Classifications

• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
  • A61K31/105—Persulfides
• • 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
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
• • 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
  • A61K31/4164—1,3-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
  • A61K31/41—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
  • A61K31/4164—1,3-Diazoles
  • A61K31/4168—1,3-Diazoles having a nitrogen attached in position 2, e.g. clonidine
• • 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
  • A61K31/4164—1,3-Diazoles
  • A61K31/417—Imidazole-alkylamines, e.g. histamine, phentolamine
• • 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
  • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
• • 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
• • 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
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• Airway epithelial cells include a mixture of predominantly multiciliated cells (MCCs) and mucus-secreting goblet cells exposed at the luminal surface and underlying basal (stem) cells. MCCs each possess 200 to 300 motile cilia that beat in a coordinated, directional manner to propel inhaled contaminants trapped by the mucus layer out of the lungs.
• MCCs multiciliated cells
• Goblet cells secrete mucus that forms a protective barrier for the respiratory epithelia, and they can increase in activity and number in response to noxious stimuli such as infection.
• CF cystic fibrosis
• PCD primary ciliary dyskinesia
• CRS chronic rhinosinusitis
• COPD chronic obstructive pulmonary disease
• asthma Id.
• CF CF transmembrane conductance regulator
• MCCs are terminally differentiated and arise from the basal cells or secretory cell types of the airway epithelium beginning in embryonic development and continuing as a regenerative process throughout life.
• MCC differentiation starts with a Notch signaling event, in which cells respond to activation of the Notch transmembrane protein to become secretory cells, whereas ligand-expressing cells not responsive to Notch are directed to the MCC fate via an MCC-specific gene expression program that drives differentiation and ultimately the production of hundreds of regulatory and structural components required for motile cilium biogenesis.
• Notch signaling event in which cells respond to activation of the Notch transmembrane protein to become secretory cells
• ligand-expressing cells not responsive to Notch are directed to the MCC fate via an MCC-specific gene expression program that drives differentiation and ultimately the production of hundreds of regulatory and structural components required for motile cilium biogenesis.
• Robust mucociliary clearance requires production of cilia of the correct number, length, beat frequency and waveform, and, importantly , correct directionality along the tissue axis.
• Notch signaling in differentiated epithelia has also been shown to shift cellular composition away from secretory and toward MCC cell fate by inducing transdifferentiation of secretory cells into MCCs (Laikas et al. Nature 2015 Dec 3;528(7580):127-31).
• Airway epithelia from patients with CF and other chronic inflammatory diseases have been shown to have sparse or absent MCCs, defective mucociliary clearance, and related decreased barrier function and regenerative capacity.
• In vitro and animal models have shown that by suppression of Notch signaling, gamma secretase inhibitors are able to restore a healthy balance of secretory and MCC cells both by driving de novo MCC differentiation and by promoting transdifferentiation of mature secretory cells into MCCs, thereby rescuing these cellular composition, barrier and regenerative phenotypes.
• Vladar EK, Nayak JV, Milla CE, Axelrod JD Airway epithelia from patients with CF and other chronic inflammatory diseases have been shown to have sparse or absent MCCs, defective mucociliary clearance, and related decreased barrier function and regenerative capacity.
• gamma secretase inhibitors are able to restore a healthy balance of secretory and MCC cells both by driving de novo MCC differentiation and
• Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation. JCI Insight. 2016;l(13);e88027). Further, transdifferentiation of mature secretory cells by gamma secretase inhibitors is relati vely rapid, as compared to new cell differentiation, which is relatively slow.
• CFTR modulators are an example of personalized medicine in that they are designed to treat individuals carrying specific CFTR mutations.
• CFTR modulators can be classed into three main classes: potentiators, correctors and premature stop codon suppressors, or read-through agents.
• CFTR potentiators increase the open probability of CFTR channels that have gating or conductance mutations.
• CFTR correctors are designed to increase the amount of functional CFTR protein delivered to the cell surface.
• CFTR read -through agents are designed to “force” read-through of premature stop codons, leading to the production of more full-length CFTR protein.
• CFTR amplifiers are a type of CFTR modulator being developed and tested, and are designed to increase the amount of CFTR protein a cell makes at the transcriptional level, thereby potentially enhancing function in patients with CFTR mutations that lead to insufficient protein at the cell surface.
• CFTR modulators improve CFTR function in patients having the corresponding CFTR mutations, the modulators do not affect the altered cellular composition, damage to epithelial cell architecture and corresponding epithelial dysfunction.
• Improved therapies are needed for restoring MCC function and improving mucociliary clearance in cystic fibrosis and other diseases characterized by mucus hypersecretion and/or inadequate mucociliary clearance.
• GPIs Gamma secretase inhibitors
• Mahman DM Meredith JE, Zaczek R, Houston JG, Albright CF: Gamma-secretase inhibitors for Alzheimer's disease: balancing efficacy and toxicity.
• Evin G Sernee MF, Masters Cl.,: Inhibition of gamma-secretase as a therapeutic intervention for Alzheimer's disease: prospects, limitations and strategies.
• Gamma secretase is a multi-unit transmembrane protease complex, consisting of four individual proteins. It is an aspartyl protease that cleaves its substrates within the transmembrane region in a process called regulated-intramembrane-proteolysis (RIP).
• RIP regulated-intramembrane-proteolysis
• GSIs can be classified into three general types based on where they bind to gamma secretase: (1) active-site binding GSIs, (2) substrate docking-site-binding GSIs, and (3) alternate binding site GSIs.
• the latter category can be further subdivided into carboxamide- and arylsufonamide-containing GSIs. (Kreft et al, at 6171).
• Alzheimer's disease clinical trials have revealed toxicities believed to be associated with gamma secretase inhibition.
• GSIs gamma secretase inhibitors
• a CFTR modulator is effective in correcting epithelial cell dysfunction in cystic fibrosis cell-based model systems (primary cells from patients), in contrast to certain prevailing concepts, and indeed the combination may be synergistic in improving CFTR ion channel function and epithelial cell correction.
• the invention therefore provides methods of treating a respiratory disease characterized by mucus hyper-secretion comprising administering to a human patient in need of such treatment a GSI, wherein the administration of low dose GSI is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in said patient's lungs.
• the methods of the invention are effective in treating a respiratory disease selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans, idiopathic pulmonary fibrosis and other fibrotic lung disorders, respiratory infection including exacerbations in chronic respiratory disorders, and mucus accumulation in response to acute infection.
• a respiratory disease selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans, idiopathic pulmonary fibrosis and other fibrotic lung disorders, respiratory infection including exacerbations in chronic respiratory disorders, and mucus accumulation in response to acute infection.
• the GSI is selected from the group consisting of semagacestat, avagacestat, GS-1, DBZ, 1.-685,458, BMS-906024, crenigascestat, MRK 560, nirogacestat, RO-4929097, MK-0752, itanapraced, LY-3056480, fosciclopirox, tarenflurbil, and begacestat.
• the GSI is selected from the group consisting of semagacestat, nirogacestat, MK-0752, RO-492907, or crenigacestat. In some embodiments, the GSI is a carboxamide based GSI.
• methods for treating respiratory diseases characterized by mucus hypersecretion comprising systemically administering semagacestat in an amount of from about 0.1 mg to about 50mg daily, wherein the administration of semagacestat is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• semagacestat is administered in an amount of from about 0.5mg to about 40mg daily.
• semagacestat is administered in an amount of from about 0.5mg to about 30mg daily, or from about 0.5mg to about 20mg daily, or from about 0.5mg to about 10mg daily.
• semagacestat may be administered in about 0.1 mg, 0.25mg, 0.5mg, 1mg, 2.5mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg or 50mg daily.
• semagacestat is administered orally.
• a method for treating a respiratory disease characterized by mucus hyper-secretion comprising systemically administering to a human patient in need of such treatment a therapeutically effective amount of semagacestat, wherein said patient's semagacestat plasma concentration at steady state following multiple dose administration comprises an AUC (area under the curve) less than 2100 ng*hr/mL, such as less than 1220 ng*hr/mL, wherein the systemic administration of semagacestat is effective in reducing mucus in such patient' s lungs or preventing mucus accumulation in such patient's lungs.
• AUC area under the curve
• said patient's steady state semagacestat plasma concentration comprises an AUC less than 1500 ng*hr/mL, less than 1200 ng*hr/mL, or less than 900 ng*hr/mL, such as an AUC less than 1220 ng*hr/mL, less than 600 ng*hr/mL, or less than 250 ng*hr/mL.
• methods for treating cystic fibrosis comprising administering an effective amount of a GSI to a human patient taking a CFTR modulator, wherein the mucus in such patient's lungs is reduced or mucus accumulation in such patient's lungs is inhibited.
• the GSI is selected from the group consisting of semagacestat, nirogacestat, MK-0752, RO-492907, or crenigacestat. In some embodiments, the GSI is semagacestat.
• the CFTR modulator is selected from the group consisting of a CFTR potentiator, a CFTR corrector, a CFTR premature stop codon inhibitor, a CFTR amplifier and combinations thereof. In some embodiments, the CFTR modulator is selected from the group consisting of ivacaftor, lumacaftor, tezacaftor, elexacaftor and combinations thereof.
• Figure 1 shows dose response data of semagacestat in primary human nasal epithelial cells (HNECs) compared to untreated cells and DAPT positive control.
• MCCs are labeled in green (acetylated tubulin) and cell junctions are labeled in red (ECAD).
• ECAD acetylated tubulin
• the percentage of MCCs increases from its baseline at 15.625 nM semagacestat to its maximum at around 125 nM. Toxicity is observed at micromolar doses.
• Figure 2 shows the ratio of MCCs to total luminal cells in HNECs treated with DAPT and various doses of semagacestat.
• Figure 3 shows method of scoring the ratio of MCCs to non-ciliated cells in airway epithelia in mice treated systemically in vivo by intraperitoneal (IP) dosing of semagacestat.
• IP intraperitoneal
• Figure 4 shows the body weight at days 9, 24 and 30 with daily systemic (IP) administration of semagacestat and vehicle control.
• IP systemic
• Figure 5 shows the ratio of MCCs to total cells at day 7 following a three-day treatment with DAPT and low and high doses of semagacestat, with vehicle control.
• Figure 6 shows the ratio of MCCs to total cells at day 31 following a three-week treatment with semagacestat, with vehicle control.
• Figure 7 shows the effect of GSI treatment during proliferation (prior to differentiation) and during differentiation of HNECs.
• Figure 8 shows the quantitation of MCCs per total luminal cells of the data of Figure 7.
• Figure 9 shows the effects of GSI treatment duration on mature (ALI+30d) HNECs, treated with DAPT and semagacestat for one (ALI+30 to +37d) or two weeks (ALI+30 to +44d).
• Figure 10 shows the quantitation of MCCs per total luminal cells of the data of Figure 9.
• Figure 11 shows the results of HNECs treated with DAPT and semagacestat during differentiation only (ALI+0 to +2 Id) from either the apical or basal surface.
• Figure 12 shows the quantitation of MCCs per total luminal cells of the data of Figure 11.
• Figure 13 shows the effect of semagacestat and DAPT treatment in an epithelial culture model of chronic airway inflammation. HNEC cultures were treated with IL- 13 from ALI+7 to 14 to induce inflammation. DAPT and semagacestat increase the percentage of MCCs in controls (left). IL- 13 treatment increases the percentage of mucin positive secretory cells and decreases the percentage of MCCs. Subsequent DAPT and semagacestat treatment rescues cell composition, increasing the percentage of MCCs and decreasing the percentage of mucin positive secretory cells.
• Figure 14 shows the quantitation of MCCs per total luminal cells of the data of Figure 13.
• Figure 15 shows representative Ussing chamber tracings of cultures treated with ETI, semagacestat, or both.
• Figure 16 shows representative tracings of Ussing-chamber short circuit currents (Isc) following treatment with semagacestat in wild-type and CF cells.
• Figure 17 shows Ussing-chamber Isc responses following treatment with semagacestat in wildtype and CF cells.
• Two wild-type control samples (WT) and two CF patient samples (CF1; a rare allelic combination and CF2; a F508 ⁇ homozygote) were studied.
• CFTR current activity were assessed by CFTR inhibitor response and were found to be as great as or greater than vehicle control currents in both wild-type controls and in CF samples. Values are normalized to the baseline current.
• Figure 18 shows that under in vitro treatment in combination with the CFTR modulator lumacaftor, semagacestat decreased mucus production in human CF samples with different CFTR mutations.
• Figure 19 shows the effect of treatment with semagacestat, lumacaftor and combinations on human CF samples with different CFTR mutations.
• Semagacestat is effective at increasing the percentage of MCCs in the presence of Lumacaftor, and the combination may be more effective for some donors than when either is applied alone.
• Figure 20 shows the quantitation of MCCs per total luminal cell data for healthy patient and CF donor 1 of Figure 19.
• Figure 21 shows the effects on primary healthy and cystic fibrosis airway epithelial cells treated with semagacestat (LY45139) during differentiation only (ALI+0 to +2 Id).
• Figure 22 shows SEM of healthy and CF primary human airway epithelial cultures, showing that multiciliated cells formed under DAPT treatment are indistinguishable from those in untreated healthy cultures.
• Figure 23 shows the result of DAPT treatment in mature cystic fibrosis HNEC cultures, demonstrating that GSI treatment induces the formation of additional multiciliated cells in mature cystic fibrosis cultures, while untreated cultures do not differentiate any more multiciliated cells.
• Figure 24 shows the results of HNECs treated during differentiation (ALI+0 to +2 Id) with DAPT and high and low concentrations of the GSIs LY45139, PF-03084014, RO-4929097 and MK-0752.
• Figure 25 shows the quantitation of MCCs per total luminal cells of the data shown in Figure 24.
• Figure 26 shows measurements of CFTR short-circuit current activity measured in Ussing chambers in cultures from CF patients treated with LY45139, Elexcaftor/Tezacaftor/Ivacaftor (or “ETI”), or both.
• Figure 27 shows measurements of CFTR current activity measured in Ussing chambers in cultures from CF patients treated with MK04752, ETI, or both.
• Figure 28 shows measurements of CFTR current activity measured in Ussing chambers in cultures from CF patients treated with MK04752, ETI, or combinations with reduced doses of Elexacaftor (E component of the ETI modulator combination).
• Figure 29 shows the results of the GSI DAPT treatment on ionocyte formation in HNECs treated with DAPT during differentiation only (ALI+0 to +2 Id).
• Figure 30 shows the effect of varying concentrations of GSI MK-0752 and Elexacaftor of ciliary beat frequency (CBF).
• Figure 31 shows ciliary beat frequency (CBF) and cilium length of HNEs treated with DAPT versus untreated controls.
• CBF ciliary beat frequency
• Figure 32 shows the airway surface liquid (ASL) reabsorption characteristic of CF HNEC cultures treated with ETI, semagacestat, or both.
• ASL airway surface liquid
• Figure 33 shows images taken from a high-speed video recorded microscopic images of latex bead movement as a reflection of mucus transport by the ciliated surface of cell cultures.
• Cultures were of HNECs from two CF donors (F508del homozygotes) under treatment with vehicle control, ETI, Semagacestat (LY) or both treatments combined.
• Figure 34 shows the calculated bead movement of the cultures shown in Figure 33.
• a “disease characterized by mucus hypersecretion” means a disease wherein at least one pathology of the disease is due to presence of mucus at an epithelial surface in excess of the amount present under normal conditions. Included are diseases in which excess mucus is located in small airway passageways in which it is not normally present, and may be due to excess goblet cell production, hypertrophy of mucus glands, decreased MCCs, or other inadequate mucociliary clearance.
• an “effective amount” or “therapeutically effective amount” refers to an amount of the compound of the present disclosure that is effective to achieve a desired therapeutic result such as. for example decreasing goblet cell production and/or increasing production of multiciliated cells, thereby improving mucociliary clearance.
• a desired therapeutic result includes reducing mucus production in such patient's lungs or inhibiting mucus accumulation in such patient's lungs. While the doses mentioned in the present disclosure are guidelines, an attending physician may adjust the dose according to the specific needs of the patient, including for example, severity of the disease, size and physical condition.
• “Gamma secretase inhibitor(s)” or “GSI(s)” means a molecule capable of inhibiting or modulating the gamma secretase enzyme, and thereby inhibiting Notch signaling. Examples include DAPT ( N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester), semagacestat, avagacestat ((2R)-2-[[(4-Chlorophenyl)sulfonylj[[2-fluoro-4-(1 ,2,4-oxadiazol-3- yl)phenyl]metliyl]amino]-5,5,5-trifluoropentanamide) (commercially available from www.tocris.com).
• the CAS Registry Number is 847925-91-1) (commercially available from www.adooq.com), MK-0752 (CAS No. 471905-41-6 (www.medchemexpress.com)); itanapraced (CAS No. 749269-83-8 (www.medchemexpress.com)); LY-3056480 (Samarajeewa, Anshula & Jacques, Bonnie & Dabdoub, Alain. (2019). Therapeutic Potential of Wnt and Notch Signaling and Epigenetic Regulation in Mammalian Sensory Hair Cell Regeneration. Molecular Therapy. 27.
• Carboxamide-based GSI means a GSI having a carboxamide group, and includes molecules formed by carboxamide substitution as well as derivatives of known carboxamide-based GSIs such as DAPT.
• Examples include DAPT ( N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester), semagacestat, avagacestat ((2R)-2-[[(4-Chlorophenyl)sulfonyl][[2-fluoro-4-(1.2,4-oxadiazol-3- yl)phenyi]methylJamino]-5,5,5-trifluoropentanamide) (commercially available from www.tocris.com), DBZ (N-[(1S)-2-[[(7S)-6,7-Dihydro-5-methyl-6-oxo-5H-dibenz[b,d] azepin-7-yl]amino]-1-methyl-2- oxoethyl]-3,5-difluorobetizeneacetamide) (commercially available from www.tocris.com),
• the CAS Registry Number is 847925-91-1) (commercially available from www.adooq.com) and BMS-708163 (Gillman, KW et al, Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable Gamma-Secretase Inhibitor. ACS Med. Chem. Lett. (2010) 1(3)120-124). See also, Sekioka, R. et al., Discovery of N-ethylpyridine-2-carboxamide derivatives as a novel scaffold for orally active gamma secretase modulators. Bioorg. & Med. Chem., (2020) 28(1): 115132.
• GSIs include any salt form, polymorph, hydrate, analog, or pro-drug that retains gamma secretase inhibiting or modulating activity.
• Treatment includes therapeutic treatments, prophylactic treatments, and ones that reduce the risk that a subject will develop a disorder or risk factor. Treatment does not require complete curing of disorder or condition, and includes the reduction in severity, reduction in symptoms, reduction of other risk factors associated with the condition and /or disease modifying effects such as slowing the progression of tire disease.
• GSIs A variety of GSIs have been developed as potential clinical candidates for Alzheimer's Disease and cancer indications. (See Kreft et al, at 6171). DAFT' was one of the earliest GSIs identified. Modifications of DAPT led to clinical candidates.
• Semagacestat is (2S)-2-hydroxy-3-methyl-N-[(lS)-l-methyl-2-oxo-2-[[(1S)-2, 3,4,5- tetrahydro-3-methyl-2-oxo- 1H-3-benzazepin- 1-yl] amino] ethyl] -butamide, a small molecule gamma secretase inhibitor that was initially developed for the treatment of Alzheimer' s Disease. (See U.S. Patent No. 7,468,365). Semagacestat is known to exist in a number of polymorphic forms, including a dihydrate and at least two anhydrate forms. (Id., See also U.S. Patent No. 8,299,059). See also, Yi et al, DMD (2010) 38:554-565; http://doi: 10.1124/dmd.109.03084,1.
• Nirogacestat (aka PF-03084014) is ((S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3- ylamino)-N-(11(2-m- ethyl-l-(neopentylamino)propan-2-yl)-lH-imidazol-4-yl)pentanamide, a small molecule gamma secretase inhibitor that was developed for cancer indications. It is available as a hydrobromide salt (www.medchemexpress.com), and exists is solid state forms. (See U.S. Patent No. 10,590,087). See Wei P, et al.
• MK-0752 is a small molecule gamma-secretase inhibitor being studied for cancer indications. Phase 1 clinical data is described in Krop I, et al. Phase I pharmacologic and pharmacodynamic study of the gamma secretase (Notch) inhibitor MK-0752 in adult patients with advanced solid tumors. J Clin Oncol. 2012;30(19):2307-2313.
• RO-4929097 is a small molecule gamma secretase inhibitor being studied for cancer indications. See, e.g., Tolcher AW, Messersmith WA, Mikulski SM et al. Phase I study of RO4929097, a gamma secretase inhibitor of Notch signaling, in patients with refractory metastatic or locally advanced solid tumors. J Clin Oncol 2012; 30: 2348-2353; Wu et al, Journal of Chromatography B, 879 (2011) 1537— 1543.
• RO4929097 In this study, patients received escalating doses of RO4929097 orally on two schedules: (A) 3 consecutive days per week for 2 weeks every 3 weeks; (B) 7 consecutive days every 3 weeks; and (C) continuous daily dosing. Toxicities included fatigue, thrombocytopenia, fever, rash, chills, and anorexia. The study concluded that RO4929097 was well tolerated at 270 mg on schedule A and at 135 mg on schedule B; and the safety of schedule C was not fully evaluated.
• Crenigascestat (aka LY3039478) is a small molecule gamma secretase inhibitor being studied for cancer indications. See Yuen E., et al., Evaluation of the effects of an oral notch inhibitor, crenigacestat (Ly3039478), on QT interval, and bioavailability studies conducted in healthy subjects. Cancer Chemother PHarmacol. 2019 Mar;83(3):483-492.In this study crenigacestat was administered to healthy subjects as single 25, 50, or 75 mg oral doses or as an intravenous dose of 350 pg 13 C 15 N 2 H-crenigacestat.
• GSIs have been developed as potential cancer therapeutics, as monotherapies or in combination with other agents. See, e.g., Takebe N, Nguyen D, Yang SX. Targeting notch signaling pathway in cancer: clinical development advances and challenges. Pharmacol Ther. 2014 Feb; 141(2): 140-9. doi: 10.1016/j.pharmthera.2013.09.005. Epub 2013 Sep 27; Shao H, Huang Q, Liu ZJ. Targeting Notch signaling for cancer therapeutic intervention. Adv Pharmacol. 2012;65:191-234. doi: 10.1016/B978-0- 12-397927-8.00007-5.
• low dose GSIs are effective in the treatment of respiratory diseases characterized by mucus hypersecretion, and are effective at doses allowing therapeutic activity while avoiding or minimizing the adverse effects previously associated with this class of molecules.
• “low dose” of a GSI refers to a dose that is an effective amount to treat the respiratory disease characterized by mucus hypersecretion and is a lower dose as compared to a dose of the GSI suitable for administering to a patient suffering from a neurodegenerative disorder, an oncology disorder, or a respiratory disease not characterized by mucus hyper-secretion.
• a low dose would be a dose yielding a peak plasma level in the submicromolar range.
• a low dose of a GSI may be administered as a single daily dose, multiple doses per day (e.g., 2 or 3 doses per day), intermittent, or weekly, with the dosing regimen dependent on the dosage form (e.g., immediate release or controlled release), and the needs of the patient. Administration may be for an extended period of time, intermittent, or may be for a limited amount of time, with administration repeated if and to the extent determined by a patient's medical provider.
• a GSI may be provided daily for 1, 3, 5, 7, 10, 14, 18, 21, 24, 28 or 30 days, and then stopped.
• the GSI is administered intermittently, such as every 3 days, or weekly.
• references to daily dosing amounts herein can be accomplished by dosing regimens other than daily, e.g., a weekly dose of 35mg would correspond to a daily dose of 5mg/day.
• slow-release formulations such as depots or patch formulations are known in the art and can be utilized to provide doses equivalent to the daily doses described herein.
• the GSI dosing regimen may be repeated if necessary.
• each of the GSIs semagacestat, nirogacestat (PF-03084014), RO-4929097 and MK- 0752 when administered in the nanomolar range to human nasal epithelial cells is effective in blocking Notch signaling, driving differentiation towards MCCs, and rescuing conditions associated with excessive goblet cell mucus secretion.
• relatively low systemic levels of GSIs may provide effective treatment for respiratory conditions associated with mucus hypersecretion, while avoiding or minimizing adverse events observed with higher doses.
• GSIs administered in combination with a CFTR modulator is effective in correcting epithelial cell dysfunction in cystic fibrosis cell-based model systems (primary cells from patients), in contrast to certain prevailing concepts, and indeed the combination may be synergistic in improving CFTR ion channel function and epithelial cell correction.
• various GSIs have now been shown to not interfere with CFTR ion channels and not inhibit effects of CFTR modulators on CFTR ion channels in cystic fibrosis airway epithelial cells.
• GSI treatment improves airway surface liquid (ASL) reabsorption of CF cells to the same degree as CFTR modulator drugs.
• ASL airway surface liquid
• GSI treatment may be synergistic with CFTR modulator treatment, thereby allowing the potential to decrease doses of either or both drugs, and further reducing potential toxicities of each.
• GSls may be administered by pharmaceutical dosage forms known in the art, including but not limited to oral solid dosages, oral liquids, injection, transdermal patch, and inhalation. Dosage forms may be formulated with excipients and other compounds to facilitate administration to a subject and to maintain shelf stability. See “Remington's Pharmaceutical Sciences” (Mack Publishing Co., Easton, PA). Oral pharmaceutical formulations include tablets, minitablets, pellets, granules, capsules, gels, liquids, syrups and suspensions.
• a GSI is administered orally, typically via oral solid dosage, although oral liquids may be desirable for certain populations that have difficulty with tablets and capsules, such as pediatric and elderly patients. Oral dosage forms may be immediate release or controlled release.
• semagacestat Tablet forms of semagacestat are known in the art. (See U.S. Patent No. 8,299,059). Upon oral administration, semagacestat is reported to have a half-life of approximately 2.5 hours. Hence, in one embodiment of the invention, semagacestat may be provided as an immediate release formulation. Immediate release semagacestat may be provided as a single daily dose, or divided into multiple daily dosages which may be administered 2, 3, 4 or more times per day. In another embodiment of the invention, semagacestat is provided as an extended release formulation. An extended release formulation may provide patient convenience by reducing daily administrations, and may improve patient compliance. Further, controlled release formulations of the present invention may be useful in reducing serum peaks and troughs, thereby potentially reducing adverse events.
• Oral controlled release formulations are known in the art and include sustained release, extended release, delayed release and pulsatile release formulations. See “Remington's Pharmaceutical Sciences” (Mack Publishing Co., Easton, PA).
• the active agent may be formulated in a matrix formulation with one or more polymers that slow release of the drug from the dosage form, including hydrophilic or gelling agents, hydrophobic matrices, lipid or wax matrices and biodegradable matrices.
• the active agent may be formulated in the form of a bead, for example with an inert sugar core, and coated with known excipients to delay or slow release of the active agent by diffusion.
• Enteric coatings are known in the art for use in delaying release of an active agent until the dosage form passes from the low pH environment of the stomach to the higher pH environment of the small intestine, and my include methyl acrylate-methacrylic acid copolymenrs, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetatate succinate, polyvinyl acetate phthatlate (PVAP), shellac, sodium alginate, and cellulose acetate trimellitate.
• CAP cellulose acetate phthalate
• PVAP polyvinyl acetate phthatlate
• shellac sodium alginate
• cellulose acetate trimellitate methyl acrylate-methacrylic acid copolymenrs
• the GSI is selected from the group consisting of semagacestat, avagacestat, GS-1, DBZ, L-685,458, BMS-906024, crenigascestat, MRK 560, nirogacestat, RO-4929097, MK-0752, itanapraced, LY-3056480, fosciclopirox, tarenflurbil, and begacestat.
• the GSI is selected from semagacestat., nirogacestat, MK-0752, RO- 492907, or crenigacestat. In one embodiment, the GSI is semagacestat.
• a method for treating a respiratory disease characterized by mucus hyper-secretion comprising systemically administering to a patient in need thereof about 0.1 mg to about 50mg semagacestat daily wherein the oral administration of semagacestat is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• semagacestat is systemically administered at dosages of from about 0.5mg to about 40mg daily, or from about 0.5mg to about 30mg daily, and most preferably of from about 0.5mg to about 20mg daily, or from 0.5mg to about 10mg daily.
• semagacestat may be administered in about O.lmg, 0.25nig, 0.5mg, Img, 2.5mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg or 50mg daily.
• methods for treating a respiratory disease characterized by mucus hypersecretion comprising systemically administering to a patient in need thereof about 5 pg to about 1 mg/kg daily, preferably from about 50 to about 100 pg/kg semagacestat daily.
• a method for treating a respiratory disease characterized by mucus hyper-secretion comprising systemically administering to a human patient in need of such treatment a therapeutically effective amount of semagacestat, wherein said patient's semagacestat plasma concentration at steady state following multiple dose administration comprises an AUC (area under the curve) less than 1220 ng*hr/mL wherein the systemic administration of semagacestat is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• said patient's steady state semagacestat plasma concentration comprises an AUC less than 1220 ng*hr/mL, less than 600 ng*hr/mL, or less than 250 ng*hr/mL.
• a method for treating a respiratory disease characterized by mucus hyper-secretion comprising systemically administering to a patient in need thereof about O.lmg to about 50mg nirogacestat daily wherein the oral administration of semagacestat is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• the nirogacestat is systemically administered at dosages of from about 0.5mg to about 40mg daily, or from about 0.5 to about 30mg, or of from about 0.5mg to about 20mg daily.
• methods are provided for treating a respiratory disease characterized by mucus hypersecretion comprising systemically administering to a patient in need thereof about 8 pg to about 0.9 mg/kg daily, preferably from about 10 to about 300 pg/kg nirogacestat daily.
• a method is provided for treating a respiratory disease characterized by mucus hyper-secretion comprising systemically administering to a patient in need thereof about 0.1 mg to about 20mg RO-4929097 daily wherein the oral administration of RO-4929097 is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• the RO-4929097 is systemically administered at dosages of from about 0.1 to about 10mg daily, or from about 0.5mg to about 10mg daily, or of from about 0.1 mg to about 5mg daily.
• methods for treating a respiratory disease characterized by mucus hypersecretion comprising systemically administering to a patient in need thereof about 5 pg to about 0.4 mg/kg daily, preferably from about 50 to about 100 pg/kg RO-4929097 daily.
• a method for beating a respiratory disease characterized by mucus hyper-secretion comprising systemically administering to a patient in need thereof about 0.1 mg to about 40mg MK-0752 daily wherein the oral administration of MK-0752 is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• the MK-0752 is administered at a dose in the range of from about 0.1 to about 30mg daily, or from about 0.1 mg to about 20 mg daily, and most preferably of from about 0.1 mg to about 10 mg daily.
• methods for treating a respiratory disease characterized by mucus hypersecretion comprising systemically administering to a patient in need thereof about 2.5 pg to about 0.6 mg/kg daily, preferably from about 2.5 to about 500 pg/kg MK-0752 daily.
• the respiratory disease characterized by mucus hypersecretion is selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesia, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans, Idiopathic pulmonary fibrosis and other fibrotic lung disorders and respiratory infection, including exacerbations in chronic respiratory disorders and mucus accumulation in response to acute infection.
• the respiratory disease is cystic fibrosis.
• the respiratory disease is chronic obstructive pulmonary disease.
• the GSI is administered by inhalation. In preferred embodiments, the GSI is administered by oral administration. In one embodiment, the GSI is provided in an immediate release solid oral dosage form. In another embodiment, the GSI is provided in a controlled release solid oral dosage from. In a further embodiment, the GSI is provided in a liquid dosage form. In a further embodiment, the GSI is provided in an inhalation dosage form.
• CFTR modulator drugs have provided a significant advance in the treatment of cystic fibrosis. They do not, however, address damage that occurs to the lung epithelium due to cystic fibrosis. Further, CFTR modulators are limited to use in patients that have the specific CFTR mutations addressed by the particular CFTR modulator drug.
• ionocyte The cell type or types that most express functional CFTR is not defined. It has been suggested that a rare cell type named ionocyte might be the major source of CFTR expression and therefore activity. Plasschaert, L.W., et. al., A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature (2018) 560: 377-381; https ://doi .org/10.1038/s41586-018-0394-6. Furthermore, it was suggested that the ionocyte population is expected to be diminished upon Notch inhibition and CFTR activity likewise decrease. (Id. at 380).
• methods of the invention address the dysfunction present in cystic fibrosis airway and other epithelial cells that lead to mucus hypersecretion (and often infection), by promoting differentiation of MCCs and reduction of mucus secreting cells, and enabling improved mucociliary clearance.
• Administration of a GSI may improve epithelial function in cystic fibrosis without regard to the CFTR mutations causing the underlying disease.
• a GSI may be administered alone or in combination with any CFTR modulator or combination of CFTR modulators.
• methods for treating cystic fibrosis comprising administering to a patient in need thereof a therapeutically effective amount of a GSI and a CFTR modulator.
• the GSI may be administered prior to, after or concurrently with the CFTR modulator.
• the GSI is administered orally to a patient taking a CFTR modulator.
• the GSI may be provided in a single course of treatment or may be provided intermitently in combination with a CFTR modulator dosing regimen.
• a CFTR modulator may be administered daily and a GSI may be administered daily for 1, 3, 5, 7, 10, 14, 18, 21, 24, 28 or 30 days, and then stopped.
• the GSI is administered intermittently, such as every 3 days, or weekly. It will be appreciated that references to daily dosing amounts herein can be accomplished by dosing regimens other than daily, e.g., a weekly dose of 35mg would correspond to a daily dose of 5mg/day.
• slow-release formulations such as depots or patch formulations are known in the art and can be utilized to provide doses equivalent to the daily doses described herein.
• the GSI dosing regimen may be repeated if necessary.
• the GSI is selected from semagacestat, nirogacestat, MK-0752, RO-492907, or crenigacestat. In one embodiment, the GSI is semagacestat.
• CFTR modulators useful in the present invention include CFTR potentiators, correctors, premature stop codon suppressors, amplifiers and combinations thereof.
• CFTR modulators include ivacaftor, lumacaftor, tezacaftor and elexacaftor and combinations.
• Ivacaftor is marketed in tablet and granule form as KALYDECO. (See U.S. Patent Nos. 7,495,103 and 8,754,224).
• Ivacaftor and tezacaftor are marketed as SYMDEKO. (See U.S. Patent Nos. 7,745,789; 7,776,905; 8,623,905 and 10,239,867).
• ORKAMBI A combination of lumacaftor and ivacaftor is marketed as ORKAMBI.
• ORKAMBI A combination of elexacaftor, ivacaftor and tezacaftor (“ETI”) is marketed as TRIKAFTA.
• ETI tezacaftor
• Additional CFTR modulators that may be used in the present invention are in development. (See, e.g., U.S. Patent Nos.
• methods are provided for treating cystic fibrosis by administration of a therapeutically effective amount of a GSI and a CFTR modulator.
• a method of treating cystic fibrosis in which a GSI is systemically administered to a patient being administered or in need of administration of a CFTR modulator.
• the GSI may be provided concurrently, prior to or after administration of the CFTR modulator.
• the GSI is provided intermittently in combination with a CFTR modulator dosing regimen.
• a method of treating cystic fibrosis in a patient being administered or in need administration of a CFTR modulator comprising systemically administering to such patient about O.lmg to about 50mg semagacestat daily wherein the oral administration of semagacestat is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• semagacestat is systemically administered at dosages of from about 0.5mg to about 40mg daily.
• semagacestat is administered at a dosage of from about 0.5mg to about 20mg daily.
• a method of treating cystic fibrosis in a patient taking a CFTR modulator comprising systemically administering semagacestat to a patient in need thereof about 5 pg to 1 mg/kg daily, preferably from about 50 to 100 pg/kg daily.
• semagacestat is provided orally to a patient taking a CFTR modulator wherein said patient's semagacestat plasma concentration at steady state following multiple dose administration comprises an AUC (area under the curve) less than 1220 ng*hr/mL, wherein the systemic administration of semagacestat is effective in reducing mucus in such patient' s lungs or preventing mucus accumulation in such patient's lungs.
• said patient's steady state semagacestat plasma concentration comprises an AUC less than 1220 ng*hr/mL, less than 600 ng*hr/mL, or less than 250 ng*hr/mL.
• Steady state semagacestat levels may be determined following about 1 week or about 2 weeks or more of administering the therapeutically effective amount of semagacestat.
• a method of treating cystic fibrosis in a patient being administered or in need administration of a CFTR modulator comprising systemically administering to a patient in need thereof about 0.1 mg to about 50 mg nirogacestat daily wherein the oral administration of nirogacestat is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• the nirogacestat is systemically administered at dosages of from about 0.5mg to about 40mg daily, or from about 0.5 to about 30mg, or of from about 0.5mg to about 20mg daily.
• a method of treating cystic fibrosis in a patient taking a CFTR modulator comprising systemically administering to a patient in need thereof about 8 pg to about 0.9 mg/kg daily, preferably from about 10 to about 300 pg/kg nirogacestat daily.
• a method of treating cystic fibrosis in a patient taking a CFTR modulator comprising systemically administering to a patient in need thereof about 0.1 mg to about 20mg RO-4929097 daily wherein the oral administration of RO-4929097 is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• the RO- 4929097 is systemically administered at dosages of from about 0.1 to about 10mg daily, or from about 0.5mg to about 10mg daily, or of from about O.lmg to about 5mg daily.
• a method of treating cystic fibrosis in a patient taking a CFTR modulator comprising systemically administering to a patient in need thereof about 5 pg to about 0.4 mg/kg daily, preferably from about 50 to about 100 pg/kg RO-4929097 daily.
• a method of treating cystic fibrosis in a patient taking a CFTR modulator comprising systemically administering to a patient in need thereof about 0.1 mg to about 40 mg MK-0752 daily wherein the oral administration of MK-0752 is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• the MK-0752 is administered at a dose in the range of from about 0.1 to about 30mg daily, or from about 0.1 mg to about 20 mg daily, and most preferably of from about 0.1 mg to about 10 mg daily.
• a method of treating cystic fibrosis in a patient taking a CFTR modulator comprising systemically administering to a patient in need thereof about 2.5 pg to about 0.6 mg/kg daily, preferably from about 2.5 to about 500 pg/kg MK-0752 daily.
• the GSI is provided by oral administration.
• the GSI may be provided as an immediate release oral dosage form, or as a controlled release oral dosage form.
• the human subject that is treated by methods of the invention e.g., as described above, is one that has been diagnosed as having a respiratory disease characterized by mucus hypersecretion.
• the respiratory disease characterized by mucus hypersecretion for which the subject is diagnosed as having is selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesia, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans.
• Idiopathic pulmonary fibrosis and other fibrotic lung disorders and respiratory infection including exacerbations in chronic respiratory disorders, and mucus accumulation in response to acute infection.
• the subject is a subject diagnosed as having cystic fibrosis. In other embodiments, the subject is a subject diagnosed as having chronic obstructive pulmonary disease.
• Air-Liquid Interface (ALT) cultures were prepared as described in Rajar EK, Nayak JV, Milla CE, Axelrod JD. Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation. J CI Insight. 2016;l(13);e88027. Human nasal epithelial cells (HNECs) were generated from human sinonasal epithelial brushings or from tissue obtained from patients undergoing endoscopic sinus surgery at Stanford Hospital and cultured as described in Vladar et al.
• HNECs Human nasal epithelial cells
• FIG. 3 demonstrates the method of evaluating airway cellular composition.
• nuclei red; DAPI
• MCCs green; GFP
• non- ciliated cells absence of GFP
• Acetylated tubulin blue marks cilia. Samples were blinded for treatment group prior to scoring.
• Figure 5 shows an increase in the ratio of ciliated to non-ciliated cells following 3 days of systemic (IP) semagacestat treatment at both the low and high doses.
• Example 3 Dependence of Multiciliated Cell Formation on Timing of Treatment in Differentiating and Mature Airway Epithelia.
• ALI HNEC cultures were prepared as described in Vladar et al. ALI cultures were treated with and without administration of 11-13 on days 7-14. DAPT (lum), semagacestat (125nm) or vehicle control were administered on days 14-21. PFA-fixed cultures were stained for Muc5AC (red; mucin producing secretory cells), Acetylated tubulin (green; MCCs) and E- Cadherin (blue to reveal cell boundaries).
• Figure 13 shows the effect of semagacestat and DAPT treatment in an ALI model of chronic inflammation.
• HNEC cultures from a CF patient were treated with IF- 13 from ALI+7 to 14 to induce inflammation.
• DAPT and semagacestat increase the percentage off MCCs in controls (left).
• IL-13 treatment increases the percentage of mucin positive secretory cells and decreases the percentage of MCCs.
• Subsequent DAPT or semagacestat treatment rescues cell composition, increasing the percentage of MCCs and decreasing the percentage of mucin positive secretory cells.
• Figure 14 shows the quantitation of MCCs per total luminal cells of the data from Figure 13.
• Example 5 Representative Ussing chamber tracings Cells grown at an air liquid interface were mounted on a holding slider and inserted on an Ussing chamber for electrophysiological short circuit current (Ise) measurements. Solutions in the serosal and mucosal bath were prepared so that a chloride gradient was established between both sides. After stable baseline current recordings were obtained, agonists were added in the following order: amiloride (10 ⁇ M) to block sodium channel activity, forskolin (10 ⁇ M) to stimulate CFTR, Ivacaftor (10 ⁇ M) to potentiate CFTR activity, and CFTRinh-172 (20 ⁇ M) to block CFTR current. For each agonist signals were monitored until a plateau in current was noted before adding the next agonist. The delta-Isc in response to CFTRinh-172 was used as our main read out for CFTR-mediated chloride transport. Results are shown in Figure 15.
• HNECs GSIs
• Semagacestat GSIs
• HNECs HNECs from CF patients and non-CF controls were collected and grown at air-liquid interface to maturity (+2 Id) according to Vladar et al. Cultures were then treated with DAPT, Semagacestat, the CFTR modulator Lumacaftor or vehicle control added to basal media 3x per week for 2 weeks. Filter inserts were then assessed for short circuit current (Isc) against a chloride gradient in Ussing chambers to assess CFTR activity.
• Figure 16 shows representative tracings.
• wild-type control and CF (F508A/F508A) HNEC cultures were grown to maturity with or without semagacestat from ALI +0-21d and cultures were treated with or without Lumacaftor (VX-809) from ALI +19-21d and Ivacaftor (VX-770) for ten minutes prior to fixation. PFA fixed membranes were then stained for Acetylated tubulin (green; MCCs) and E-Cadherin (red). Results are shown in Figure 19. Note that combination treated cultures differentiated MCCs as well as or better than cultures treated with semagacestat alone.
• Figure 20 shows the quantitation of data for healthy patient and CF donor 1 in Figure 19.
• the semagacestat-induced increase in MCC/total cell ratio is further increased by Lumacaftor.
• Example 9 GSI treatment induces multiciliated cell formation in cystic fibrosis epithelia.
• Example 10 GSI treatment induces structurally normal cilia in healthy and CF epithelia.
• Figure 22 shows SEM of healthy and CF primary human airway epithelial cultures, showing that multiciliated cells formed under DAPT treatment are indistinguishable from those in untreated healthy cultures.
• Example 11 Multiciliated Cell Formation is induced by a variety of GSIs
• Figure 25 shows the quantitation of MCCs per total luminal cells of the data shown in Figure 24.
• Example 12 CFTR current is not diminished by GSI treatment
• Example 13 GSI treatment has no impact on ionocyte formation.
• ionocytes are of particular interest due to prior assertions about CFTR expression, in addition to measuring current, we assayed ionocyte prevalence with GSI treatment.
• Primary healthy airway epithelial cells were treated with DAPT during differentiation only (ALI+0 to +2 Id) and labeled at ALI+21d with anti-FOXIl (green; an ionocyte specific marker), and acetylated a-Tubulin (red) antibodies and stained with DAPI (blue) to mark nuclei.
• Figure 29 shows that both untreated and DAPT treated cultures contained a similar small number of FOXI1 positive nuclei, indicative of ionocytes. Therefore, the prior suggestion that ionocyte numbers would decrease with Notch inhibition appears not to be correct.
• Example 14 Effect of low concentrations of the GSI MK-0752 and the CFTR modulator Elexacaftor on ciliary beat frequency (CBF).
• Figure 31 shows ciliary beat frequency (CBF) and cilium length of primary human epithelial cells treated with DAPT versus untreated controls.
• CBF ciliary beat frequency
• Example 15 Airway surface liquid (ASL) reabsorption characteristic of CF is attenuated by GSI treatment to the same degree as CFTR modulator drugs.
• ASL Airway surface liquid
• Example 16 Dramatic Improvement in mucus transport with combined GSI and CFTR modulator treatment.
• Results shown in Figure 34 show that control cells showed little movement of beads, reflective of poor mucus transport. Significant increases in movement were observed with either ETI or semagacestat treatment (p ⁇ 0.01 for either treatment vs control). Remarkable increases in transport were noted with the combination of ETI and semagacestat (p ⁇ 0.05 for comparisons against either treatment alone). This can be taken as demonstrating strong enhancement effects in mucociliary transport upon combination treatment, and predicts substantial benefit to patients on CFTR modulator therapy by adding GSI treatment.
• a method of treating a respiratory disease characterized by mucus hyper-secretion comprising: administering a low dose of a GSI to a human patient in need of such treatment; and wherein the mucus in such patient's lungs is reduced or mucus accumulation in such patient's lungs is inhibited.
• the respiratory disease is selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans, idiopathic pulmonary fibrosis and other fibrotic lung disorders and respiratory infection, including exacerbations in chronic respiratory disorders and mucus accumulation in response to acute infection.
• GSI is selected from the group consisting of semagacestat, avagacestat, GS-1, DBZ, L-685,458, BMS-906024, crenigascestac, MRK 560, nirogaeestat, RO-4929097, MK-0752, itanapraced, LY-3056480, fosciclopirox, tarenflurbil, and begacestat.
• GSI is selected from the group consisting of semagacestat., nirogaeestat, MK-0752, RO-492907, or crenigacestat.
• a method of treating a respiratory disease characterized by mucus hyper-secretion comprising: systemically administering to a human patient in need of such treatment a therapeutically effective amount of semagacestat, wherein said patient's semagacestat plasma concentration at steady state following about 1 week or after about 2 weeks or more of administering the therapeutically effective amount has an AUC less than 1220 ng*hr/mL, and wherein the administration of semagacestat is effective in reducing mucus in such patient's lungs or inhibiting mucus accumulation in such patient's lungs.
• the respiratory disease is selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease, primary ciliary dyskinesis, chronic bronchitis, asthma, idiopathic and secondary bronchiectasis, bronchiolitis obliterans, idiopathic pulmonary fibrosis and other fibrotic lung disorders and respiratory infection, including exacerbations in chronic respiratory disorders and mucus accumulation in response to acute infection.
• a method of treating cystic fibrosis comprising: administering an effective amount of a GSI to a human patient being administered or need administration of one or more CFTR modulators, and wherein the mucus in such patient's lungs is reduced or mucus accumulation in such patient's lungs is inhibited upon administration of the GSI.
• GSI is selected from the group consisting of semagacestat, avagacestat, GS-1, DBZ, L-685,458, BMS-906024, crenigascestat, MRK 560, nirogacestat, RO-4929097, MK-0752, itanapraced, LY-3056480, fosciclopirox, tarenflurbil, and begacestat.
• a method of treating cystic fibrosis comprising: administering an effective amount of a GSI to a human patient taking a CFTR modulator; wherein the GSI is selected from the group consisting of semagacestat, avagacestat, GS-1, DBZ. L-685,458, BMS-906024, crenigascestat, MRK 560, nirogacestat, RO-4929097, MK-0752, itanapraced,
• LY-3056480 fosciclopirox, tarenflurbil, and begacestat; wherein the CFTR modulator is selected from the group consisting of ivacaftor, lumacaftor, tezacaftor, elexacaftor and combinations thereof; and wherein the mucus in such patient's lungs is reduced or mucus accumulation in such patient's lungs is inhibited upon administration of the GSI.
• GSI is selected from the group consisting of semagacestat., nirogacestat, MK-0752, RO-492907, or crenigacestat.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Life Sciences & Earth Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Epidemiology (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Pulmonology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• Nutrition Science (AREA)
• Physiology (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Priority Applications (10)

Applications Claiming Priority (2)

Publications (3)

Family

ID=80323184

Family Applications (1)

Country Status (11)

Cited By (1)

Families Citing this family (1)

Citations (24)

Family Cites Families (2)

• 2021
  • 2021-08-19 WO PCT/US2021/046742 patent/WO2022040447A2/en not_active Ceased
  • 2021-08-19 EP EP21859146.9A patent/EP4199914A4/en active Pending
  • 2021-08-19 BR BR112023002651A patent/BR112023002651A2/pt unknown
  • 2021-08-19 AU AU2021329507A patent/AU2021329507A1/en active Pending
  • 2021-08-19 IL IL300405A patent/IL300405A/en unknown
  • 2021-08-19 CA CA3188234A patent/CA3188234A1/en active Pending
  • 2021-08-19 US US18/018,999 patent/US20230277550A1/en active Pending
  • 2021-08-19 JP JP2023512428A patent/JP2023538125A/ja active Pending
  • 2021-08-19 KR KR1020237009414A patent/KR20230052954A/ko active Pending
  • 2021-08-19 MX MX2023001993A patent/MX2023001993A/es unknown
  • 2021-08-19 CN CN202180051358.5A patent/CN116033895A/zh active Pending

Patent Citations (28)

Non-Patent Citations (40)

Also Published As

Similar Documents

Legal Events