hydrophobic residues such as phenylalanine (F), cyclohexylalanine (F x ), aminooctaarginine (Hex), diphenylalanine (F 2 ) and (1 - naphthyl)-L-alanine (Nap), may be incorporated within the MPPs to impart lipophilicity.
  • hydrophobic residues such as phenylalanine (F), cyclohexylalanine (F x ), aminooctaarginine (Hex), diphenylalanine (F 2 ) and (1 - naphthyl)-L-alanine (Nap) may be incorporated within the MPPs to impart lipophilicity.
  • the MPPs may comprise alternating charged and hydrophobic residues to increase the level of lipophilicity within the MPP.
  • MPPs according to the invention may be made using well-established techniques of peptide synthesis, including automated or manual techniques, as one of skill in the art will appreciate.
  • the length of the present MPPs is not particularly restricted but will generally be of a length suitable for transport across plasma and mitochondrial membranes, either alone or conjugated to another entity such as a biological agent as will be described. Generally, the MPPs will be comprised of 4-20 residues.
  • the MPPs may include one or more residues modified to impart on the MPP desirable properties, for example, increased intracellular stability.
  • the MPPs may include d-stereoisomers, and terminal modifications such as amide termini.
  • the MPP can traverse the inner membrane of the mitochondria, preferably in a potential dependent manner.
  • the MPP comprises a charge of +3 and a log P value of at least about -1.7. In other embodiments, the MPP comprises a charge of +5 and a log P value of at least about -2.5.
  • the MPP is any one of SEQ ID NOs. 1-7.
  • the compound described herein for treating infection by a microbe preferably bacteria, further preferably Gram-positive bacteria.
  • a pharmaceutical composition comprising the compound described herein and a pharmaceutically acceptable carrier.
  • a library of compounds comprising a plurality of the compounds described herein.
  • a method of treating an infection by a microbe, preferably bacteria, further preferably Gram-positive bacteria, in a subject comprising administering to the subject a therapeutically effect amount of the composition described herein.
  • a use of the compound described herein in the preparation of a medicament for the treatment of infection by a microbe preferably bacteria, further preferably Gram-positive bacteria.
  • a use of the composition described herein for the treatment of infection by a microbe preferably bacteria, further preferably Gram- positive bacteria.
  • a method of optimizing delivery of an antimicrobial to a microbe, preferably bacteria, further preferably Gram-positive bacteria, comprising conjugating the antimicrobial with a MPP.
  • pharmaceutically acceptable carrier means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.
  • therapeutically effective amount refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.
  • the following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
  • EXAMPLES METHODS Cell Culturing Conditions. HeLa cells were cultured in MEM alpha (Invitrogen, Carlsbad) supplemented with 10% (v/v) FBS at 37°C with 5% C0 2 . Escherichia coli strain JW5503 from the Keio collection 12 (F-, A(araD-araB)567, AlacZ4787(::rmB-3), ⁇ " , AtolC732::kan, rph-1, A(rhaD-rhaB)568, hsdR514) was grown on solid LB-agar or in LB liquid media supplemented with 50 ⁇ g/mL kanamycin at 37°C.
  • Strains harboring the pCA24N plasmid were additionally grown with chloramphenicol at 20 ⁇ g/mL.
  • B. subtilis was grown in LB media.
  • S. aureus and E. faecalis were grown in Trypticase Soy Broth (TSB).
  • S. pneumoniae were grown in TSB + 5% defibrinated sheep's blood at 37°C in an atmosphere of 5% C0 2 .
  • DIPEA N,N- diisopropylethylamine
  • DMF N,N- dimethyl formamide
  • Methotrexate was coupled onto the peptide N-terminus as described. 15 Briefly, Fmoc-L-glutamic acid-a-tert butyl ester was coupled to on resin petide as described above. APA (4 eq) was coupled using N-hydroxybenzotriazole (HOBt, 2 eq), HBTU (4 eq) and DIPEA (8 eq) for 1 hour at room temperature. APA was allowed to activate for 5 minutes prior to addition to resin.
  • MtxA was synthesized by using a Fmoc-L-glutamic acid-y-tert butyl ester that had been coupled to rink amide resin, and cleaved to yield an amide on the alpha carbon and a deprotected gamma carbon. This product was precipitated in cold ether, confirmed by electrospray ionization mass spectroscopy and used without futher purification. Peptides were deprotected and cleaved from the resin using TFA:triisopropylsilane:H 2 0 (95:2.5:2.5) and precipitated in cold ether.
  • HeLa cells were cultured as described above. Cells were seeded in 96-well flat bottom tissue culture plates (Starsted, NC) at a density of 1500 cells per well. The culture media was removed and cells were washed with MEM- alpha (minus nucleotides). Concentrated peptide stocks were diluted in MEM-alpha (minus nucleotides) + 10% dialyzed FBS and incubated with cells for 72-96 hours. Cellular viability was analyzed after the indicated times using the CCK-8 viability dye (Dojindo, Rockville, MD) at an absorbance of 450 nm. Analysis of Toxicity (Bacteria).
  • MIC Minimum inhibitory concentration
  • Mem-alpha minus nucleotides (Invitrogen, Carlsbad CA) was used to control for variability in thymidine content of undefined media. The final dilution into media was at 1 :2000 for S. pneumoniae, and E. faecalis. 3% laked horse blood was added to the media for S. pnemoniae.
  • Mtx concentrations were determined using a modification of a protocol described previously. 16 HeLa cells. Cells were seeded in a 12-well plate at a density of 100,000 cells per well one day prior to experiments. Cells were treated with indicated amounts of compound in OPTI-MEM for 4 hours at 37°C. Treated cells were washed 2x with PBS and lysed with RIPA buffer. To a 100 ⁇ sample the following was added: 30 ⁇ acetic acid/Na acetate buffer (pH 5.0, 0.5M) ,15 ⁇ . KMn0 4 (1 mM), and 5 ⁇ xL ascorbic acid (60 ⁇ g/mL) and incubated 20 minutes in the dark.
  • Mtx concentrations were determined as described above. Uptake in S. aureus and E. faecalis were analysed similarly with the exception that TSB media was used for growth and lysis occurred in 0.4% SDS for 10 minutes at 100°C. Measurement of Cellular ATP levels. HeLa cells were seeded at a density of 50,000 cells per well one day prior to experiments. Cells were treated for 16 hours with compounds Mtx-Fxr3 (16 ⁇ ) and Mtx (16 ⁇ ) and 1 hour for rotenone (100 nM) in OPTI-MEM media at 37°C. After treatment, cells were washed with PBS, harvested with trypsin/ EDTA for 7 min at 37°C.
  • HeLa cells were seeded in at 6,250 cells per well in 24 well plates. Cells were treated with Mtx-Fxr3 (16 ⁇ ) and Mtx (16 ⁇ ) for 72 hours in Mem Alpha media with nucleotides and 10% FBS. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was added at 10 ⁇ for 6 minutes prior to analysis to disrupt the mitochondrial membrane potential.
  • FCCP Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone
  • the mitchondrial membrane potential sensitive dye JC-1 (Invitrogen, Carlsbad CA) was added at 5 ⁇ g/mL for 10 minutes at 37°C. Cells were washed with PBS and harvested as described above.
  • Mtx-Fxr3 Elimination from Cells. Hela cells were incubated with 5 ⁇ of a fluorophore labeled version of Mtx-Fxr3 (Mtx-Fxr3-to) in OPTI-MEM for 45 minutes. After incubation, cells were washed and the OPTI-MEM media was refreshed. Cells were analyzed at the indicated times for retention of the Mtx-Fxr3-to compound by flow cytometry on a BD FACSCanto flow cytometer (BD Biosciences). The excitation wavelength of 488 nm and emmission wavelength of 530 nm was used. A minimum of 10,000 cells were analyzed per sample. The fluorescence median of the live population was used to determine intracellular compound levels. Table 2. List of Peptide Conjugates
  • the developed drug delivery system utilizes a peptide transporter to effectively increase the accumulation of antibacterial agents into bacteria, potentiating their antibacterial activity as well as preventing host toxicity by preventing the antibacterial agent from interacting with potential human targets. These goals have been attained though the use of a peptide transporter that shows efficient cellular uptake and specific mitochondrial localization. 8 Mitochondrial penetrating peptides are described in Horton, K.L., Stewart, K.M., Fonseca, S.B., Guo, Q. & Kelley, S.O. Mitochondria
  • the drug-peptide conjugates described in this invention have two unique features:
  • the peptide domain augments the uptake of drug into bacteria to increase its effective concentration; and 2.
  • the peptide also decreases host cell toxicity by sequestering drugs in the mitochondria away from its cytoplasmic target.
  • Methotrexate was delivered to each of bacteria and human cells. Methotrexate is an anti-neoplastic agent used in cancer chemotherapy and has appreciable human toxicity. In addition, this molecule only has minimal antibacterial activity against Gram-positive bacteria. These two reasons limit its use to applications that do not include antimicrobial therapy. Two molecules were chemically synthesized to test the utility of MPPs in increasing methotrexate's antimicrobial prospects and are depicted in Figure 2.
  • Mtx-Fxr3 is a covalent conjugation of a MPP to methotrexate, 9 ' 10 while MtxA-Fxr3 is a modified version engineered to increase cellular uptake.
  • methotrexate the enzyme dihydrofolate reductase
  • the target of methotrexate is only found in the cytoplasm and nucleus of human cell and not within the mitochondrion. Sequestration of methotrexate within the mitochondrion should abolish the toxicity of the methotrexate.
  • cellular viability of a HeLa cell line after 72-hour incubation with these molecules was evaluated. It was found that through conjugation of methotrexate to an MPP, the toxicity of methotrexate was reduced by 1000-fold (Figure 4). Mtx had a 50% lethal dose of 0.015 ⁇ while the 50% lethal dose for Mtx-Fxr3 was greater than 16 ⁇ .
  • Mtx-Fxr3 In order to evaluate the intracellular localization of Mtx-Fxr3 in human cells, a fluorescently labelled compound was synthesized Mtx-Fxr3-to. This molecule incorporates the fluorophore thiazole orange. As shown in Figure 6A, the Mtx-Fxr3 localizes preferentially to the mitochondria of human cells, sequestered away from the cytoplasmic target DHFR.
  • Escherichia coli JW5503 that lacks the tolC gene that can cause drug efflux was used as a model strain. The use of this strain presented the opportunity to observe where the drug conjugate would localize once inside a bacterial cell in the absence of competing efflux. When introduced into E. coli JW5503, the drug exhibited a distinct cytoplasmic localization ( Figure 6B).
  • Mtx-Fxr3 The impact of Mtx-Fxr3 on mitochondrial function and health was assessed. Decreases in mitochondrial membrane potential and decreases in cellular ATP levels are phenotypes associated with mitochondrial toxicity. Treatment of HeLa cells with Mtx- Fxr3 did not affect mitochondrial membrane potential or cellular ATP levels ( Figure 9 A and 9B). Moreover, upon removal of compound from the cell media intracellular levels of the compound decreased in a time dependent manner ( Figure 9C), with > 50% of the compound cleared from the cell in 24 hours. A series of bacterial species, many of which are clinically relevant, were then evaluated. A panel of Gram-positive strains was selected, and activity was observed across a variety of organisms.
  • Mtx-Fxr3 In Enterococcus faecalis, Mtx-Fxr3 exhibited comparable activity to the parent compound, but given the attenuated activity in human cells, its therapeutic index was > 10. This represents a > 3000-fold improvement over the parent drug (Figure 8A). In Bacillus subtilis, Mtx-Fxr3 was significantly more toxic than unmodified Mtx, which produced > 10,000 fold improvement in the therapeutic index of the DHFR inhibitor ( Figure 8B). In Streptococcus pneumoniae, Mtx was very active, but the MIC was in the range where significant toxicity occurs in human cells, leading to a therapeutic index ⁇ 1.
  • mitochondrial localization was rendered non-toxic to mammalian cells.
  • Mitochondrial localization was imparted to methotrexate by attaching a mitochondria-penetrating peptide, which sequestered the drug from its enzymatic target.
  • Mtx remained active when conjugated to a mitochondrial targeting vector, and exhibited high levels of activity in the presence of clinically-relevant Gram-positive pathogens.
  • the combination of the antibacterial activity with lowered mammalian cell toxicity produced large improvements in the therapeutic indices for the DHFR inhibitor.
  • 5-fluorouracil is a thymidine analogue that inhibits the cytoplasmic target thymidylate synthase, and is used in cancer therapy. As thymidylate synthase is only found in the cytoplasm and the nucleus, conjugation to an MPP shows great promise to repurpose this molecule as an antibacterial agent.
  • Azidothymidine is a therapeutic used in the treatment of HIV that requires enzymatic activation within the cytoplasm of cells to act as an inhibitor of DNA synthesis. Sequestering azidothymidine in the mitochondria will prevent cytoplasmic phosphorylation reactions necessary for activation decreasing the toxicity of the molecule in human cells. Each of these molecules is a promising candidate for conjugation to a mitochondria-penetrating peptide for the basis of antimicrobial design.
  • MTX antineoplastic methotrexate

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Abstract

La présente invention concerne des composés comprenant un peptide pénétrant dans les mitochondries (MPP) conjugué à un antimicrobien et leur procédé d'utilisation.
PCT/CA2011/000609 2010-05-30 2011-05-27 Peptides pénétrant dans les mitochondries comme vecteurs d'antimicrobiens WO2011150493A1 (fr)

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CA2800730A CA2800730C (fr) 2010-05-30 2011-05-27 Peptides penetrant dans les mitochondries comme vecteurs d'antimicrobiens
EP11789002.0A EP2576594B1 (fr) 2010-05-30 2011-05-27 Peptides pénétrant dans les mitochondries comme vecteurs d'antimicrobiens

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US20160151446A1 (en) * 2013-06-27 2016-06-02 Stealth Biotherapeutics Corp Peptide therapeutics and methods for using same

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WO2013169060A1 (fr) 2012-05-11 2013-11-14 주식회사 카엘젬백스 Composition pour la prévention ou le traitement d'une sepsie
US10967000B2 (en) 2012-07-11 2021-04-06 Gemvax & Kael Co., Ltd. Cell-penetrating peptide, conjugate comprising same and composition comprising same
KR102201430B1 (ko) * 2012-09-19 2021-01-12 주식회사 젬백스앤카엘 세포 투과성 펩티드, 그를 포함한 컨쥬게이트 및 그를 포함한 조성물
JP6517692B2 (ja) 2012-09-19 2019-05-22 ジェムバックス アンド カエル カンパニー，リミティド 細胞透過性ペプチド、それを含んだコンジュゲート、及びそれを含んだ組成物
ES2716870T3 (es) 2013-04-19 2019-06-17 Gemvax & Kael Co Ltd Composición para el tratamiento y prevención de lesión isquémica
CA2912557A1 (fr) 2013-06-07 2014-12-11 Gemvax & Kael Co., Ltd. Marqueurs biologiques utiles en immunotherapie du cancer
KR102166545B1 (ko) 2013-06-21 2020-10-16 주식회사 젬백스앤카엘 호르몬 분비 조절제, 이를 포함하는 조성물, 및 이를 사용한 호르몬 분비 조절 방법
WO2015009678A2 (fr) * 2013-07-15 2015-01-22 The Scripps Research Institute Ciblage spécifique de séquences répétées étendues d'arn
KR101691479B1 (ko) 2013-10-23 2017-01-02 주식회사 젬백스앤카엘 전립선 비대증 치료 및 예방용 조성물
KR102494803B1 (ko) 2013-11-22 2023-02-06 주식회사 젬백스앤카엘 혈관 신생 억제 활성을 가지는 펩티드 및 이를 포함하는 조성물
ES2809251T3 (es) 2013-12-17 2021-03-03 Gemvax & Kael Co Ltd Composición para tratar cáncer de próstata
CN106456697B (zh) 2014-04-11 2019-12-17 珍白斯凯尔有限公司 具有纤维化抑制活性的肽和含有其的组合物
ES2962532T3 (es) 2014-04-30 2024-03-19 Gemvax & Kael Co Ltd Composición para el trasplante de órganos, tejidos o células, kit y procedimiento de trasplante
KR102413243B1 (ko) 2014-12-23 2022-06-27 주식회사 젬백스앤카엘 안질환 치료 펩티드 및 이를 포함하는 안질환 치료용 조성물
EP3263122B1 (fr) 2015-02-27 2020-05-06 Gemvax & Kael Co., Ltd. Peptide pour prévenir la perte de l'audition et composition le comprenant
JP7000161B2 (ja) 2015-05-26 2022-01-19 ジェムバックス アンド カエル カンパニー，リミティド 新規ペプチド及びこれを含む組成物
US11015179B2 (en) 2015-07-02 2021-05-25 Gemvax & Kael Co., Ltd. Peptide having anti-viral effect and composition containing same
EP3441082B1 (fr) 2016-04-07 2023-06-21 Gemvax & Kael Co., Ltd. Peptide ayant des effets d'augmentation de l'activité de la télomérase et d'extension de télomère, et composition le contenant
EP4365590A2 (fr) 2019-03-20 2024-05-08 Juntendo Educational Foundation Agent ciblant l'adn d'organite à double membrane

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009036092A2 (fr) * 2007-09-10 2009-03-19 University Of Massachusetts Agents antitumoraux ciblant les mitochondries

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009036092A2 (fr) * 2007-09-10 2009-03-19 University Of Massachusetts Agents antitumoraux ciblant les mitochondries

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CHEM BIOL, vol. 15, 2008, pages 375 - 82
ELLERBY, H.M. ET AL.: "Anti-cancer activity of targeted pro-apoptotic peptides.", NATURE MEDICINE., vol. 5, no. 9, September 1999 (1999-09-01), pages 1032 - 1038, XP002929457 *
HORTON, K. L. ET AL.: "Mitochondria-penetrating peptides.", CHEMISTRY & BIOLOGY, vol. 15, no. 4, April 2008 (2008-04-01), pages 375 - 382, XP022613220 *
PEREIRA, M. P. ET AL.: "Maximizing the therapeutic window of an antimicrobial drug by imparting mitochondrial sequestration in human cells.", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 133, no. 10, 16 March 2011 (2011-03-16), pages 3260 - 3263, XP055101330 *
See also references of EP2576594A4 *
UEHARA, Y.: "Natural product origins of Hsp90 inhibitors.", CURRENT CANCER DRUG TARGETS., vol. 3, no. 5, October 2003 (2003-10-01), pages 325 - 330, XP009021074 *
WIPF, P. ET AL.: "Mitochondrial targeting of selective electron scavengers: synthesis and biological analysis ofhemigramicidin-TEMP conjugates.", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 127, no. 36, 14 September 2005 (2005-09-14), pages 12460 - 12461, XP055018693 *
YOUSIF L. F. ET AL.: "Mitochondria-penetrating peptides: sequence effects and model cargo transport.", CHEMBIOCHEM : A EUROPEAN JOURNAL OF CHEMICAL BIOLOGY., vol. 10, no. 12, 17 August 2009 (2009-08-17), pages 2081 - 2088, XP055101341 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160151446A1 (en) * 2013-06-27 2016-06-02 Stealth Biotherapeutics Corp Peptide therapeutics and methods for using same
US10293020B2 (en) * 2013-06-27 2019-05-21 Stealth Biotherapeutics Corp. Peptide therapeutics and methods for using same

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