WO2011150494A1 - Peptides pénétrant dans les mitochondries comme vecteurs de composés anticancéreux - Google Patents

Peptides pénétrant dans les mitochondries comme vecteurs de composés anticancéreux Download PDF

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WO2011150494A1
WO2011150494A1 PCT/CA2011/000610 CA2011000610W WO2011150494A1 WO 2011150494 A1 WO2011150494 A1 WO 2011150494A1 CA 2011000610 W CA2011000610 W CA 2011000610W WO 2011150494 A1 WO2011150494 A1 WO 2011150494A1
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compound
cbl
mpp
cells
anticancer
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PCT/CA2011/000610
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English (en)
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Shana Kelley
Mark Pereira
Sonali Fonseca
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The Governing Council Of The University Of Toronto
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Priority to CA2800741A priority Critical patent/CA2800741C/fr
Priority to EP11789003.8A priority patent/EP2576595B1/fr
Priority to US13/700,970 priority patent/US9132198B2/en
Priority to CN201180037346.3A priority patent/CN103097397B/zh
Publication of WO2011150494A1 publication Critical patent/WO2011150494A1/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 anticancer compounds.
  • the energy-producing capacity of mitochondria is contingent on the preservation of a barrier limiting the permeation of ions or other small molecules.
  • the highly hydrophobic, densely packed structure of the inner mitochondrial membrane is impenetrable to most molecular species - a property critical for the proton pumping that directs oxidative phosphorylation 1 .
  • the impermeability of the inner membrane has impeded the delivery of drug molecules that could target the other important biological role of mitochondria - apoptotic triggering . Given that apoptotic resistance is
  • a compound comprising a mitochondrial penetrating peptide (MPP) conjugated to an anticancer compound.
  • MPP mitochondrial penetrating peptide
  • composition comprising the compound described herein and a pharmaceutically acceptable carrier.
  • a library of compounds comprising a plurality of compounds described herein.
  • a method of treating cancer comprising administering to the subject a therapeutically effect amount of the composition described herein.
  • composition described herein for the treatment of cancer.
  • a method of inducing apoptosis in a cancer cell comprising administering a therapeutically effect amount of the composition described herein.
  • Figure 1 shows mitochondrial localization and toxicity of mt-Cbl.
  • A The structure of fluorescently labeled mt-Cbl conjugate used in studies of mitochondrial drug localization. In all other assays, an acetyl group replaced the thiazole orange (to) fluorophore on the peptide N-terminus.
  • B Localization of MPP in mitochondrial matrix as observed by immunogold staining and TEM imaging of isolated mitochondria. Gold nanoparticles corresponding to the locations of biotinylated peptides within isolated mouse mitochondria are observed (note that the electron dense, darker regions represent the mitochondrial matrix under the conditions used for staining).
  • C Quantitation of the results for 100s of mitochondria provide quantitative evidence for predominant matrix localization.
  • FIG. 2 shows Cbl and mt-Cbl induce DNA damage in different organelles. Relative amplification of 17.7 kb nuclear and 8.9 kb mitochondrial DNA segments. HL60 cells were treated with Cbl (150 ⁇ ) or mt-Cbl (3 ⁇ ) for 2 hr prior to PCR analysis. Lesions/ 10 kb values are included above the graph bars.
  • Figure 3 shows differing gene expression profiles in response to DNA damage by Cbl and mt-Cbl.
  • B Results from qPCR array highlighting hits with greater than l-fold change in expression.
  • Figure 4 shows the evaluation of MPP-Cbl activity and therapeutic window in primary CLL cells.
  • Cbl and MPP-Cbl activity evaluated at LC25 and LC50 in red blood cells (RBCs) (healthy donors), peripheral blood stem cells (PBSCs) (healthy donors), mononuclear cells (healthy donors) and CLL patient cells. Both Cbl and MPP-Cbl were more selectively toxic to CLL patient samples cells compared to those derived from healthy donors. Percent viability was determined by FACS analysis of Annexin V/Sytox Red cell staining. Hemolytic activity of RBCs with Cbl and MPP-Cbl was found to be minimal at the concentrations used in this experiment.
  • Figure 5 shows the toxicity of MPP-Cbl in cell lines exhibiting drug resistance and apoptotic resistance.
  • A Toxicity of MPP-Cbl towards a panel of leukemia cell lines (M2, K562, HL60 and U937). Mean values plotted, n>3, error bars are s.e.m.
  • Inset FACS analysis of HL60 Annexin V-FITC (A-FITC) / Sytox Red (SR) cell staining after treatment with 6 ⁇ MPP-Cbl.
  • A-FITC-/SR- Alive
  • FITC+/SR- Apoptotic
  • A-FITC-/SR+ Necrotic
  • A-FITC+/SR+ Dead
  • Dead Toxicity of Cbl with leukemia cell lines showed two distinct populations with HL60 and U937 being sensitive to Cbl and K562 and M2 being resistant to Cbl. Inset same as part (a) but with 34 ⁇ Cbl.
  • C Apoptotic resistance in Cbl-resistant cell lines. Response of leukemia cell lines to staurosporine as measured by FACS analysis of Annexin V / Sytox Red stained cells (black bars). Graphed are percent viable cells.
  • Figure 6 shows a Western blot of Bcl XL and /3-actin levels in leukemia cell lines. Western blot analysis was performed as described above. Total protein levels were determined via BCA assay and equal protein was loaded in each well as seen with ⁇ - actin loading control.
  • Figure 7 shows a comparison of LC50 in A2780 wildtype cells between Cbl and MPP- Cbl. A2780 wildtype cells were treated with Cbl (right curve) and MPP-Cbl (left curve) as described above. Toxicity was analyzed with the CCK8 assay.
  • FIG. 8 shows DNA alkylation by mt-Cbl.
  • Cbl retains the MPP in mitochondria after cellular fixation. Fluorescently labeled mt-Cbl and the control peptide (MPP) were incubated with live HeLa cells and mitochondrial localization was observed in unfixed cells. Under fixation and permeabilization conditions, mt-Cbl maintained mitochondrial localization while the MPP diffused to the nucleus and cytoplasm. Following this, cells were incubated with DNase (10 units) to catalyze the cleavage of DNA. Treatment with DNase results in diffusion of mt-Cbl localization.
  • B Alkylation activity of mtCbl.
  • a compound comprising a mitochondrial penetrating peptide (MPP) conjugated to an anticancer compound.
  • MPP mitochondrial penetrating peptide
  • Anticancer compounds includes any substance administered for the treatment of cancer. Typically, the majority of chemotherapeutic drugs can be divided in to alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumour agents.
  • Preferable anticancer agents according to the disclosed aspects and used in connection with MPPs include the following.
  • the anticancer agent is conjugated to the C-terminus of the MPP. In other embodiments, the anticancer agent is conjugated to the N-terminus of the MPP.
  • the compound is F x r3-Cbl.
  • 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 (Fx) aminooctaarginine (Hex), diphenylalanine (F 2 ) and (1- naphthyl)-L-alanine (Nap), may be incorporated within the MPPs to impart lipophilicity.
  • phenylalanine (F), cyclohexylalanine (Fx) 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.
  • 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.
  • composition comprising the compound described herein and a pharmaceutically acceptable carrier.
  • a library of compounds comprising a plurality of compounds described herein.
  • a method of treating cancer comprising administering to the subject a therapeutically effect amount of the composition described herein.
  • composition described herein for the treatment of cancer.
  • a method of inducing apoptosis in a cancer cell comprising administering a therapeutically effect amount of the composition described herein.
  • 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 Tefers 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.
  • HeLa cells were cultured in MEM alpha (Invitrogen, Carlsbad) supplemented with 10% (v/v) FBS at 37 °C with 5% C0 2 .
  • U937 cells were cultured in RPMI 1640 + 10% FBS and Iscove's Modified Dulbecco's Media + 10% FBS was used for OCI-AML2, HL60, K562, OCI-M2, LY17 and Daudi.
  • A2780 Wildtype and Cbl-Resistant Lines were cultured in RPMI 1640 + 10% FBS at 37 °C with 5% C0 2 and the Cbl-resistant line was treated with 100 ⁇ Cbl once a week for 1 hour to maintain resistance.
  • DIPEA N,N- diisopropylethylamine
  • DMF N,N- dimethyl formamide
  • Chlorambucil Sigma-Aldrich, St. Louis
  • HBTU 4 eq
  • DIPEA 4 eq
  • DMF dimethyl formamide
  • the N-terminus of unlabeled peptides was capped using acetic anhydride, pyridine and DCM (1 :5: 10, Sigma).
  • Peptides were deprotected and cleaved from the resin using TFA:triisopropylsilane:H 2 0 (95:2.5:2.5) and precipitated in cold ether.
  • Mitochondria were isolated from fresh mouse liver as previously described 1 . Functionality was confirmed using respirometry. The isolated organelles were used only when the levels of oxygen consumption in state III respiration (presence of ADP) were >4 fold greater than in state II respiration, indicating well-coupled mitochondria. Mitochondrial protein concentration was determined by BCA assay (Sigma). Mitochondria were diluted to 0.5 mg/mL in PBS and incubated for 20 minutes at 25°C with biotin-F x rF x rF x r. Cold PBS was added and mitochondria were pelleted by centrifugation.
  • the pellet was fixed in 1% glutaraldehyde in PBS for 90 min at room temperature, washed with PBS, then fixed with 1% osmium tetroxide for 2 hr at 4°C.
  • the pellets were dehydrated using graded ethanol, followed by stepwise infiltration with propylene oxide and Epon-Araldite resin.
  • the pellets were cured in resin for 48 hr at 60°C .
  • the blocks were sectioned to 60 nm and the sections adhered to nickel grids for 30 min at 60°C.
  • the grids were floated on saturated aqueous sodium metaperiodate for an hour at room temperature, washed, then blocked with 1% BSA, and labeled with Anti-biotin (Jackson Immunolabs) followed by Protein A-gold (Aurion, 10 nm). The grids were rinsed with water and stained with 2% uranyl acetate for 5 minutes. To quantitate gold labeling, 200 gold particles (for more densely labeled samples) or 400 mitochondria (for less densely labeled samples) were counted for each counting event. A minimum of three counting events was performed per sample. Counting was performed over different sections. Analysis of Toxicity.
  • HeLa cells were seeded in 96-well flat bottom tissue culture plates (Starstedt, Germany) at a density of 12,000 cells per well.
  • Leukemic cell lines K562, OCI-M2, U937, HL60, AML2, LY17, Daudi
  • A2780 wildtype and A2780 Cbl-resistant cells were plated in 96-well flat bottom tissue culture plates (Starstedt, Germany) at 25,000 cells per well. The culture media was removed and cells were washed.
  • Peptide incubations were conducted in cell appropriate media, HeLa cell incubations were conducted in OPTI-MEM media.
  • HeLa cells were plated at 100,000 per well of a 24-well plate 24 hours prior to experiment and treated with Cbl or MPP-Cbl in OPTI-MEM (Invitrogen, Carlsbad) for 1 hour. Media was removed and cells were incubated with MitoSox (Invitrogen, Carlsbad) according to manufacturer's instructions. Cells were washed with PBS, trypsinized and analyzed via flow cytometry with FACSCanto (BD, Franklin Lakes).
  • Annexin V Apoptosis Assay Leukemic cell lines (K562, OCI-M2, U937, HL60) were seeded at 200,000 cells per well of a 24-well plate (Greiner Bio-one, Germany). A2780 WT and Cbl-resistant cells were plated in 24-well plate at a density of 75,000 cells per well (BD, Franklin Lakes). Healthy donor mononuclear cells were obtained by Ficoll separation from peripheral blood. CLL patient samples, PBSCs, and healthy donor mononuclear cells were plated at 200,000 per well (Greiner Bio-one, Germany). Cells were incubated in triplicate with peptides at concentrations indicated in cell appropriate media.
  • Leukemia cells were cultured as above and were washed twice with PBS prior to lysis (10 mM Tris, 200 mM NaCl, ImM EDTA (pH 7.4), 1 mM PMSF, 0.5% NP-40, 1% Triton X-100, IX Protease Inhibitor Cocktail (Bioshop, Burlington, ON)) at 4°C, 30 min. Cells were then centrifuged at 1,200 rcf, 4 °C, 5 min and protein levels were quantified using bicinchoninic acid (BCA) assay (Pierce, Rockford). 15 ⁇ g of total protein was diluted in 8x sample buffer and heated to 42°C for 5 min prior to loading on 15% gel.
  • BCA bicinchoninic acid
  • Red blood cells obtained during Ficoll separation of healthy donor peripheral blood was used for this assay. Cells were washed with PBS until the supernatant was clear. Peptide solutions were made in Iscove's media and al :2 dilution was made across a 96-well plate. To each well, 2 of red blood cells were added, mixed and then incubated for 1 hour at 37 °C with 5% C0 2 . For 100% lysis, 0.1% Triton X-100 was added to three wells and for 0% lysis, cells from three wells without peptide were used. Plates were spun at 1000 x g for 10 min and 50 of supernatant was then transferred to a new plate, mixed with 50 ⁇ , of PBS and read at 415 nm.
  • CLL patient samples, PBSCs and healthy donor B cells were seeded at 200,000 cells per well in triplicate in a 24-well plate in Iscove's media. Cells were then incubated with 5 ⁇ thiazole orange-labeled peptide for 15 min, washed with PBS and analyzed on FACSCanto (BD, Franklin Lakes) to determine relative intracellular peptide concentrations. Mitochondrial Membrane Potential. HeLa cells were seeded at 50,000 cells per well 24 hours prior to experiment. CLL patient samples, PBSCs and healthy donor B cells were seeded at 200,000 cells per well in triplicate in a 24-well plate in Iscove's media.
  • HeLa cells were treated with Cbl or MPP-Cbl for 1 hour in OPTI-MEM (Invitrogen, Carlsbad) prior to incubation with JC-1. Cells were washed with PBS, trypsinized and analyzed as above.
  • Crosslinking of Isolated D A Crosslinking of isolated pBR322 DNA was determined from a modification of a published method. 30 Briefly, pBR322 DNA was incubated with compounds at concentrations and times indicated in 25 mM triethanolamine (pH 7.2) and 1 mM EDTA. Reactions were terminated by the addition of 50 mM EDTA and 150 /ig/ml excess short oligonucleotide DNA. Samples were denatured at 95°C in denaturation buffer (30% DMSO, 1 mM EDTA, bromophenol blue, xylene cyanol, and 0.4% SDS) for 3 min and flash frozen in a dry ice/ethanol bath.
  • denaturation buffer 30% DMSO, 1 mM EDTA, bromophenol blue, xylene cyanol, and 0.4% SDS
  • Electrophoresis was carried out in 0.8% agarose in TAE buffer and stained post- run with ethidium bromide. Quantitative Real-Time PCR. Six hundred thousand HL60 cells were incubated in Iscove's media (Invitrogen) with Cbl, mt-Cbl, or MPP at the LC25 dose (17, 3, and 3 ⁇ , respectively) for either 2 or 24 hr as indicated. For Lig3, cells were incubated at an LC50 dose (34, 6, and 6 ⁇ , respectively) for 1 hr. KNA was then isolated using the RNeasy Mini-Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.
  • Collection of Patient Samples Peripheral blood cells from normal individuals and patients with CLL were collected following written informed consent according to a research ethics board (REB) approved protocol. Mononuclear cells were isolated by Ficoll-Hypaque centrifugation. The cells were either used fresh or stored in a viable state at -150°C in 10% DMSO, 40% FBS, and alpha medium. PBSCs were excess filgrastim-mobilized cells obtained from stem cell transplant donors obtained according to an REB approved protocol.
  • REB research ethics board
  • Cbl nitrogen mustard chlorambucil
  • Cbl was selected as a preferable drug for mitochondrial delivery because it exhibits rapid reaction kinetics and does not require cellular activation.
  • the carboxylic acid moiety provides an ideal functional group for facile attachment to an MPP.
  • This peptide accesses the mitochondrial matrix ( Figure IB) and therefore is a suitable vector for delivery of Cbl for mitochondrial DNA alkylation.
  • This peptide is comprised entirely of artificial amino acids, which make it resistant to intracellular degradation.
  • the peptide sequence was designed based on previous work indicating that the inclusion of cyclohexylalanine units within a sequence introduces sufficient hydrophobicity to allow penetration of the mitochondrial membranes, whereas cationic units drive uptake across the energized barrier enclosing mt 4 .
  • the peptide was generated by conventional solid-phase synthesis, and the drug was then attached by coupling to a C-terminal lysine residue. Retention of the alkylation activity and DNA crosslinking activity of Cbl in the mitochondrially targeted conjugate were confirmed in vitro ( Figure 8B and 8C). It was also confirmed that placing the drug at the N- terminus resulted in comparable effects with little change in activity of the MPP-drug conjugate (data not shown).
  • the alkylating activity of MPP-Cbl within mitochondria was also assessed by incubating HeLa cells with the conjugate, allowing alkylation to occur, and then fixing the cells and permeabilizing their membranes (Figure 8A).
  • the MPP-Cbl conjugate maintained its mitochondrial localization, even upon membrane permeabilization, while the MPP control peptide diffused from the mitochondria to the cytoplasm and nucleus upon membrane disruption. Following fixation and permeabilization, cells were treated with DNase to fragment the DNA and this resulted in diffusion of MPP-Cbl (Figure 8A).
  • the cytotoxicity of MPP-Cbl towards HeLa cells was compared to the parent peptide and unconjugated Cbl.
  • a 100-fold increase in potency was observed with MPP-Cbl compared to Cbl ( Figure IE).
  • the parent peptide did not show appreciable toxicity in the concentration range tested, confirming that the cell death resulted from the activity of the drug and not the vector.
  • organellar membrane potential and superoxide levels were assessed. Mitochondrial DNA lesions have been shown to increase mitochondrial superoxide levels and depolarize this organelle's membrane 8 .
  • the alkylation of nuclear versus mitochondrial DNA was quantitatively assessed by comparing the efficiency of PGR amplification of the two genomes.
  • HL60 cells were treated with either Cbl or mt-Cbl, and damage of the nuclear and mitochondrial genomes was assessed independently.
  • a 17.7kb segment of nuclear DNA at the ⁇ - globin gene and an 8.8kb fragment of mitochondrial DNA were analyzed.
  • Cbl primarily damaged nuclear DNA with very few mitochondrial lesions, whereas mt-Cbl caused a significant reduction of mitochondrial DNA amplification with minimal effect on the nuclear genome (Figure 2).
  • GADD Growth Arrest and DNA Damage-inducible family of genes are known to be involved in apoptosis and cell-cycle arrest 32 , and the p21 protein is also known to play an important role in DNA damage sensing. p21 has been shown to interact with GADD45G 33 , which can activate p38 or INK pathways 34 . The activation of these pathways was assessed with immunoblotting following treatment with Cbl or mt-Cbl, and interestingly, differ- ential activation was observed, with Cbl activating p38 and mt-Cbl activating ⁇ ( Figure 3D). These results show that different cellular responses are mounted when the same compound is targeted to distinct intracellular sites.
  • Ligase III The levels of Ligase III were also investigated in Cbl and mt-Cbl treated cells.
  • This ligase present in both the nucleus and mt, is the only ligase in the latter organelle and is involved in the repair of most forms of DNA damage 35 . Therefore, in response to mt- Cbl induced damage of the mitochondrial genome, an increase in expression of this gene should be observed. Indeed, this was detected in cells treated with mt-Cbl ( Figure 3A; Figure 3C).
  • Table 3 List of mitochondrially-targeted proteins included in qPCR array.
  • Cbl is a clinically used therapeutic indicated for the treatment of leukemia. 6
  • MPP-Cbl conjugate would show enhanced activity over the parent compound in primary cancer cells.
  • CLL chronic lymphocytic leukemia
  • PBSCs peripheral blood stem cells
  • mononuclear cells from healthy donors were used to evaluate the therapeutic window of MPP-Cbl.
  • MPP-Cbl treatment we observed activity against CLL patient cells that was significantly lower in healthy cells, indicating that a therapeutic window exists for the peptide conjugate (Figure 4A).
  • MPP- Cbl showed nominal hemolysis levels at the concentrations used in this study ( Figure 4A) suggesting a lack of toxicity to red blood cells at concentrations where leukemic cells were ablated.
  • MPP uptake in CLL cells was higher than in healthy cells, indicating that higher drug concentrations would be achieved in these cells ( Figure 4B).
  • a higher relative mitochondrial membrane potential was observed with CLL cells ( Figure 4B), indicating that there would be a greater driving force for mitochondrial accumulation in this cell type.
  • This difference in mitochondrial membrane potential between cancer and healthy cells has been widely reported 13 and differential drug toxicity due to this characteristic has also been previously observed, such as in studies with Rhodamine 123 and MKT-077 14"16 .
  • a drug is being delivered that is not a delocalized lipophilic cation; and thus, the peptide carrier is providing the specificity. Therefore, MPPs present a general vector for mitochondrial drug delivery that preserves therapeutic window.
  • BCIXL has been shown previously to be over-expressed in certain cancers and its anti-apoptotic activity is thought to contribute to drug resistance 21 . Indeed, in these two Cbl-resistant lines, we observed a much higher level of BCIXL than in the sensitive lines ( Figure 5C, Figure 6). This suggests that BCIXL overexpression, and the resulting suppression of apoptosis, may underlie the resistance to Cbl. When the activity of MPP-Cbl was tested in this panel, potentiation was observed in all lines tested (Figure 5A). In addition, the mechanism of cell death for Cbl and the MPP conjugate appeared similar, with early apoptotic cells apparent by flow cytometry (Inset, Figure 5A and 5B).
  • MPP-Cbl conjugate still exhibits high levels of cytotoxicity in these apoptosis-resistant cell lines indicates that the delivery of a toxic drug to mitochondria presents an effective means to overcome this mechanism commonly employed by cancer cells to resist the action of drugs.
  • Another major form of drug resistance in cancer cells results from the overexpression of enzymes or other factors that facilitate chemical deactivation of pharmacophores.
  • Cbl inactivation via glutathione modification is a common mechanism of resistance.

Abstract

La présente invention concerne des composés comprenant un peptide pénétrant dans les mitochondries (MPP) conjugué à un composé anticancéreux et leur procédé d'utilisation.
PCT/CA2011/000610 2010-05-30 2011-05-27 Peptides pénétrant dans les mitochondries comme vecteurs de composés anticancéreux WO2011150494A1 (fr)

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Application Number Priority Date Filing Date Title
CA2800741A CA2800741C (fr) 2010-05-30 2011-05-27 Peptides penetrant dans les mitochondries comme vecteurs de composes anticancereux
EP11789003.8A EP2576595B1 (fr) 2010-05-30 2011-05-27 Peptides pénétrant dans les mitochondries comme vecteurs de composés anticancéreux
US13/700,970 US9132198B2 (en) 2010-05-30 2011-05-27 Mitochondrial penetrating peptides as carriers for anticancer compounds
CN201180037346.3A CN103097397B (zh) 2010-05-30 2011-05-27 作为抗癌化合物载体的线粒体穿透肽

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US34988210P 2010-05-30 2010-05-30
US34988110P 2010-05-30 2010-05-30
US61/349,881 2010-05-30

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US9540419B2 (en) 2012-05-11 2017-01-10 Gemvax & Kael Co., Ltd. Anti-inflammatory peptides and composition comprising the same
US9572858B2 (en) 2013-10-23 2017-02-21 Gemvax & Kael Co., Ltd. Composition for treating and preventing benign prostatic hyperplasia
US9572900B2 (en) 2012-09-19 2017-02-21 Gemvax & Kael Co., Ltd. Cell penetrating peptide, conjugate comprising same, and composition comprising conjugate
EP3022219A4 (fr) * 2013-07-15 2017-07-26 The Scripps Research Institute Ciblage spécifique de séquences répétées étendues d'arn
US9730984B2 (en) 2012-05-11 2017-08-15 Gemvax & Kael Co., Ltd. Composition for preventing or treating rheumatoid arthritis
US9907838B2 (en) 2013-04-19 2018-03-06 Gemvax & Kael Co., Ltd. Composition and methods for treating ischemic damage
US9937240B2 (en) 2014-04-11 2018-04-10 Gemvax & Kael Co., Ltd. Peptide having fibrosis inhibitory activity and composition containing same
US10034922B2 (en) 2013-11-22 2018-07-31 Gemvax & Kael Co., Ltd. Peptide having angiogenesis inhibitory activity and composition containing same
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US10463708B2 (en) 2014-12-23 2019-11-05 Gemvax & Kael Co., Ltd. Peptide for treating ocular diseases and composition for treating ocular diseases comprising same
US10561703B2 (en) 2013-06-21 2020-02-18 Gemvax & Kael Co., Ltd. Method of modulating sex hormone levels using a sex hormone secretion modulator
US10662223B2 (en) 2014-04-30 2020-05-26 Gemvax & Kael Co., Ltd. Composition for organ, tissue, or cell transplantation, kit, and transplantation method
US10676507B2 (en) 2015-05-26 2020-06-09 Gemvax & Kael Co., Ltd. Peptide and composition containing the same for anti-inflammation, anti-fibrosis, wound healing, and anticancer treatment
WO2020189779A1 (fr) 2019-03-20 2020-09-24 千葉県 Agent ciblant l'adn d'organelles à double membrane
US10835582B2 (en) 2015-02-27 2020-11-17 Gemvax & Kael Co. Ltd. Peptide for preventing hearing loss, and composition comprising same
US10898540B2 (en) 2016-04-07 2021-01-26 Gem Vax & KAEL Co., Ltd. Peptide having effects of increasing telomerase activity and extending telomere, and composition containing same
US10967000B2 (en) 2012-07-11 2021-04-06 Gemvax & Kael Co., Ltd. Cell-penetrating peptide, conjugate comprising same and composition comprising same
US11015179B2 (en) 2015-07-02 2021-05-25 Gemvax & Kael Co., Ltd. Peptide having anti-viral effect and composition containing same
US11058744B2 (en) 2013-12-17 2021-07-13 Gemvax & Kael Co., Ltd. Composition for treating prostate cancer

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US9907838B2 (en) 2013-04-19 2018-03-06 Gemvax & Kael Co., Ltd. Composition and methods for treating ischemic damage
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