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WO2019156946A2 - Compositions et procédés pour améliorer la fonction cftr dans des cellules affectées par la fibrose kystique - Google Patents

Compositions et procédés pour améliorer la fonction cftr dans des cellules affectées par la fibrose kystique Download PDF

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Publication number
WO2019156946A2
WO2019156946A2 PCT/US2019/016581 US2019016581W WO2019156946A2 WO 2019156946 A2 WO2019156946 A2 WO 2019156946A2 US 2019016581 W US2019016581 W US 2019016581W WO 2019156946 A2 WO2019156946 A2 WO 2019156946A2
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cftr
ivacaftor
cells
tezacaftor
autophagy
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WO2019156946A3 (fr
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Benjamin Kopp
Susan Reynolds
Larry Schlesinger
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Nationwide Childrens Hospital Inc
Ohio State Innovation Foundation
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Nationwide Childrens Hospital Inc
Ohio State Innovation Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/443—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines

Definitions

  • Cystic fibrosis is an autosomal recessive disorder, caused by mutations of one single protein, the cystic fibrosis transmembrane conductance regulator (CFTR).
  • CFTR is a cAMP-regulated chloride channel that is primarily located at the apical membrane of epithelial cells. Greater than 1,500 different disease-associated mutations have been identified, however a single codon deletion, F508del, occurs in approximately 90% of patients with CF. This F508del gene mutation results in the deletion of phenylalanine in position 508 of the CFTR. The F508del mutation causes the CFTR to be misfolded and lose its ion channel activity at the plasma membrane, resulting in local inflammation, increased susceptibility to respiratory bacterial infections, and progressive pulmonary and digestive insufficiency.
  • the clinical condition caused by mutations of one single protein, the cystic fibrosis transmembrane conductance regulator (CFTR).
  • Autophagy is an evolutionary conserved biological process, in which cytoplasmic material is enclosed in a double-membrane autophagosome. Fusion with lysosomes results in the formation of an autophagolysosome, the contents of which are subjected to lysosomal degradation. Starvation is a regulator of autophagy which induces this complex process through the inhibition of the mammalian target of rapamycin (mTOR), which resides in the mTORCl complex.
  • mTOR mammalian target of rapamycin
  • mTORCl In response to growth factors that stimulate the phosphatidylinositol 3-kinase-AKT pathway, mTORCl negatively regulates a complex consisting of UNC-5 l-like kinase 1 (ULK1), ATG13, ATG101, and FIP200. Nutrient depletion or energy exhaustion inhibits mTORCl, which permits activation of ULK1, an initiating step in autophagy.
  • the regulatory Beclin-l- interacting complex consists of Beclin-l, BCL-2, the class III phosphatidylinositol 3-kinase (VPS34), and ATG14L. Activation of the Beclin l-interacting complex generates
  • LC3 light chain 3
  • LC3-ATG8 microtubule associated protein light chain 3
  • LC3-I cytosolic truncated form of LC3
  • LC3-II autophagosomal membrane- associated, phosphatidylethanolamine-conjugated form
  • Autophagy contributes to the suppression of inflammation, including the down-regulation of both interferon responses and proinflammatory cytokine responses to invading pathogens, and the inhibition of inflammasome-dependent maturation and secretion of proinflammatory cytokines such as interleukin- 1b and interleukin- 18. Autophagy also has a crucial role in the adaptive immune response including antigen presentation and lymphocyte development.
  • Rapamycin an inhibitor of mTOR markedly decreases B. cenocepacia infection in vitro by enhancing the clearance of B. cenocepacia via enhancement of autophagy; in vivo , rapamycin decreases B. cenocepacia burden in the lungs of CF mice and reduced the signs of lung inflammation (7).
  • Similar in vitro findings with CFBE4lo- (CFBE) homozygous F508del CF bronchial epithelial cells infected with Pseudomonas aeruginosa have been observed, where rapamycin through the induction of autophagy restores bacterial clearance (13).
  • Enhancing autophagy has been proposed as a potential strategy for facilitating the therapeutic action of potentiators on the F508del CFTR, as preclinical data indicates that prior restoration of autophagy in CF airways can enable the action of CFTR potentiators in reducing lung inflammation (23). If induction of autophagy results not only in a reduction of infections and associated pulmonary inflammation, but also in the enhancement of the therapeutic effect of CFTR potentiators or CFTR correctors, this could have a beneficial clinical impact for patients with CF.
  • AR-12 (previously known as OSU-03012) is a celecoxib derivative that was discovered at The Ohio State University. The compound was initially developed in the oncology setting, and a phase I study demonstrated an acceptable safety profile with long term oral exposures up to 33 weeks. The AR-12 oncology dose most likely substantially exceeds the exposure needed in other settings such as the treatment of infectious diseases.
  • AR-12 (a.k.a. OSU-03012) has been previously shown to exhibit anti-tumor and anti-bacterial activity. It is thought that AR-12 induces autophagy of cells harboring intracellular bacteria. AR-12 is known to induce autophagy in human and mammalian immune effector cells. Supportive preclinical studies demonstrated that AR-12 has rapid blood brain barrier penetration and appreciable accumulation in tissues, exceeding the blood level concentrations several fold (24).
  • AR-13 an AR-12 analog
  • AR-12 has also been shown to have antibacterial activity. See e.g.,
  • compositions and methods for increasing CFTR expression in CF-affected cells and restoring autophagy function in CF-affected cells are provided.
  • CF-affected cells are cells having deficient CFTR function.
  • CF cells include, but are not limited to, epithelial cells (e.g., upper and lower airway, gastrointestinal mucosa, pancreas, and genitourinary system), neuronal cells, and immune effector cells (e.g., macrophages and neutrophils).
  • epithelial cells e.g., upper and lower airway, gastrointestinal mucosa, pancreas, and genitourinary system
  • neuronal cells e.g., fibroblasts and neutrophils
  • immune effector cells e.g., macrophages and neutrophils.
  • Autophagy function refers to the process of disassembling intra-cellular components (organelles, bacteria, viruses, misfolded proteins, etc.) as part of a normal cellular process.
  • Airway epithelial cells are a major host defense against pulmonary bacterial infections. CF epithelial cells have decreased CFTR function, resulting in the production of thick mucus, abnormal mucus clearance, and deficient autophagy which contribute to various host pathologies including frequent difficult to treat pulmonary infections.
  • aspects described herein provide methods of enhancing autophagy function in CF- affected cells by administering AR-13 to the host wherein the expression of CFTR is increased by at least about two-fold.
  • aspects described herein also provide methods of improving autophagy function in B. cenocepacia- infected macrophages having the F508del CFTR mutation by administering AR-13 to the host.
  • co-localization of B. cenocepacia with LC3-II increases by at least about 20% with AR-13 treatment compared to untreated B. cenocepacia- infected CF
  • Further aspects provide methods of improving autophagy function in patients with CF having the F508del CFTR mutation with B. cenocepacia- infections including infected macrophages having the F508del CFTR mutation by administering AR-13 to the host.
  • the level of Beclin-l increases by at least about four- fold compared to untreated B.
  • cenocepacia- infected CF macrophages cenocepacia- infected CF macrophages.
  • compositions comprising AR-13 and a CFTR modulator.
  • methods of increasing the expression of CFTR in CF epithelial cells in a host by administering AR-13 to the host wherein the expression of CFTR is increased by at least about two-fold are provided.
  • Figure 1 shows an exemplary Western blot of CF and non-CF human macrophage derived monocytes (MDMs) after 24-hour exposure to infection or treatment with AR-13 as indicated;
  • Figure 2 shows densitometric analysis of the results of Figure 1 for non-CF and CF MDMs
  • Figure 3 shows the results of an exemplary confocal microscopy analysis of CF and non-CF MDMs infected with B. cenocepacia for 24 hours with and without treatment with AR- 13;
  • Figure 4 shows quantitative scoring for the experiment depicted in Figure 3;
  • Figure 5 shows an exemplary Western blot analysis of human CF airway epithelial cells treated with DSMO (control), AR-13, or AR-12 as indicated and densitometry analysis of all replicates;
  • Figure 6 shows an exemplary Western blot analysis of human CF F508del/F508del airway epithelial cells treated with VX-661 ⁇ AR-12 or AR-13 and densitometry analysis of all replicates;
  • Figure 7 shows and exemplary CFTR function assay performed on human CF F508del/F508del airway epithelial cells treated with AR-12, AR-13, VX-661, or a combination of two of the compounds.
  • compounds suitable for use alone or in combination with CFTR restoration agents as described herein include, for example, AR-13 (N- ⁇ 4-[5-(Phenanthren-2-yl)- 3-(trifluoromethyl)-lH-pyrazol-l-yl]phenyl]sulfuric diamide), having the following structure:
  • this pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the CFTR modulator is selected from the group consisting of ivacaftor, lumacaftor, ataluren, VX-661, FDL169, N91115, QBW251, Riociguat, QR-010, lumafactor and ivacaftor, GLPG222, VX-152, VX-152 and tezacaftor and ivacaftor, VX-440, VX-440 and tezacaftor and ivacaftor, VX-445, VX-445 and tezacaftor and ivacaftor, VX-561 (aka CTP-656), VX-659, VX-659 and tezacaftor and ivacaftor, PTI-428, PTI-801, and PTI-808.
  • the CFTR modulator is VX-661.
  • the CFTR current is increased by at least about three-fold.
  • the CFTR modulator can be selected from the group consisting of ivacaftor, lumacaftor, ataluren, VX-661, FDL169, N91115, QBW251, Riociguat, QR-010, lumafactor and ivacaftor, GLPG222, VX-152, VX-152 and tezacaftor and ivacaftor, VX-440, VX-440 and tezacaftor and ivacaftor, VX-445, VX-445 and tezacaftor and ivacaftor, VX-561 (aka CTP-656), VX-659, VX-659 and tezacaftor and ivacaftor, PTI-428, PTI-801, and PTI-808.
  • the CFTR modulator is VX-661.
  • the CFTR current is increased by at least about three-fold.
  • the at least one CFTR modulator can be selected from the group consisting of ivacaftor, lumacaftor, ataluren, VX-661, FDL169, N91115, QBW251, Riociguat, QR-010, lumafactor and ivacaftor, GLPG222, VX-152, VX-152 and tezacaftor and ivacaftor, VX-440, VX-440 and tezacaftor and ivacaftor, VX-445, VX-445 and tezacaftor and ivacaftor, VX-561 (aka CTP-656), VX-659, VX-659 and tezacaftor and ivacaftor, PTI-428, PTI-801, and PTI-808.
  • the CFTR modulator is VX-661.
  • AR-13 is provided to a subject in an amount to achieve a blood or tissue concentration of AR-13 from about 2.5 mM to about 20 mM.
  • the subject is infected with B. cenocepacia.
  • the epithelial cells are airway epithelial cells.
  • the immune effector cells are macrophages.
  • CF-affected cells refers to cells whose normal function is disrupted, inhibited, or reduced by cystic fibrosis, including but not limited to epithelial cells (e.g., upper and lower airway, gastrointestinal mucosa, pancreas, and genitourinary system), neuronal cells, and immune effector cells (e.g., macrophages and neutrophils).
  • epithelial cells e.g., upper and lower airway, gastrointestinal mucosa, pancreas, and genitourinary system
  • neuronal cells e.g., a cell that kill cells
  • immune effector cells e.g., macrophages and neutrophils.
  • the epithelial cells are airway epithelial cells.
  • the immune effector cells are macrophages.
  • Such cells may not exhibit normal autophagy function due to impact of the genetic mutations interfering with the function of the CFTR and a subsequent impact on cellular functions including autophagy.
  • the mutation is a single codon mutation, F508del, in the CFTR which occurs in about 90% of CF patients.
  • CF-affected cells can include cells having other mutations. Over 1,500 different CF disease-associated mutations have been identified.
  • the gene mutations causing CF may be somatic, and all cells in the body may be affected.
  • the clinical symptomatology is manifested by dysfunction in the affected cells, including but are not limited to, the upper and lower airway, gastrointestinal mucosa, pancreas, and genitourinary system and immune effector cells (e.g., macrophages and neutrophils).
  • AR-13 can be administered directly to the host by any suitable route of administration that delivers a sufficient amount of AR-13 to have a therapeutic effect on the CF-affected cells.
  • Administration of AR-13 can improve autophagy function of the CF-affected cells. Improvement of autophagy function with AR-13 treatment can also result in increased expression of CFTR in CF cells compared to untreated CF controls.
  • AR-13 can be administrated to a patient having CF-affected cells to improve autophagy function of the cells, or to reduce the risk of CF patients developing CF symptoms.
  • Further aspects provide methods of increasing CFTR current in CF-affected cells by administering AR-13 to the cells. In this aspect, the CFTR current is increased by at least about three-fold.
  • a CFTR modulator e.g., ivacaftor, lumacaftor, ataluren, VX-661, FDL169, N91115, QBW251, Riociguat, QR-010, lumafactor and ivacaftor, GLPG222, VX-152, VX-152 and tezacaftor and ivacaftor, VX-440, VX-440 and tezacaftor and ivacaftor, VX-445, VX-445 and tezacaftor and ivacaftor, VX-561 (aka CTP-656), VX-659, VX-659 and tezacaftor and ivacaftor, PTI-428, PTI-801, and PTI-808) can also be administered to the cells.
  • the CFTR modulator is VX-661.
  • AR-13 can be provided in an amount to achieve
  • aspects described herein provide methods of treating a subject with CF and a bacterial infection, comprising administering AR-13 to the patient. Further aspects comprise administering at least one CFTR modulator (e.g., ivacaftor, lumacaftor, ataluren, VX-661, FDL169, N91115, QBW251, Riociguat, QR-010, lumafactor and ivacaftor, GLPG222, VX-152, VX-152 and tezacaftor and ivacaftor, VX-440, VX-440 and tezacaftor and ivacaftor, VX-445, VX-445 and tezacaftor and ivacaftor, VX-561 (aka CTP-656), VX-659, VX-659 and tezacaftor and ivacaftor, PTI-428, QR-010, PTI-801, and PTI-80
  • the relative dose of AR-13 and the CFTR restoration agent can be adjusted to further limit the risk of developing resistance from treatment.
  • AR-13 is provided in an amount to achieve a blood or tissue concentration in a subject from about 5pm to about 20pM and the CFTR restoration or CFTR modulator agent is provided in an amount to achieve a blood or tissue concentration in a subject from about 5pg/ml to about lOOpg/ml.
  • AR- 13 can be provided to a subject in an amount sufficient to achieve a blood or tissue concentration in the subject from about 0.5pM to about 5pM.
  • treatment of CF-affected cells with AR-12 and AR-13 was assessed by exposing human CF airway epithelial cells (i.e., cells derived from nasal brushings from patients with known CF) to B. cenocepacia , and treating the cells with a control (DMSO), AR- 12, or AR-13 for 24 hours.
  • CFTR and LC3 expression was assessed.
  • AR-13 treatment alone increased CFTR and LC3-II expression compared to untreated cells or those treated with AR-12.
  • AR-12 is known as an autophagy inducer, it is not effective with respect to treatment of CF-affected cells.
  • FIG. 1 shows densitometric analysis of Figure 1 for non-CF and CF MDMs under the following conditions: No treatment (NT), infection with B. cenocepacia (Be), treatment with AR-13, infection with B. cenocepacia (Be) plus treatment with AR-13 (Bc/AR-l3), or infection with B. cenocepacia (Be) plus treatment with AR-13 and bafilomycin.
  • B. cenocepacia infected CF macrophage cells having the F508del mutation with AR-13 increases the level of Beclin-l by at least about four-fold compared to untreated B. cenocepacia- infected (Bc-infected) CF macrophages (e.g., compare lane 2 (Be) to lane 4 (Bc/ARl3) in the CF panel).
  • LC3-II also show a significant increase in Bc-infected CF cells treated with AR-13 ( Figure 2, right panel).
  • FIG. 3 shows images from confocal microscopy of CF and non-CF MDMs infected with B. cenocepacia for 24 hours ⁇ treatment with AR-13.
  • Figure 4 shows the summed quantification of bacterial co-localization from Figure 3.
  • Figure 5 shows that AR-13 increases autophagy and CFTR expression in human CF nasal epithelial cells.
  • Well-differentiated Human CF F508del/F508del airway epithelial cells were derived from nasal brushings. At passage 2 the cells were differentiated using the air-liquid-interface method. On differentiation day 21, the well differentiated cells were treated with DMSO, AR-12, or AR-13 for 48 hours and LC3 and CFTR were assayed by
  • Figure 5 shows a two-fold increase in CFTR and LC3 II expression resulting from treatment of human CF airway epithelial cells with AR-13 compared to either a control or treatment with AR-12. LC3 II levels correlate with autophagy activity. Thus, AR-13 increases autophagy in these cells by at least about two-fold.
  • Figure 5 (rights panel) shows the corresponding densitometry for Figure 5 left panel normalized to calreticulin or GAPDH for all replicates.
  • AR-13 increases CFTR current (i.e., indicative of CFTR function) in CF epithelial cells by at least three-fold while AR-12 did not increase CFTR current.
  • Statistical significance for Fig. 7 comparisons was determined by an unpaired t-test.
  • AR-l3 s parent compound, AR-12, has known autophagy-inducing properties.
  • AR-12 does not increase CFTR current. Macrophages from patients with CF are deficient in autophagy leading to failed bacterial clearance. (2,7). Therefore, AR-l3’s ability to induce autophagy in CF macrophages compared to the parent compound AR-12 was examined. Western blotting demonstrated an increase in LC3-II expression in CF macrophages treated with AR-13 during infection in comparison to infection or treatment alone ( Figure 1). The addition of 3-MA (3-methyladenine autophagy inhibitor) to block autophagy reduced this effect. Beclin-l expression was reduced basally in CF
  • the CFTR modulator is selected from the group consisting of ivacaftor, lumacaftor, ataluren, VX-661, FDL169, N91115, QBW251, Riociguat, QR 010, lumafactor and ivacaftor, GLPG222, VX-152, VX-152 and tezacaftor and ivacaftor, VX-440, VX-440 and tezacaftor and ivacaftor, VX-445, VX-445 and tezacaftor and ivacaftor, VX-561 (aka CTP-656), VX-659, VX-659 and tezacaftor and ivacaftor, PTI-428, PTI-801, and PTI-808.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises AR-13 and the CFTR modulator VX-661 (also known as
  • compositions provided herein can further comprise an antibiotic, an anti-viral, or an anti-fungal drug to treat patients who are also afflicted with bacterial, viral, or fungal infections.
  • administer or“administered” refers to providing the compositions described herein to a patient including by the patient, a healthcare professional, a caretaker, and also includes prescribing the compositions described herein to the patient.
  • compositions described herein can be administered orally, parenterally
  • IV intravenously [IV], intramuscularly [IM], depot- IM, subcutaneously [SQ], and depot-SQ), sublingually, intranasally, by inhalation, intrathecally, topically, or rectally.
  • Dosage forms known to those of skill in the art are suitable for delivery of the compositions described herein.
  • AR-13 can be formulated into suitable pharmaceutical preparations such as creams and gels, for topical application; suspensions, tablets, granules, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration; suspensions or solutions appropriate for inhalation (e.g., metered dose inhalers, dry powder inhaler,
  • AR-13 can be formulated into pharmaceutical compositions using techniques and procedures well known in the art.
  • about 0.1 to 1000 mg, about 5 to about 200 mg, or about 10 to about 50 mg of the AR-13, or a physiologically acceptable salt or ester can be compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice.
  • the amount of active substance in compositions or preparations comprising AR-13 is such that a suitable dosage achieving the therapeutic range indicated is obtained.
  • compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 1000 mg, about 1 to about 500 mg, or about 10 to about 200 mg of the active ingredient.
  • unit dosage from refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • AR-13 is mixed with a suitable pharmaceutically acceptable carrier to form compositions.
  • the resulting mixture may be a cream, gel, solution, suspension, emulsion, or the like.
  • Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for lessening or ameliorating at least one symptom of the disease, disorder, or condition treated and may be empirically determined.
  • compositions suitable for administration of AR-13 and compositions described herein include any such carriers suitable for the particular mode of administration.
  • the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action.
  • the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • methods for solubilizing may be used. Such methods are known and include, but are not limited to, using co-solvents such as ethanol (EtOH) or dimethylsulfoxide (DMSO), using surfactants (e.g., anionic, cationic, zwitterionic, and non-ionic). Specific suitable surfactants include, but are not limited to, TWEEN, poloxamer, sodium lauryl sulfate, aluminum
  • compositions are formulated for single dosage administration.
  • AR-13 and compositions described herein may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings.
  • Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems.
  • the active compound can be included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective dose may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder.
  • AR-13 and compositions described herein can be enclosed in multiple or single dose containers.
  • the enclosed compounds and compositions can be provided in kits, for example, including component parts that can be assembled for use.
  • AR- 13 in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use.
  • a kit may include AR-13 and a second therapeutic agent for co administration.
  • a kit may include a plurality of containers, each container holding one or more unit doses of AR- 13 described herein.
  • the containers can be adapted for the desired mode of administration, including, but not limited to suspensions, tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampoules, vials, and the like for parenteral administration; and patches, medipads, gels, suspensions, creams, and the like for topical administration.
  • composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • the active ingredient(s) may be administered at once or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules.
  • the active compound or compounds can be incorporated with excipients and used in the form of tablets, granules, capsules, or troches.
  • Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum
  • tragacanth acacia, com starch, or gelatin
  • an excipient e.g., any suitable filler/bulking agent
  • a disintegrating agent such as, but not limited to, alginic acid and com starch
  • a lubricant such as, but not limited to, magnesium stearate
  • a glidant such as, but not limited to, colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.
  • the dosage unit form when it is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
  • dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the compounds can also be administered as a component of an elixir, suspension, symp, wafer, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.
  • the active materials can also be mixed or co-administered with other active materials that do not impair the desired action, or with materials that supplement the desired action.
  • AR-13 can be used, for example, in combination with a CFTR restoration agent or modulator agent, antibiotic, antiviral, antifungal, pain reliever, or a cosmetic.
  • solutions or suspensions used for parenteral, intradermal, subcutaneous, inhalation, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, alcohols, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oil
  • suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, ethanol, N- methylpyrrolidone, surfactants and mixtures thereof.
  • PBS phosphate buffered saline
  • suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, ethanol, N- methylpyrrolidone, surfactants and mixtures thereof.
  • Liposomal suspensions including tissue- targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known in the art.
  • AR-13 may be prepared with carriers that protect the compound against rapid elimination from the body, such as time-release formulations or coatings.
  • carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like. Methods for preparation of such formulations are known to those skilled in the art.
  • compounds employed in the methods of the disclosure may be administered enterally or parenterally.
  • compounds employed in the methods of the disclosure can be administered in usual dosage forms for oral administration as is well known to those skilled in the art.
  • These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs.
  • the solid dosage forms can be of the sustained release type so that the compounds employed in the methods described herein need to be administered only once or twice daily.
  • the dosage forms can be administered to the patient 1, 2, 3, or 4 times daily.
  • AR-13 as described herein can be administered either three or fewer times, or even once or twice daily.
  • the terms "therapeutically effective amount” and “therapeutically effective period of time” are used to denote treatments at dosages and for periods of time effective to restore autophagy function.
  • administration can be parenteral, oral, sublingual, transdermal, topical, intranasal, or intrarectal.
  • the therapeutic composition when administered systemically, can be administered to a subject at a sufficient dosage to attain a blood or tissue level of the compounds of from about 0.1 pM to about 20 pM. For localized administration, much lower concentrations than this can be effective, and much higher concentrations may be tolerated.
  • MDMs were infected at a MOI (multiplicity of infection) of 10 for immunoblotting experiments. After removal of cell supernatants, plated cells were lysed in lysis buffer (HEPES, MgCl, EGTA, KCL, NP-40) with protease inhibitor (Roche Applied Science, 10-519-978-001). Then, 30 pg of protein was separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were immunoblotted for calreticulin (Enzo Life Sciences, ADI-SPA-600), LC3, beclin-l, and p62. Protein bands were detected with HRP-conjugated secondary antibodies and visualization performed using ECL reagents (Life Sciences, RPN2106).
  • Macrophages were cultured on 12 mm glass cover slips in 24-well tissue culture plates and infected synchronously with bacteria at an MOI of 2. Macrophage nuclei were stained blue with DAPI (4',6-diamidine-2'-phenylindole dihydrochloride). Macrophages were fluorescently labeled with LC3. Microscopy was performed using an Axiovert 200M inverted epifluorescence microscope equipped with the Apotome attachment for improved fluorescence resolution and an Axiocam MRM CCD camera (Carl Zeiss Inc., Thomwood, NY). Five independent fields with at least 10 macrophages were scored for each condition. All experiments were performed in triplicate.
  • AR compounds (AR-13, AR-12) were used at concentrations ranging from 1-5 mM for all experiments.
  • CFTR modulators included VX-770, VX-809, and VX-661 (Selleck Chem).
  • CF and non-CF healthy controls donated heparinized blood samples. Subjects were excluded if using chronic immunosuppressants, CFTR modulators, or had a history of transplantation.
  • Peripheral monocytes were separated from whole blood using Lymphocyte Separation Medium (Corning, 25-072-CV). Isolated monocytes were re-suspended in RPMI (Gibco, 22400-089) plus 10% human AB serum (Coming, 35-060-C1) and differentiated for 5 days at 37°C into monocyte-derived macrophages (MDMs). MDMs were then placed in a monolayer culture, and infected at bacterial multiplicity of infection (MOI) of 10. CF neutrophils were isolated from human blood and purified by negative selection.
  • RPMI Lymphocyte Separation Medium
  • MDMs monocyte-derived macrophages
  • blood was transferred to 50 ml conical tube and 50 pl of an antibody cocktail added per ml of blood (Stem Cell Technologies, Vancouver, BC, Canada), plus 50 m ⁇ of magnetic beads per ml of blood.
  • the sample was incubated 5 min at RT, then PBS-EDTA was added up to 50ml, the tube was placed in the magnet for 10 min, the supernatant was transferred to a new tube and magnetic beads were added at the same amount of the previous step.
  • the sample was placed 5 min in the magnet and the supernatant was collected.
  • the cells were centrifuged at 1600 rpm (revolutions per minute), and re-suspended in lml of HBSS plus 1% of FBS before further experimentation.
  • the human THP-l monocyte line was also used.
  • THP-l cells were grown in 10% fetal bovine serum (Thermo Scientific) in RPMI. In order to differentiate THP-l cells into macrophage they were treated with 200nM PMA
  • GM-CSF 20ng/mL GM-CSF
  • R&D Systems 415-ML-050
  • Media was replenished with 20ng/mL GM-CSF the following day and the THP-l derived macrophages were matured for 5 days before experimentation.
  • THP-l cells were then treated with the CFTR inhibitor Inh-l72 (Sigma, C2992) for 24 h prior to experimentation to create CF- like macrophages.
  • Cells were recovered from the nasal respiratory epithelium of patients homozygous for the F508del mutation via gentle brushing with a cytology brush. Cells are removed from the brush by agitation and cultured on irradiated fibroblast feeder layers in medium as recently published.25 At 80% confluence, the epithelial cells are recovered by double trypsinization and re-plated on collagen-coated transwells in proliferation medium.25 At confluence, the cultures are transitioned to Pneumacult (Stem Cells, Inc) medium. Cultures were allowed to differentiate for 21 days and are then treated with vehicle or drug.
  • Pneumacult Stemacult
  • Ringer’s buffer (115 mM NaCl, 25 mM NaHC03, 2.4 KH2P04, 1.24 K2HP04, 1.2 CaCl2, 1.2 mM MgQ2, and 10 mM D-glucose; pH 7.4) was added in the basal compartment of the Ussing chamber whereas low chloride (1.2 mM NaCl and 115 mM Na gluconate replacing 115 mM NaCl) was added to the apical chamber. Bath solutions were vigorously stirred and gassed with 5% C02. Solutions and chambers were maintained at 37°C. Amiloride (IOOmM) was first added to inhibit the epithelial sodium channel ENaC.
  • Forskolin (10 mM) was then added to the apical chamber to evaluate c AMP- stimulated currents.
  • the specific CFTR inhibitor Inh-l72 (10 mM) was added to the apical side at the end of the experiment to block CFTR channels.
  • IFN-gamma Stimulates Autophagy-Mediated Clearance of Burkholderia cenocepacia in Human Cystic Fibrosis Macrophages. PLoS One 20l4;9(5):e9668l.

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Abstract

L'invention concerne des compositions et des procédés pour améliorer la fonction CFTR dans des cellules affectées par la fibrose kystique avec des modulateurs AR-13 et CFTR.
PCT/US2019/016581 2018-02-06 2019-02-05 Compositions et procédés pour améliorer la fonction cftr dans des cellules affectées par la fibrose kystique Ceased WO2019156946A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116601158A (zh) * 2020-10-07 2023-08-15 弗特克斯药品有限公司 囊性纤维化跨膜传导调控因子的调节剂
WO2024180475A1 (fr) * 2023-02-27 2024-09-06 Dmf Pharma Foodar S.R.L. Composition lipidique pour le traitement combiné de la fibrose kystique
EP4277703A4 (fr) * 2021-01-15 2025-02-12 Children's Hospital Medical Center Méthodes d'optimisation d'une thérapie par modulateur cftr

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US20120232059A1 (en) * 2005-11-08 2012-09-13 Vertex Pharmaceuticals Incorporated Modulators of ATP-Binding Cassette Transporters
US10022352B2 (en) * 2006-04-07 2018-07-17 Vertex Pharmaceuticals Incorporated Modulators of ATP-binding cassette transporters
EP3024455A1 (fr) * 2013-07-25 2016-06-01 Bayer Pharma Aktiengesellschaft Stimulateurs de sgc ou activateurs de sgc et inhibiteurs de pde5 en combinaison avec un autre traitement pour la thérapie de la fibrose kystique
US20160108406A1 (en) * 2014-10-08 2016-04-21 University Of Iowa Research Foundation Method of regulating cftr expression and processing
US10028933B2 (en) * 2015-07-22 2018-07-24 Ohio State Innovation Foundation Compositions and methods for inhibiting the growth of multi-drug resistant microbes
CA3028966A1 (fr) * 2016-06-21 2017-12-28 Proteostasis Therapeutics, Inc. Composes de phenyle-isoxazole-carboxamide substitues et utilisation connexe pour accroitre l'activite de regulateurs de la conductance transmembranaire de la fibrose kystique

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116601158A (zh) * 2020-10-07 2023-08-15 弗特克斯药品有限公司 囊性纤维化跨膜传导调控因子的调节剂
EP4277703A4 (fr) * 2021-01-15 2025-02-12 Children's Hospital Medical Center Méthodes d'optimisation d'une thérapie par modulateur cftr
WO2024180475A1 (fr) * 2023-02-27 2024-09-06 Dmf Pharma Foodar S.R.L. Composition lipidique pour le traitement combiné de la fibrose kystique

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