WO2012170084A1 - Composition et procédé pour le traitement d'une maladie des poumons et de la métaplasie à cellules mucineuses - Google Patents

Composition et procédé pour le traitement d'une maladie des poumons et de la métaplasie à cellules mucineuses Download PDF

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WO2012170084A1
WO2012170084A1 PCT/US2012/025964 US2012025964W WO2012170084A1 WO 2012170084 A1 WO2012170084 A1 WO 2012170084A1 US 2012025964 W US2012025964 W US 2012025964W WO 2012170084 A1 WO2012170084 A1 WO 2012170084A1
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bik
cell
cells
composition
antibody
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Yohannes Tesfaigzi
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Yohannes Tesfaigzi
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR

Definitions

  • Patent Application Serial No. 61/444,957 entitled “COMPOSITION AND METHOD FOR TREATFNG LUNG DISEASE AND MUCUS CELL METAPLASIA", filed on February 21, 2011, and the specification and claims thereof are incorporated herein by reference.
  • the embodiments of the present invention relate generally to the field of lung disease, pulmonary disease and chronic lung disease, mucous cell metaplasia (MCM) in airways of the lung, methods of modulating Bik in airway epithelial cells (AECs) and methods of treating mucous cell metaplasia and mucous hypersecretion.
  • MCM with mucous hypersecretion is a prominent manifestation of many common inflammatory diseases of the upper and or lower airways. Excess production of mucus is a problem in both minor and serious illnesses, ranging from the common cold to fatal exacerbations of chronic bronchitis, asthma, and cystic fibrosis for example.
  • MCM mucous cells in airways that are normally devoid of these cells. This condition is called MCM.
  • MCM mucous cells in airways that are normally devoid of these cells.
  • MCM mucous cells in airways that are normally devoid of these cells.
  • MCM mucous cells in airways that are normally devoid of these cells.
  • MCM mucous cells in airways that are normally devoid of these cells.
  • MCM mucous cells in airways that are normally devoid of these cells.
  • MCM is the foundation for hypersecretion that can obstruct the airway lumen.
  • MCM poses a serious risk of reducing airflow in the lung due to acutely secreted mucus that can sometimes completely obstruct the airways and lead to death of asthmatics for example.
  • epidemiological studies suggest that patients with chronic MCM are at a higher risk of developing lung cancer. Therefore, reducing hyperplastic cells in patients with chronic MCM, such as chronic bronchitics, may reduce their risk of developing lung cancer.
  • Cigarette smoking is the leading cause of disease for 25-27 million individuals with chronic obstructive pulmonary disease (COPD) in the United States alone and for over 350 million people world-wide. Cigarette smoke alone or in combination with environmental pollutants or allergens can induce an inflammatory response in the lungs of individuals. With 1.25 billion people smoking cigarettes daily worldwide, these diseases are expected to reach epidemic proportions in the next decade. In humans, goblet cell metaplasia is a frequent finding in the large and small airways of cigarette smokers, as are increases in the number and size of mucus-producing glands in the large airways.
  • the amount of intraluminal mucus in the small airways is responsible for reduced lung function and represents a major difference between subjects with moderate to severe COPD (GOLD stages 3 and 4) and those with a lesser degree of airflow obstruction (GOLD stages 1 and 2).
  • excess mucous secretions in the airways are important in the pathogenesis of acute exacerbations of COPD.
  • IFNy and STAT1 signaling are required for the resolution of allergen-induced
  • compositions for modulating Bik as a therapeutic treatment for patients in need thereof.
  • Bik protein levels to determine the health of an individual with and without airway disease.
  • Bik peptide to a target cell in vitro.
  • Fig. 1 illustrates that Exposure to CS decreases Bik expression
  • Fig. 2 illustrates that Exposure to CS leads to increased epithelial cell hyperplasia
  • Fig. 3 illustrates that Restoration of Bik Expression Reduces CS-induced MCM and ECH
  • Fig. 4 illustrates that Bik expression reduces nuclear localization of CS-activated
  • ER l/2 and phopho-ERKl/2 enhances Bik-induced cell death
  • Fig. 5 illustrates that Bik causes cell death by interacting with DAPK to inhibit nuclear localization of activated ERKl/2;
  • Fig. 6 illustrates that Activated Bak is part of the Bik/phospho-ERK/DAPK
  • Fig. 7 illustrates a Proposed IFNy-induced pathway that results in killing of
  • Fig. 8 illustrates that Peptide SEQ ID NO. 1 is a moderate killer of primary
  • FIG. 9 illustrates that Peptide SEQ ID NO. 17 is the most active peptide to cause cell death in cells with low Bcl-2 levels in comparison to Peptides SEQ ID NO. 1, 2, 16, and 18.
  • the intrinsic apoptotic pathway that is responsible for removing excess numbers of mucous cells may be dysfunctional in individuals with asthma and cystic fibrosis.
  • AECs airway epithelial cells
  • the cell death of AECs during the resolution of metaplastic mucous cells is regulated by the Bcl-2 family of proteins, and involves the intrinsic apoptotic pathway.
  • mice [0034] During the resolution of allergen-induced epithelial and mucous cell hyperplasia that is mediated by IFNy, STAT1 and Bik, (a Bcl-2 family member), participate in clearing cell hyperplasia because unlike wild-type mice STAT1- and Bik-deficient mice fail to resolve MCM. Consistent with these findings, mouse airway epithelial cells (MAECs) from STAT1- and Bik-deficient mice are resistant to IFNy-induced cell death. Following inflammatory responses to LPS, allergen or cigarette smoke, up to 30% of hyperplastic cells undergo death to eliminate excess mucous cells and to return to the original cell numbers.
  • MAECs mouse airway epithelial cells
  • Disruption of this recovery process may lead to persistent elevation of mucous cell numbers and contribute to chronic mucous hypersecretion and airway obstruction found in chronic lung diseases such as cystic fibrosis, asthma, and chronic bronchitis.
  • the cell death of AECs during the resolution of metaplastic mucous cells is regulated by the Bcl-2 family of proteins and involves the intrinsic apoptotic pathway.
  • Bronchial brushings were obtained from 11 subjects each with chronic bronchitis, and 9 controls with no evidence of lung disease (Table 1). Screening for dysregulated expression of Bcl-2 family of mRNAs identified Bik levels to be significantly reduced in bronchial brushings of patients with chronic bronchitis compared to non-diseased controls as is illustrated in figure 1A.
  • exposure to cigarette smoke reduces Bik expression.
  • Bik expression was not only suppressed in the biopsy and autopsy lung tissues of cigarette smokers compared to non-smokers, but also in lung tissues of former smokers with chronic bronchitis compared to former smokers without.
  • Bik is a useful biomarker that predicts susceptibil ity to chronic bronchitis. Factors that differentially regulate Bik expression in subjects with chronic bronchitis will aid in management of individuals who may hav e chronic bronchitis and those that were former smokers.
  • FIG. 2A 2B shown are airway epithelial cell numbers in bik+/+ and bik-/- mice exposed to 250 mg/m3 CS or filtered air for 6 h/d, 5 d/wk for 10 wks (A), or 3 wks followed by 8 wks of recovery in filtered air (B). The left lungs were fixed under constant pressure perfusion, cut into 4-mm slices from distal to caudal and slices embedded in paraffin.
  • Mucus cell numbers per mm basal lamina were increased compared to nontreated controls as is illustrated in figure 2F, in which Primary HAECs placed in culture in an ALI culture were left untreated or treated with 1000 ng/ml CS for 24 h and 3 d later membranes were embedded in paraffin and stained with AB/PAS. The number of mucous cells was significantly increased in CS-treated culture compared to untreated controls. Representative photomicrographs of AB/PAS stained culture showing increased number of mucous cells in primary differentiated HAECs treated with 1000 ng/ml CSE for 24 h and harvested 3 d later, compared to untreated groups. These findings suggest that in mice CS exposure suppresses Bik expression in a permanent manner.
  • mice were sacrificed one day later and lungs from HA-Ad-Bik- and Ad-GFP-instilled mice were analyzed by Western blotting.
  • the HA-tagged Bik was detected in mice instilled with Ad-Bik but not in those instilled with Ad-GFP as is illustrated in figure 3B.
  • CS-induced ECH was significantly reduced in the airways of mice instilled with Ad-Bik compared to those instilled with Ad-GFP or PBS as is illustrated in figure 3C.
  • HAECs were treated with another stimulus that is known to activate ERKl/2. HAECs were treated with different concentrations of IGF- 1 for 0-30 min and protein lysates analyzed for p-ERKl/2, ERKl/2 and actin by Western blotting. ERKl/2 was activated earlier and with increasing intensity when cells were treated with increasing IGF-1 concentrations as is illustrated in figure 4D.
  • HAECs were infected with 100 MOI Ad-Bik or Ad-BikL6io and treated with different concentrations of IGF- 1. Cell viability was quantified by trypan blue exclusion assay. IGF-1 enhanced Ad-Bik-induced cell death in a dose-dependent manner as is illustrated in figure 4E. HAECs were treated with 1 ⁇ ERKl/2 inhibitor, U0126, which effectively reduced the levels of activated ERKl/2 as detected by Western blotting as is illustrated in figure 4F.
  • Bik retains activated ERKl/2 in the cytosol to cause cell death.
  • Bik-induced cell death was enhanced by CS- or IGF- 1 -induced ERKl/2 activation. Blocking ERKl/2 activation using U0126 suprisingly allev iated Bik-induced cell death suggesting that ERK l /2 activation may be an integral functional part of Bik- induced cell death.
  • ERKl/2 stimulates apoptosis in cells expressing DAPK and serum-induced phosphorylation of DAPK by ERK enhancing its kinase activity and death promoting effects.
  • Bik is a link between ERKl/2 and DAPK to lead toward death pathways.
  • Activation of EGFR immediately and efficiently activates ERKl/2. Cancer cells that have strong activation of ERKl/2 due to persistent activation of EGFR would be highly susceptible to Ad-Bik.
  • Bik is linked to cell death by interacting with DAPK to inhibit nuclear localization of activated ERKl/2. Lung adenocarcinoma cell lines that have wild-type EGFR (HI 299) or mutations in EGFR (H1975) were selected. Both H1299 and H1975 cells showed increased activated ERKl/2 when cultured in serum-containing medium as is illustrated in figure 5A.
  • HI 975 was selected for further study as to the mechanism by which Ad-Bik induces cell death.
  • Bik overexpression inhibited nuclear localization of activated ERKl/2 in serum-treated but not in serum- starved HI 975 cells as is illustrated in figure 5D, where Nuclear and cytosolic protein preparations 24 h after infection of HI 975 cells with 100 MOI Ad-Bik or Ad-BikL61G were analyzed for phospho-ERKl/2, total ERKl/2, Bik, lamin, and actin, by Western blotting.
  • FIG 5E immunoprecipitation assays with proteins extracted from HI 975 cells that were infected with Ad-Bik, or Ad-BikL61G were performed.
  • the cell lysates (input) and immunoprecipitates were resolved by SDS-PAGE and probed by Western blotting using antibodies to Bik, phospho-ERKl/2, total ERKl/2. Increased phospho-ERKl/2 was detected in pull-down products from Ad-Bik compared to Ad-BikL61G-infected cells despite increased levels of Ad-BikL61G being present as is illustrated in figure 5E.
  • Ad-Bik was over expressed in HI 975 cells and protein lysates were
  • the 60 kDa DAPK protein is a DAPK breakdown product that interacted with
  • ERKl/2 and Bik contributes to clearage of DAPK.
  • the 160 kDa DAPK protein levels in HI 975 cells expressing Ad-Bik or Ad-BikL61G were compared. It was found that DAPK levels were reduced in cells expressing wild-type compared to mutant Bik as is illustrated in figure 5G, where Protein lysates from non-treated HI 975 cells or cells infected with Ad-Bik or Ad-BikL61G for 24 h were resolved by SDS-PAGE and probed with antibodies to DAPK, Bik and actin. bik+/+ and bik-/- MAECs were treated with 1000 ng/ml CS extract for 4 or 8 hours.
  • DAPK levels were not affected at 4 hours but were reduced in bik+/+ but not in bik-/- MAECs 8 hours after CS treatment. Similarly, DAPK levels were also reduced in bik+/+ but not in bik-/- MAECs that were incubated in serum-containing medium for 24 hours; conditions that favor activation of ERKl/2 as is illustrated in figure 51.
  • ER l/2 antibodies from cells maintained with or without serum and infected with Ad-Bik showed that Bik and the 60 kDa DAPK product were detected more in cells cultured with serum compared to those without. Similar to the previous finding, the 100 but not the 60 kDa DAPK protein was detected in the input.
  • shRNA short hairpin RNA
  • the peptides that constitute the BH3 domain bind to the minor groove of the anti-apoptotic proteins Bcl-2 and Bcl-xL to inactivate their anti-apoptotic function, or they can activate pro-apoptotic genes, such as Bax and Bak.
  • Peptides that encompass the BH3 domain linked to the TAT protein, tagged with polyarginine peptide or myristylated to facilitate entry into NHBECs or hydrocarbon staples to ensure stability were designed to modulate Bik activity. Our results indicate that hydrocarbon stapled peptides enter MTECs and NHBECs. These peptides are also expressed using adenoviral vectors. Mutation of the Leu residue within the BH3 domain abrogates the pro-apoptotic function of Bik.
  • Peptides SEQ ID NO. 1 and 2 were compared in their ability to cause death in primary airway epithelial cells. The results showed a higher reduction in cell number with the Peptide SEQ ID. NO. 1 treatment.
  • ATLAXIGDX-Nle-DVSLRA (SEQ ID NO: 11);
  • ALARLAXIGX-Nle-DV (SEQ ID NO: 12);
  • RLAXIGDX-Nle-DVSLRA (SEQ ID NO: 13);
  • RLAXIGDX-Nle-DV (SEQ ID NO: 14);
  • LAXIGDX-Nle-D (SEQ ID NO: 15);
  • Peptides SEQ ID NO. 1, 2, 16, 17, and 18 were used to treat primary airway epithelial cells from 5 individuals that have different levels of Bcl-2, an anti-apoptotic protein that inhibits Bik-induced cell death. Persons 1 and 2 have low and persons 3-5 have higher levels of Bcl-2. The cells were cultured on 12-well dishes using BGEM medium as described previously. Cells were then treated with 5 micromolar of peptide and maintained in culture for 3 days. Cells were then harvested and cell counts determined for each individual. Results show that all peptides were effective in reducing cell numbers in the samples with low Bcl-2 levels. Peptide SEQ ID NO. 17 was the most effective in reducing the number to below 50%. For the persons with higher levels of Bcl- 2 only peptide SEQ ID NO. 17 showed an effective reduction in cells numbers. The other two samples with higher levels of Bcl-2 were unaffected by these peptides.
  • Bak can be activated to cause cell death following the interaction of Bik, phosphor-ER l/2, and DAPK.
  • DAPK expression was knocked down in HI 975 cells through stable expression of short hairpin DAPK (shDAPK).
  • HI 975 cells expressing shDAPK and cells expressing a control hairpin (shCon) were infected with 100 MOI Ad-Bik expressing Bik tagged with the HA epitope.
  • Cell lysates were
  • BIK mimetic therapies can be formulated in a pharmaceutical composition for administration to a mammalian patient.
  • a pharmaceutical composition includes the active agent and a pharmaceutically acceptable carrier, excipient or diluent.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce a severe allergic or similar untoward reaction when administered to a mammal.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particular in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or other aqueous solutions, saline solutions, aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
  • the pharmaceutical compositions including the active agents, will be prepared in accordance with good manufacturing process (GIMP) standards as set by the Food & Drug Administration (FDA).
  • GIMP good manufacturing process
  • FDA Food & Drug Administration
  • Quality assurance (QA) and quality control (QC) standards will include testing for purity and function, homogeneity and function, and/or other standard measures.
  • the pharmaceutical composition hereof is administered by any route that will permit delivery of the active agent to the affected cells. Since it is believed that Bik polypeptide or mimetic therapy does not harm normal cells, systemic administration of the active agent is acceptable. Preferably, administration is intraperitoneal and also including, but not limited to, inhalation, intra-arteriole, intramuscular, intradermal, subcutaneous, perenteral, intraventricular, and intracranial administration. Alternatively, the active agent may be delivered locally to the affected cells by any suitable means.
  • a therapeutically effective amount of the pharmaceutical composition is administered to a mammalian patient.
  • the term "therapeutically effective amount” means an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the patient.
  • a therapeutically effective amount will cause one or more of the following: decreased edema; decreased mucous cell metaplasia, decreased mucous production, or a decrease in any other markers as discussed herein or that would be known to one of ordinary skill in the art as it relates to lung disease, airway obstruction, ECH or MCM.
  • the frequency and dosage of the therapy can be titrated by the ordinary physician or veterinarian using standard dose-to-response techniques that are well known in the art.
  • BIK polypeptides or mimetic therapy as an efficacious treatment of lung disease or symptoms or treatment of mucous cell metaplasia in the airways or cells for example AECs or other cells in vitro described herein or as known to one of ordinary skill in the art.
  • a pharmaceutically effective amount of a pharmaceutical composition for treatment of lung disease or symptom is administered to a mammalian patient.
  • composition is administered to a patient.
  • the pharmaceutical composition includes an active agent composed of at least one cell delivery agent and at least one cell death agent.
  • the cell delivery agent is a cell-penetrating peptide (CPP).
  • CPP is a peptide vector that can traverse through the plasma membrane barrier without breaching the integrity of the cell, and deliver a desired cargo inside the cell.
  • the range of cargoes that can be delivered intracellularly by CPPs encompasses a broad variety of hydrophilic molecules, such as peptides, proteins, antibodies, imaging agents, DNA and even nanosized entities, including polymer-based systems, solid nanoparticles and liposomes.
  • the CPP hereof is preferably a cationic amphipathic peptide leader sequence and, more preferably, a TAT sequence.
  • the CPP is selected from the group consisting of TAT, penetratin, VP22, transportan, synthetic oligoarginines, and combinations thereof.
  • a Bik polypedtide or a derivative of a Bik polypeptide can be expressed in cells through a viral vector such as adenovirus, lentivirus, or retrovirus or as part of an acceptable expression vector.
  • a viral vector such as adenovirus, lentivirus, or retrovirus or as part of an acceptable expression vector.
  • adenoviral expression vector FIG. 3
  • the same approach would be taken to express parts of Bik (the peptides listed above and other parts of Bik) that show the highest effective killing of mucous cells.
  • the pharmaceutical composition for treatment of an immune-mediated disorder hereof also includes a cell death agent capable of stimulating cell death.
  • the mutation in the BH3 domain of Bik will lack the translocation of Bax or Bak to the ER. Identification of the mutation that fails to translocate Bax or Bak to the ER and/or to interact with phosphorylated ER l/2 or DAPkinase may be useful in the future to identify which protein(s) may be involved in directly interacting with Bik to cause Bax or Bak translocation to the ER.
  • instillation of the active peptides into allergic mice will cause a reduction of MCM, similar to what we observed in mice instilled with Ad-Bax or Ad-Bik (see Preliminary Results).
  • Noxa was found to enhance Bik induced cell death, we will consider using a combination of Bik and Noxa to possibly have a synergistic reduction of MCM and thereby reduce the amount of peptide used. Noxa enhances Bik-induced cell death. 100911 Cigarette smoke exposure destabilized Bik mRNA rather than affecting promoter activity. Similarly, I Ny-induced Bik expression does not involve the promoter activity.
  • TGF- ⁇ induces apoptosis by TGF-P-activated Smad transcription factor complexes being recruited to a consensus Smad-binding element in the Bik promoter and directly inducing Bik transcription.
  • Other studies have reported that Bik protein degradation is modified by anti-cancer agents. Bortczomib, a proteasome inhibitor, induces cell death in various cancer cells and primary HA EC's by inhibiting Bik protein degradation. Therefore, Bik expression can be regulated at both the transcriptional and post-transcript ional levels.
  • Bik Since Bik remains suppressed in the lungs of mice even 60 days after recov ery then miRNA or the RNa.se that is activated by CS once induced, remains intact ev en after cessation of CS exposure.
  • Expression of Bik is dependent on a polymorphism variant in the ntronic region of the Bik gene.
  • the common variant encoded by guanine causes reduced expression of Bik while the rare v ariant encoded by adenine causes increased expression. Therefore, this variant can be used to determine the susceptible person who develops mucous cell metaplasia and mucous hypersecretion.
  • treatment with peptide can be specifically optimized by genotyping indiv iduals who are prone to expressing low levels of Bik due to their genetic polymorphism in the intronic region of Bik .
  • Bik may modify phospho-ER K 1 /2 to interact to DAPK .
  • ERKl/2 or DAPK may phosphorylate Bik to enhance the formation of the B i k-E Rk 1 2- D A PK complex.
  • Bik disrupts the interaction of Bcl-2 and nutrient-deprivation autophagy factor- 1 (NAF-1) to regulate Bik-initiated autophagy, but not Bik-initiated activation of caspases.
  • Bik may promote autophagy by displacing NAF-1 from Bcl-2
  • Bik promotes an apoptotic cell death by being modified by phospho-ERK 1 /2 to interact with DAPK.
  • Expression vectors containing different length peptides of the Bik protein were constructed. Constructs will express either the BH3 domain of Bik or will include 5, 10, or 15 amino acids on the N- or C-termini of the BH3 domain. Constructs in which the conserved Leu residue in the BH3 domain is mutated to Ala by site-directed mutagenesis is presented. The constructs for mutant Bik proteins are tested for cell killing along with the construct expressing the wild-type Bik. Constructs that show effective cell killing are then tested by immunofluorescence for their effect on translocating Bax to the ER and their interaction with Bcl-2 or BI-1, depending on results obtained.
  • Peptides are either hydrocarbon stapled to ensure stability, myristylated, or tagged with polyarginine or the TAT protein to ensure entry into cells.
  • the control peptide is randomized to have a sequence different from any of the known BH-3 peptides known to date.
  • Stock solutions at 10-20 mM concentrations are prepared in DMSO.
  • NHBECs are treated with 1, 10, 20, or 100 ⁇ concentrations of BH3 peptides and analyze the extent of cell death that is induced in NHBECs by this BH3 peptide at 24, 48, and 72 hours. Cell death is assessed by cell counts and by Annexin V staining followed by flow cytometry. The number of cells showing co-localization of Bax with ER are quantified, as denoted by the yellow color as a result of the green calnexin stain overlapping with the red stain for Bax.
  • Explant cultures from bronchioles and from nasal midseptum are used to successfully identify the most effective antisense oligonucleotides to Bcl-2 mRNA and to introduce Bax by adenoviral infection.
  • Explant cultures are treated with IL- 9 and IL- 13 at 1 ng/ml each for 6 days, and the BH-3 peptides at 1, 10, 20, or 100 um concentrations is added on day 7 of culture as previously described. After an additional 24 and 48 hours of culture, explants are harvested and processed for quantification of MCM as described previously. Furthermore, tissue sections are analyzed for Bax localization to the ER by immunofluorescence as described in Preliminary Studies.
  • mice are immunized and exposed to OVA for 5 days as described above to induce maximum MCM.
  • mice will be instilled with the active peptide that causes cell death in NHBECs at 10 or 100 ⁇ in 50 ⁇ saline.
  • Mice are instilled with control peptide or with saline only.
  • mice are sacrificed, and the extent of MCM is compared between the groups of mice instilled with active Bik peptide, control peptide, or saline only.
  • Plasmids, adenoviral constructs, and reagents Plasmids, adenoviral constructs, and reagents. Adenoviral expression vectors for
  • Retroviral silencing vector encoding for DAPK shRNA and the control vector were purchased from Origene Technologies, Inc. The suppressing effect of the shRNA was established in HA EC ' s and H 1975 cells, and amplification and purification of pi asm id DNA and packaging of the retroviral particles in Phoeni cells were performed as specified by the manufacturer's instructions.
  • the MAPK extracellular signal-regulated kinase inhibitor 4-diamino-2,3- dicyano-l,4-bis(2-ammophenyltbio) butadiene (U0126) was purchased from EMD
  • Retroviral silencing with DAPK shRNA Retroviral silencing vectors encoding for DAPK shRNA and the control shRNA (Origene Technologies, Inc. Vectors,
  • Bronchial brushings were obtained by bronchoscopy at the University of New Mexico Health Sciences Center. All participants were recruited by advertising in local newspapers and in the University newspaper. Chronic bronchitis was defined as a daily cough with phlegm production for 3 consecutive months, 2 years in a row. Bronchial brushings were obtained from 11 subjects each with chronic bronchitis, and 9 controls with no evidence of lung disease. Demographics of these subjects is described in table 1. Bronchial brushings, performed under local anesthesia with 1% lidocaine, contained 0.4-2 million epithelial cells. At least 60,000 cells from each subject were used for qRT-PCR as described previously.
  • LTRC Research Consortium
  • NHLBI NHLBI.
  • the subjects were categorized into four groups based on records obtained by questionnaires administered. For the subgroups classified by chronic bronchitis, the definition of "signs with chronic bronchitis", which is cough and phlegm for 3 consecutive months and for at least 2 years was used. All, who did not answer yes for these questions and answered "do not usually have cough, and do not usually have phlegm" were defined as subjects with no signs of chronic bronchitis.
  • mice and adenoviral infection Male-specific pathogen-free wild-type C57BL/6 mice were purchased from The Jackson Laboratory. Mice were housed in isolated cages under specific pathogen-free conditions. After a 14-day quarantine period, mice were acclimatized for 8 days and entered into the experimental protocol at 8 - 10 weeks of age. bik+/- mice on C57BL/6 background were provided by Andreas Strasser (Walter and Eliza Hall Institute, Melbourne, Australia). bik+/+ with bik-l- littermates were bred from the respective heterozygote mice at the Lovelace Respiratory Research Institute under specific pathogen- free conditions and genotyped as described previously.
  • mice were exposed to 250 mg/m3 CS or filtered air for 6 hours/day, 5 days/week for 3 weeks or were allowed to recover in air for an additional 8 weeks following 3 weeks of CS exposure.
  • Preparation of lung tissues for histopatho logical examination was performed as described previously. After 3 weeks of exposure to CS mice were anesthetized with isoflurane and intranasally instilled with HA-Ad-Bik or Ad-GFP as a control in a volume of 50ul saline on day 1 and 2 after the last day of exposure.
  • mice from each group were euthanized and right lung tissue harvested and immediately examined for expression of HA protein via Western blotting of protein extracted from lung homogenate. Left lungs were inflated and fixed at 25 mm pressure with zinc formalin for preparing tissue sections and evaluating ECH and MCM.
  • Tissue sections were stained with Alcian blue (AB) and periodic acid Schiff or hematoxylin and eosin as described previously.
  • AB Alcian blue
  • the number of AB positive cells per millimeter of basal lamina was quantified using a light microscope (BH-2; Olympus) equipped with the Image analysis system (National Institutes of Health) as described previously.
  • MAECs were harvested and cultured on Transwell membranes (Corning) after seeding with 4 x 104 or 9 x 104 cells as previously described.
  • Primary HAECs were purchased from Cambrex Bio Science Walkersville, Inc.
  • the immortalized HAECs, AALEB cells were provided by S. Randell (University of North Carolina Chapel Hill, Chapel Hill, NC) were described previously.
  • the lung cancer cell lines with wild-type or mutant K-ras, NCI-H1299 and NCI-H1975 were purchased from ATCC (HAECs were maintained in bronchial epithelial growth medium supplemented with growth factors as described previously.
  • HI 975 and HI 299 cells were maintained in RPM medium supplemented with 10% serum, 0.01% L-glutamine and 0.01% antibiotics. Cell viability was assessed by trypan blue exclusion.
  • Cytosolic and nuclear fractions were prepared by lysing cells in NP-40 to obtain the cytosolic fraction and extracting the nuclear proteins with a hypertonic extraction buffer (50 mM Hepes, pH 7.8, 50 mM KC1, and 300 mM NaCl) in the presence of protease and phosphatase inhibitors as described previously.
  • the following antibodies were used: goat anti-Bik polyclonal antibody (Santa Cruz
  • Immunofluorescence was imaged using Axioplan 2 (Carl Zeiss, Inc.) with a Plan-Neofluor 40x/0.75 air objective and a charge-coupled device camera (Hamamatsu Photonics, Japan) with the acquisition software Slidebook 5.0 (Intelligent Imaging Innovation).
  • Bik-associated proteins were immunoprecipitated by incubating protein lysates prepared from Ad-Bik-, or Ad-BikL61G- infected AALEB or HI 975 cells with gentle mixing at 4°C overnight.
  • the primer/probe sets (Applied Biosystems) were distributed into each well in duplicates, and target mRNAs were amplified by PCR in 20- ⁇ reactions.
  • Preamplification efficiency was assessed by performing amplification of non-amplified cDNA with TaqMan Gene Expression Assays (Applied Biosystems). For all reactions, CT values >37 were eliminated for evaluation of preamplification efficiency.
  • MCF7 cells were transfected with BIK promoter reporter plasmids and a control plasmid expressing the Renilla luciferase (Promega) and incubated in the presence or absence of 100 nmol/L fulvestrant for 48 hours, and expression of reporter genes was determined by the Dual Luciferase assay (Promega).
  • Bik a protein localized to the endoplasmic reticulum (ER) is the major regulator of the IFNy-induced cell death pathway. Noxa enhances Bik-induced apoptosis in proliferating AECs. Modulating Bik is useful to control allergen induced MCM and help reduce symptoms associated with excessive mucous secretion in chronic pulmonary/lung diseases. Bik expression is a biomarker for the health of an individual and biomarker for treatment of lung disease and MCM.
  • Bcl-2 sustains increased mucous and epithelial cell numbers in metaplastic airway epithelium. Am J Respir Crit Care Med 171 :764-772.
  • Endoplasmic reticulum BIK initiates DRP1 -regulated remodelling of mitochondrial cristae during apoptosis. Embo J 24: 1546-1556.
  • nontumorigenic human epithelial cells as a new model for studying K-ras mediated
  • TGF-beta induces apoptosis in human B cells by transcriptional regulation of BIK and BCL-XL. Cell Death Differ 16:593-602.
  • Bik/NBK accumulation correlates with apoptosis-induction by bortezomib (PS- 341, Velcade) and other proteasome inhibitors. Oncogene 24:4993-4999.

Abstract

Des modes de réalisation de la présente invention concernent des peptides Bik biologiquement et pharmaceutiquement actifs destinés à une utilisation comme agents thérapeutiques pour le traitement d'une maladie des poumons, d'une maladie pulmonaire et d'une maladie pulmonaire chronique, et de la métaplasie à cellules mucineuses (MCM) dans les voies aériennes des poumons. Ils concernent également des procédés de modulation de Bik dans les cellules épithéliales des voies aériennes (AEC) et des procédés de traitement de la métaplasie à cellules mucineuses.
PCT/US2012/025964 2011-02-21 2012-02-21 Composition et procédé pour le traitement d'une maladie des poumons et de la métaplasie à cellules mucineuses WO2012170084A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6600024B1 (en) * 1998-03-31 2003-07-29 Thomas Jefferson University Blk genes, gene products and uses thereof in apoptosis
US20080234201A1 (en) * 2002-09-09 2008-09-25 Korsmeyer Stanley J BH3 peptides and method of use thereof
US20080234178A1 (en) * 2007-02-14 2008-09-25 Saint Louis University Compositions and methods for treating immune system mediated diseases

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6600024B1 (en) * 1998-03-31 2003-07-29 Thomas Jefferson University Blk genes, gene products and uses thereof in apoptosis
US20080234201A1 (en) * 2002-09-09 2008-09-25 Korsmeyer Stanley J BH3 peptides and method of use thereof
US20080234178A1 (en) * 2007-02-14 2008-09-25 Saint Louis University Compositions and methods for treating immune system mediated diseases

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MEBRATU Y.A. ET AL.: "Cigarette smoke suppresses Bik to cause epithelial cell hyperplasia and mucous cell metaplasia", AM J RESPIR CRIT CARE MED, vol. 183, no. 11, 1 February 2011 (2011-02-01), pages 1531 - 1538 *
YOHANNES A. MEBRATU ET AL.: "The BH3-only protein BikBlk/Nbk inhibits nuclear translocation of activated ERK1/2 to mediate IFNy-induced cell death", J. CELL BIOL., vol. 183, no. 3, 3 November 2008 (2008-11-03), pages 429 - 439 *

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