Use of GADD34 inhibitors such as sephin1 for the treatment of fibrotic airways disease

Abstract

This disclosure relates to the unexpected discovery that agents capable of inhibiting GADD34 are effective in treating or alleviating the symptoms of fibrotic airways diseases. In patients who have been diagnosed with a fibrotic airways disease, the methods of this disclosure can, e.g., reverse or inhibit the worsening of symptoms related to such condition and / or prevent development of new symptoms.

Description

[0001] Docket No.: 6-25007 / 161118.08401

[0002] TREATMENT OF FIBROTIC AIRWAYS DISEASE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 696,569, filed September 19, 2024. The foregoing application is incorporated by reference herein in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under ES033290 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] FIELD OF THE INVENTION

[0008] This invention relates to methods and agents for treating fibrotic airways disease.

[0009] BACKGROUND OF THE INVENTION

[0010] Bronchiolitis obliterans (BO) is a devastating lung disease of the small airways. The gold standard for diagnosing BO is lung biopsy, in which progressive luminal narrowing and subsequent obliteration of the airway lumen are identified on lung histopathology. Although BO was previously thought to occur primarily after organ transplant, it has become increasingly associated with other insults, including autoimmune diseases, severe respiratory tract infections, and toxic inhalation exposures. In adults, organ transplants remain the leading cause of BO, with nearly 50% of all lung transplants affected by BO at 5 years post-transplant. In addition, autoimmune disorders such as rheumatoid arthritis (RA) and graft-versus-host disease (GVHD) following bone marrow transplant (BMT) are also associated with BO. In children, BO is most frequently seen following a severe respiratory tract infection. Common respiratory tract infections associated with BO include adenovirus, mycoplasma, and respiratory syncytial virus (RSV). However, the mechanisms contributing to BO induction after exposure remain underexplored.

[0011] Among adults without a history of organ transplantation, inhalation exposure to certain toxic chemicals is also associated with BO. Chemical inhalation exposures associated with BO include exposure to chemical warfare agents such as sulfur mustard, deployment-related exposures to bum pits and sulfur mines, as well as occupational inhalation exposures to certain volatile Docket No.: 6-25007 / 161118.08401 organic compounds. Specific to occupational exposures, sentinel cases of flavorings-related lung disease or ‘popcorn lung’ were described in young workers following repetitive inhalation of the flavoring chemicals diacetyl (2,3 -butanedione) and 2,3-pentanedione at occupationally relevant concentrations. Four of the nine (44%) initially reported cases of flavorings-related lung disease were listed for lung transplant, highlighting the debilitating nature of this disease with no FDA- approved treatment.

[0012] SUMMARY OF THE INVENTION

[0013] This disclosure addresses the need mentioned above in a number of aspects. In one aspect, the disclosure provides a method of treating a fibrotic airways disease in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an agent that inhibits a growth arrest and DNA-damage inducible protein 34 (GADD34) in a cell of the subject.

[0014] In some embodiments, the fibrotic airways disease is associated with the GADD34 pathway. In some embodiments, inhibition of GADD34 results in down-regulation of the integrated stress response (ISR) pathway. In some embodiments, the fibrotic airways disease is selected from bronchiolitis obliterans, constrictive bronchiolitis, lymphocytic bronchiolitis, tracheal stenosis, an infection, an autoimmune disease, and other inflammatory diseases.

[0015] In some embodiments, the subject has an increased expression level of the GADD34 compared to a reference subject. In some embodiments, the method comprises identifying the subject having an increased expression level of the GADD34 compared to a reference subject. In some embodiments, the reference subject does not have the fibrotic airways disease.

[0016] In some embodiments, the method comprises analyzing a GADD34 gene or a GADD34 protein of a biological sample from the subject or the reference subject.

[0017] In some embodiments, the agent is an antibody, an aptamer, a protein, a peptide, a nucleic acid, or a small molecule. In some embodiments, the agent comprises Sephinl, a stereoisomer thereof, a derivative thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0018] In some embodiments, Sephin 1 has a chemical structure below: Docket No.: 6-25007 / 161118.08401

[0019] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0020] In some embodiments, the agent is administered to the subject at one or more doses of from about 0.01 to about 10 mg / kg of body weight of the subject. In some embodiments, the agent is administered at one or more doses of from about 0.1 to about 5 mg / kg of body weight of the subject. In some embodiments, the agent is administered at one or more doses of from about 0.1 to about 1 mg / kg of body weight of the subject.

[0021] In some embodiments, the one or more doses of the agent are administered at least every 6 hours, 12 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0022] In some embodiments, the agent is administered to the subject intratumorally, intravenously, subcutaneously, intraosseously, orally, transdermally, sublingually, intratracheally, by nebulization, in sustained release, in controlled release, in delayed release, or as a suppository.

[0023] In some embodiments, the agent is administered to the subject via inhalation or via direct instillation into lower airways. In some embodiments, the inhalation is carried out with a nebulizer, an inhaler, a sprayer, a powder dispenser, or a dry powder generator. In some embodiments, the direct instillation is carried out by intratracheal or mucosa. In some embodiments, the direct instillation is carried out with an intrapulmonary aerosolizer or a sub-miniature aerosolizer.

[0024] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent or therapy.

[0025] In another aspect, this disclosure provides use of an agent that inhibits a GADD34 in the manufacture of a medicament for treating a fibrotic airways disease in a subject in need thereof.

[0026] In yet another aspect, this disclosure provides an agent that inhibits a GADD34 for use in treatment of a fibrotic airways disease in a subject in need thereof. Docket No.: 6-25007 / 161118.08401

[0027] The foregoing summary is not intended to define every aspect of the disclosure, and additional aspects are described in other sections, such as the following detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, or paragraph, or section of this document. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIGS. 1A, IB, and 1C show inhalation vapor exposure to diacetyl for inducing airway scarring in Sprague Dawley rats. FIG. 1 A shows Kaplan-Meier survival curve of Sprague-Dawley rats exposed to 200 parts-per-million (ppm) diacetyl (DA; 2,3-butanedione; red; n=43) vapor versus air-exposed control rats (blue, n=20; Mantel-Cox regression, ***p<0.0001). FIG. IB shows oxygen saturations in DA (red; n=27) and air controls (blue; n=12). Significant hypoxemia developed in DA-exposed rats and differed significantly from air-exposed controls until the end of the study (t-test; *p<0.05). FIG. 1C shows a percent (%) weight change in DA (red; n=27) and air controls (blue; n=12). Percent weight change differed significantly in DA animals from air for all time points (Holm-Sidak t-test; *p<0.05). Representative Trichrome stained sections of air controls (FIGS. 1 A and IB) and DA-exposed (FIGS. 1C and ID) rat airways at the study end (Day 19; scale bars: 500 pm (FIGS. 1A and 1C) and 200 pm (FIGS. IB and ID). Solid arrows identify intrapulmonary bronchial airways, and the hollow arrow identifies adjacent bronchial vessels.

[0030] FIG. 2 shows rat lung tissue fixed and stained for GADD34 in room air- (left) and DA- exposed (right) at two weeks following diacetyl (DA) exposure (scale bar: 500 pm and 200 pm, respectively). Increased staining of GADD34 centered around intrapulmonary airways (right images).

[0031] FIG. 3 shows rat lung tissue fixed and stained for GADD34 and nuclear stain DAPI (blue) in room air- (left) and DA-exposed (right) at two weeks following diacetyl (DA) exposure (bar: 200 pm). Docket No.: 6-25007 / 161118.08401

[0032] FIG. 4 shows higher magnification images of DA-exposed airways stained with a-SMA (left) and a-tubulin (right) (bar: 50 pm). GADD34 expression is observed in epithelial cells located above a-SMA-positive cells (left) and below a-tubulin-positive cells (right).

[0033] FIG. 5 shows hematoxylin and eosin (H&E)-stained lung section from a 6-year-old boy with post-infectious bronchiolitis obliterans (PIBO) who died of respiratory failure (bar: 5 mm). The black arrow highlights airway affected by airway remodeling (left image). Immunofluorescent staining on distal lungs with PIBO for GADD34, pan-cytokeratin (CK), and nuclear stain DAPI. Arrows highlight co-staining of GADD34 with pan-cytokeratin positive cells (bar: 200 pm).

[0034] FIG. 6 shows the experimental outline of 7-8 week-old Sprague-Dawley rats exposed to diacetyl (DA) vapors 200 parts-per-million (ppm) for 6 hours / day x 5 days (‘exposure period,’ bar) and then monitored for up to 2 weeks (Day 19; ‘post-exposure monitoring’). Syringes depict intraperitoneal injection of sephinl (GADD34 inhibitor; 5mg / kg / dose) administered daily starting immediately after the exposure period and continued until the study end. Four total groups undergoing experimentation: (1) DA+Sephinl, (2) DA + Dimethylsulfoxide (DMSO), (3) Air- exposed+Sephinl, and (4) Air + DMSO.

[0035] FIG. 7 shows a Kaplan-Meier survival curve of Sprague-Dawley rats exposed to 200 parts- per-million (ppm) diacetyl (DA) or air and monitored for 2 weeks post-exposure (Day 19). Groups include DA+Sephinl (n=8), DA+DMSO (n=8), Air+Sephinl (n=6), and Air + DMSO (n=6). Survival was significantly worse in those animals exposed to DA and treated with DMSO (DA+DMSO, Mantel-Cox, **p=0.0014), but did not differ significantly in those rats exposed to DA+Sephinl compared to Air-exposed and treatment matched controls.

[0036] FIG. 8 shows a percentage (%) weight change from those groups previously described in Figure 7. Groups include DA+Sephinl (n=8), DA+DMSO (n=8), Air+Sephinl (n=6), and Air+DMSO (n=6). Percent weight change differed significantly in DA-exposed animals from air for all time points. Percent weight change was significantly improved in DA+Sephinl vs. DA+DMSO at Days 13, 15, 17, and 19 (two-way ANOVA, *p<0.05).

[0037] FIG. 9 shows oxygen saturations at 1 week s / p diacetyl (DA) exposure (Day 12) in DA+Sephinl rats (n=8) and Diacetyl + DMSO (n=7). Oxygen saturations were significantly worse in DA+DMSO versus DA+Sephinl (t-test; **p<0.01). Docket No.: 6-25007 / 161118.08401

[0038] FIG. 10A shows representative Trichrome-stained images of airways from rat lung tissue sections obtained following exposure and treatment. The samples are grouped into four conditions: (1) Air exposure with vehicle control (dimethyl sulfoxide; DMSO) (top left panel); (2) Air exposure with Sephinl treatment (top right panel); (3) Diacetyl (DA) exposure with DMSO (bottom left panel); and (4) DA exposure with Sephinl treatment (bottom right panel). FIG. 10B shows quantitative measurements of lung collagen content determined using a hydroxyproline assay, reported as micrograms per milliliter (pg / mL) of homogenized lung tissue. The data represent the following groups: Air+DMSO (closed circles; mean = 6.5, SD = 34.0), Air+Sephinl (half circles; mean = 12.4, SD = 7.0), DA+DMSO (closed circles; mean = 49.8, SD = 19.5), and DA+Sephinl (half circles; mean = 28.9, SD = 14.7) (n = 6 per group). Statistical analysis using two-way ANOVA revealed a significant interaction between exposure and treatment (****p < 0.0001). Collagen content was significantly increased in the DA+DMSO group relative to the Air+DMSO group (Tukey’s post hoc test, ****p < 0.0001), and significantly decreased in the DA+Sephinl group compared to the DA+DMSO group (*p = 0.048).

[0039] FIG. 11A shows representative images of rat airway epithelial organoids generated from tracheal epithelial cells isolated from rats exposed to air and treated with DMSO. Organoids were cultured ex vivo in Matrigel for seven days, followed by fixation and staining with hematoxylin and eosin (H&E, upper image) or immunofluorescent staining with antibodies against keratin 5 (K5) and nuclear counterstaining with 4' ,6-diamidino-2-phenylindole (DAPI). Brightfield and fluorescence images are shown (scale bar = 50 pm). FIG. 1 IB shows lower magnification images of airway organoids from four exposure and treatment groups — Air+DMSO (top left), DA+DMSO (bottom left), Air+Sephinl (top right), and DA+Sephinl (bottom right) — stained with calcein AM (viable cells) and BOBO-3 Iodide (non-viable cells) (scale bar = 500 pm). FIG. 11C shows the number of viable cells per organoid culture well after seven days in Matrigel. The cells were dissociated from tracheal epithelium of rats previously exposed to Air (control, circles) or DA (squares) (n = 5-6 per group). Two-way ANOVA identified a significant interaction between exposure and treatment (**p = 0.0066). The number of viable cells in the DA+Sephinl group (mean = 3.5 x 106, SD = 1.9 x 106cells / well) was significantly greater than in both the Air+Sephinl group (mean = 1.6 x 106, SD = 0.7 x 106cells / well; **p = 0.007) and the DA+DMSO group (mean Docket No.: 6-25007 / 161118.08401

[0040] = 1.6 x 1 o6, SD = 0.3 x i o6cells / well; **p = 0.008), based on ANOVA followed by Fisher’s correction.

[0041] FIG. 12A shows representative Western blot images showing expression of phosphorylated eukaryotic translation initiation factor 2 alpha (p-eIF2a; ~38 kDa) and total eIF2a (—65 kDa), with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as a loading control (~37 kDa). Protein samples were obtained from primary rat airway epithelial cells cultured in organoid conditions and harvested seven days post-culture. The four experimental groups include: (1) airexposed cells treated with dimethyl sulfoxide (Air + DMSO; first three lanes from the left), (2) air-exposed cells treated with Sephinl (Air + Sephinl; second set of three lanes), (3) diacetyl- exposed cells treated with DMSO (DA + DMSO; third set of three lanes), and (4) di acetyl -exposed cells treated with Sephinl (DA + Sephinl; final three lanes on the right). FIG. 12B shows semi- quantitative analysis of the western blot results shown in FIG. 12A. The left panel shows normalized p-eIF2a expression levels relative to GAPDH, and the right panel shows normalized total eIF2a levels relative to GAPDH. Data are shown for the following groups: Air + DMSO (closed circles; mean ± SD: 0.76 ± 0.15), Air + Sephinl (half circles; 0.44 ± 0.05), DA + DMSO (closed circles; 0.67 ± 0.12), and DA + Sephinl (half circles; 1.50 ± 0.34) (n = 3-4 per group). Statistical comparisons were conducted using two-way ANOVA with interaction between exposure and treatment conditions. A significant increase in p-eIF2a expression was observed in the DA + Sephinl group compared to DA + DMSO (***p = 0.0003) and Air + Sephinl (****p < 0.0001). Total eIF2a expression did not differ significantly among the groups (two-way ANOVA, p = 0.23).

[0042] FIG. 13 shows representative high-resolution, mass-accurate MSI spectra showing mass- to-charge ratio (m / z) measurements with sub-3 ppm mass accuracy. The spectra include signals from a solvent (DMSO) blank (unlabeled dots), rat lung homogenates + DMSO (black dots), a Sephinl solution standard (light grey dots and corresponding curve), and rat lung homogenates obtained from animals treated with nebulized Sephinl (dark grey dots and corresponding curve). A peak corresponding to Sephinl is observed at an estimated m / z value of approximately 197.

[0043] FIG. 14A shows quantitative measurements of Sephinl concentration in homogenized rat lung tissue collected immediately after the end of Sephinl dosing. FIG. 14A illustrates the concentration of Sephinl measured in homogenized rat lung tissue harvested immediately Docket No.: 6-25007 / 161118.08401 following the final nebulized treatment on Day 19. Data are shown for each exposure-treatment group: Air + DMSO (open circles), DA + DMSO (open circles), Air + Sephinl (closed circles), DA + Sephinl (closed circles), and Air + intraperitoneal (IP) Sephinl injection (black circles / gray bar) (n = 4-5 per group; two-way ANOVA, ***p = 0.001). Lung Sephinl concentrations were significantly increased in rats exposed to Air and treated with nebulized Sephinl compared to Air + nebulized DMSO (92.1 ± 33.4 ng / ml vs. 0.7 ± 0.3 ng / ml, *p = 0.02). Similarly, lung Sephinl concentrations were significantly increased in rats exposed to DA and treated with nebulized Sephinl compared to DA + nebulized DMSO (51.5 ± 12.9 ng / ml vs. 0.8 ± 0.2 ng / ml, **p = 0.004). Lung Sephinl concentration in the Air + IP Sephinl group (positive standard) was 186.1 ± 34.1 ng / ml. FIG. 14B shows the concentration of Sephinl measured in rat plasma collected immediately following the final nebulized treatment on Day 19 from the same exposure-treatment groups described in FIG. 14A (n = 4-5 per group; two-way ANOVA, **p = 0.004). Plasma Sephinl concentrations did not differ significantly between rats exposed to Air and treated with nebulized Sephinl versus Air + nebulized DMSO (2.5 ± 1.4 ng / ml vs. 0.02 ± 0.03 ng / ml, p = 0.08). Plasma Sephinl concentrations were significantly increased in rats exposed to DA and treated with nebulized Sephinl compared to DA + nebulized DMSO (2.4 ± 1.1 ng / ml vs. 0.3 ± 0.4 ng / ml, *p = 0.048); however, these levels were approximately 25-fold lower than the lung Sephinl concentrations observed in FIG. 14A. Plasma Sephinl concentration in the Air ± IP Sephinl group (positive standard) was 35.4 ± 16.4 ng / ml.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] This disclosure is based at least on an unexpected discovery that agents capable of inhibiting GADD34 are effective in treating or alleviating the symptoms of fibrotic airways diseases. In patients who have been diagnosed with a fibrotic airways disease, the methods of this disclosure can, e.g., reverse or inhibit the worsening of symptoms related to such condition and / or prevent development of new symptoms.

[0046] Methods for Treating Fibrotic Airways Diseases

[0047] Accordingly, this disclosure provides methods of treating or ameliorating a symptom of a fibrotic airways disease in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an agent that inhibits a growth Docket No.: 6-25007 / 161118.08401 arrest and DNA-damage inducible protein 34 (GADD34) in a cell of the subject. In some embodiments, the fibrotic airways disease is associated with the GADD34 pathway.

[0048] GADD34 (e.g., Uniport Accession No. 075807), also known as protein phosphatase 1 regulatory subunit 15A (PPP1R15A), is a member of a group of genes whose transcript levels are increased following stressful growth arrest conditions and treatment with DNA-damaging agents. GADD34 is primarily involved in the cellular response to stress. It’s induced by various stressors like DNA damage, oxidative stress, and nutrient deprivation. GADD34 interacts with protein phosphatase 1 (PPI) to regulate protein synthesis. It plays a crucial role in the integrated stress response (ISR) by controlling the phosphorylation state of eukaryotic initiation factor 2 alpha (eIF2a). GADD34 has been implicated in both cell survival and death pathways. It can promote cell survival under certain stress conditions, but it can also contribute to cell death in other contexts.

[0049] In some embodiments, inhibition of GADD34 results in down-regulation of the integrated stress response (ISR) pathway. The integrated stress response (ISR) pathway is a signaling pathway that helps cells, tissues, and organisms adapt to different environments and maintain health. It is activated in response to a variety of stressors, including protein misfolding, nutrient deficiencies, heme abundance deficiencies, viral infections, hypoxia, and ultraviolet light. The ISR pathway is important for cellular quality control, translation regulation, and disease implications. It can lead to adaptation, survival, autophagy, and cell death. The ISR restores balance by reprogramming gene expression, which can include transcriptomic adaptation, proteomic adaptation, and metabolic adaptation. The ISR pathway also allows for communication between organelles, including the mitochondria and the nucleus.

[0050] Following protein damage, the endoplasmic reticulum (ER) of a cell plays a critical role in protein quality control. With the appropriate cellular response, damaged proteins are cleared, and protein synthesis re-initiated. Conversely, ER stress persists when damaged proteins accumulate following significant or repeated protein damage. A common adaptive pathway for sustained ER stress is activation of the integrated stress response (ISR). The central signaling event that results from ISR activation is phosphorylation of eukaryotic translational initiation factor 2 alpha (eIF2a). With phosphorylation, eIF2a halts global protein synthesis as a cytoprotective strategy to prevent further protein accumulation. In parallel, p-EIF2a activates the transcription factor ATF4 to promote cellular recovery. Although the ISR is primarily ‘pro-survival’, sustained ISR activation Docket No.: 6-25007 / 161118.08401 can also result in a maladaptive response. With sustained ISR activation, the cell upregulates the DNA damage-inducible transcript 3 gene (DDIT3; equivalent protein: CCAAT / enhancer-bind protein homologous protein (CHOP)). Conversely, ISR termination occurs through an inducible feedback loop when ATF4 and / or CHOP upregulates growth arrest and DNA damage-inducible protein (GADD34; gene: PPP1R15A). GADD34’s primary function is to dephosphorylate p- EIF2a with protein phosphatase 1 (PPI). Dephosphorylation of p-EIF2a shuts off ISR activation, allowing for re-initiation of translation.

[0051] To date, few studies have evaluated the ISR in humans affected by fibrotic airways diseases such as bronchiolitis obliterans. In a limited study of human airway sections from 6 subjects affected by bronchiolitis obliterans syndrome (BOS) compared to 4 non-transpl anted, age-matched control lungs, ER stress was a prominent feature in BOS subjects with severe airway remodeling versus matched control airways. These remodeled airways stained positive for ATF6 and CHOP expression, two proteins associated with ER stress and the ISR, respectively, with >40% of the airway epithelia in BOS subjects positive for both proteins. Hence, these human airway findings support ISR activation occurring in human subjects post-transplant with BOS.

[0052] As used herein, the terms “subject” and “patient” are used interchangeably irrespective of whether the subject has or is currently undergoing any form of treatment. As used herein, the terms “subject” and “subjects” may refer to any vertebrate, including, but not limited to, a mammal (e.g, cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and a human). The subject may be a human or a non-human. In more exemplary aspects, the mammal is a human. As used herein, the expression “a subject in need thereof’ or “a patient in need thereof’ means a human or non-human mammal that exhibits one or more symptoms or indications of disorders, and / or who has been diagnosed with fibrotic airways diseases. In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[0053] As used herein, the term “disease” is intended to be generally synonymous and is used interchangeably with, the terms “disorder” and “condition” (as in medical condition) in that all reflect an abnormal condition (e.g, fibrotic airways diseases) of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life. Docket No.: 6-25007 / 161118.08401

[0054] Fibrotic airways diseases, such as pulmonary fibrosis, are lung diseases that occur when lung tissue becomes damaged and scarred. This thickened, stiff tissue makes it harder for the lungs to work properly. Pulmonary fibrosis worsens over time. Some people can stay stable for a long time, but the condition gets worse faster in others.

[0055] In some embodiments, the fibrotic airways disease is selected from bronchiolitis obliterans, constrictive bronchiolitis, lymphocytic bronchiolitis, tracheal stenosis, an infection, an autoimmune disease, and other inflammatory diseases.

[0056] As used herein, the term “inhibit,” “decrease,” “reduced,” “reduction,” or “decrease” generally mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced,” “reduction,” “decrease,” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example, a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0057] In some embodiments, the subject has an increased expression level of GADD34 compared to a reference subject. In some embodiments, the method comprises identifying the subject having an increased expression level of GADD34 compared to a reference subject. In some embodiments, the reference subject does not have a fibrotic airways disease.

[0058] As used herein, the terms “increased,” “increase,” or “elevated” means an increase of at least 10% as compared to a reference level e.g., an expression level in a reference subject), for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0059] In some embodiments, the method comprises analyzing a GADD34 gene or a GADD34 protein of a biological sample from the subject or the reference subject. Docket No.: 6-25007 / 161118.08401

[0060] “Sample,” “test sample,” “biological sample,” and “patient sample” may be used interchangeably herein. The sample can be a sample of serum, urine plasma, amniotic fluid, cerebrospinal fluid, cells (e.g.. antibody-producing cells) or tissue. Such a sample can be used directly as obtained from a patient or can be pre-treated, such as by fdtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art. The terms “sample” and “biological sample,” as used herein, generally refer to a biological material being tested for and / or suspected of containing an analyte of interest. The sample may be any tissue sample from the subject. The sample may comprise protein from the subject.

[0061] In some embodiments, the level or activity of GADD34 may be measured by determining or estimating a protein level or mRNA level. Methods for determining or estimating a protein level or mRNA level are well known in the art. For example, the protein level (e.g., protein expression level) of GADD34 can be determined by SDS-PAGE, Western blot, or an immunoassay (e.g., immunoblotting assay, immunoprecipitation assay). The mRNA level may be determined by RT- PCR.

[0062] The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a “therapeutic agent,” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

[0063] The terms “therapeutic agent,” “therapeutic capable agent,” and “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.

[0064] In some embodiments, the agent is an antibody, an aptamer, a protein, a peptide, a nucleic acid, or a small molecule. In some embodiments, the agent comprises (E)-2-(2-Chlorobenzylidene) Docket No.: 6-25007 / 161118.08401 hydrazinecarboximidamide (also referred to herein as Sephinl or Icerguastat; CAS Number: 951441-04-6; SMILES: C1C1=CC=CC=C1 / C=N / NC(N)=N), a stereoisomer thereof, a derivative thereof or a pharmaceutically acceptable salt thereof, or a combination thereof.

[0065] In some embodiments, Sephin 1 has a chemical structure below:

[0066] In some embodiments, the agents used herein may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. “Enantiomer” means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms. “R” and “S” represent the configuration of substituents around one or more chiral carbon atoms. Thus, “R*” and “S*” denote the relative configurations of substituents around one or more chiral carbon atoms. The symbol in a structural formula represents the presence of a chiral carbon center.

[0067] “Racemate” or “racemic mixture” means a compound of equimolar quantities of two enantiomers, wherein such mixtures exhibit no optical activity, i.e., they do not rotate the plane of polarized light.

[0068] “Geometric isomer” means isomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a cycloalkyl ring, or to a bridged bicyclic system. Atoms (other than H) on each side of a carbon-carbon double bond may be in an E (substituents are on opposite sides of the carbon-carbon double bond) or Z (substituents are oriented on the same side) configuration. “R,” “S,” “St,” “R*,” “E,” “Z,” “cis,” and “trans” indicate configurations relative to the core molecule. Docket No.: 6-25007 / 161118.08401

[0069] The compounds of the invention may be prepared as individual isomers by either isomerspecific synthesis or resolved from an isomeric mixture. Conventional resolution techniques include forming the salt of a free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming the salt of the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each of the isomers of an isomeric pair using an optically pure acid, amine or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of either a starting material or a final product using various well known chromatographic methods.

[0070] When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure relative to the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight optically pure. Percent optical purity by weight is the ratio of the weight of the enantiomer over the weight of the enantiomer plus the weight of its optical isomer.

[0071] When a disclosed compound is named or depicted by a structure without indicating the stereochemistry, and the compound has at least one chiral center, it is to be understood that the name or structure encompasses one enantiomer of compound free from the corresponding optical isomer, a racemic mixture of the compound and mixtures enriched in one enantiomer relative to its corresponding optical isomer.

[0072] When a disclosed compound is named or depicted by a structure without indicating the stereochemistry and has at least two chiral centers, it is to be understood that the name or structure encompasses a diastereomer free of other diastereomers, a pair of diastereomers free from other diastereomeric pairs, mixtures of diastereomers, mixtures of diastereomeric pairs, mixtures of diastereomers in which one diastereomer is enriched relative to the other diastereomer(s) and mixtures of diastereomeric pairs in which one diastereomeric pair is enriched relative to the other diastereomeric pair(s).

[0073] The term “derivative” as used herein refers to a chemical substance related structurally to another, / .<?., an “original” substance, which can be referred to as a “parent” compound. A “derivative” can be made from the structurally related parent compound in one or more steps. The Docket No.: 6-25007 / 161118.08401 phrase “closely related derivative” means a derivative whose molecular weight does not exceed the weight of the parent compound by more than 50%. The general physical and chemical properties of a closely related derivative are also similar to the parent compound. “Pharmaceutically active derivative” refers to any compound that, upon administration to the recipient, is capable of providing directly or indirectly, the activity disclosed herein.

[0074] As used herein, the terms “treating,” “treat,” and “treatment” include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving or ameliorating the disease, pathologic or medical condition; and / or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms “treat,” “treatment,” and “treating” can extend to prophylaxis and can include preventing, prevention, lowering, stopping, or reversing the progression or severity of the condition or symptoms being treated. As such, the term “treatment” can include medical, therapeutic, and / or prophylactic administration, as appropriate. The term “treating” or “treatment” thus can include reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject’s condition.

[0075] The term “ameliorate,” as used herein, refers to the effects of administering an agent to a patient e.g., a myotonic dystrophy patent) that result in any indicia of success in the prevention, reduction, or reversal of one or more symptoms related to the condition. Reduction may be indicated in lesser severity, delayed onset of symptoms, or a slowing of disease progression. The prevention, reduction, or reversal of symptoms can be measured based on objective parameters, such as the results of a physical examination or laboratory test (z.e., blood test), decreased need for medication, decreased need for supportive measures (i.e., use of a ventilator), or increase in mobility. The prevention, reduction, or reversal of symptoms can also be measured based on subjective parameters, such as a reduction in pain or stiffness or an increase in a patient’s mobility and sense of well-being.

[0076] As used herein, the term “administering” refers to the delivery of cells by any route including, without limitation, oral, intranasal, intraocular, intravenous, intraosseous, intraperitoneal, intraspinal, intramuscular, intra-articular, intraventricular, intracranial, Docket No.: 6-25007 / 161118.08401 intralesional, intratracheal, intrathecal, subcutaneous, intradermal, transdermal, or transmucosal administration.

[0077] In some embodiments, the agent is administered to the subject intratumorally, intravenously, subcutaneously, intraosseously, orally, transdermally, sublingually, in sustained release, in controlled release, in delayed release, or as a suppository.

[0078] In some embodiments, the agent is administered to the subject intratumorally, intravenously, subcutaneously, intraosseously, orally, transdermally, sublingually, intratracheally, by nebulization, in sustained release, in controlled release, in delayed release, or as a suppository.

[0079] In some embodiments, the agent is administered to the subject via inhalation or via direct instillation into lower airways. In some embodiments, the inhalation is carried out with a nebulizer, an inhaler, a sprayer, a powder dispenser, or a dry powder generator. In some embodiments, the direct instillation is carried out by intratracheal or mucosa. In some embodiments, the direct instillation is carried out with an intrapulmonary aerosolizer or a sub-miniature aerosolizer.

[0080] An “effective amount” refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term “effective amount” is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an “effective amount” generally means an amount that provides the desired effect. A “therapeutically effective amount” of a compound with respect to the subject method of treatment refers to an amount of the compound in a preparation which, when administered as part of a desired dosage regimen (to a mammal, e.g., a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated, e.g. , at a reasonable benefit / risk ratio applicable to any medical treatment.

[0081] The compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg / m2, e.g., 10 to 750 mg / m2, e.g., 50 to 500 mg / m2of active ingredient per unit dosage form. In some embodiments, a dose may be presented in a single dose, or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per Docket No.: 6-25007 / 161118.08401 day. The sub-dose itself may be further divided, e.g., into a number of discrete, loosely spaced administrations.

[0082] The actual dosage amount of a composition of the present invention administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0083] In some embodiments, the agent is administered to the subject at one or more doses of from about 0.01 to about 10 mg / kg (e.g., 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg,

[0084] 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, or any intermediate value therebetween) of body weight of the subject.

[0085] In some embodiments, the agent is administered at one or more doses of from about 0.1 to about 5 mg / kg (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg,

[0086] 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3 mg / kg, 3.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 4 mg / kg, 4.1 mg / kg, 4.2 mg / kg, 4.3 mg / kg, 4.4 mg / kg, 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5 mg / kg, or any intermediate value therebetween) of body weight of the subject.

[0087] In some embodiments, the agent is administered at one or more doses of from about 0. 1 to about 1 mg / kg (0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.55 mg / kg, 0.6 mg / kg, 0.65 mg / kg, 0.7 mg / kg, 0.75 mg / kg, 0.8 mg / kg, 0.85 mg / kg, 0.9 mg / kg, 0.95 mg / kg, 1 mg / kg, or any intermediate value therebetween) of body weight of the subject.

[0088] In some embodiments, one or more doses of the agent are administered at least every 6 hours, 12 hours, 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, Docket No.: 6-25007 / 161118.08401 every 14 days, every 15 days, every 16 days, every 17 days, every 18 days, every 19 days, every 20 days, every 21 days, every 22 days, every 23 days, every 24 days, every 25 days, every 26 days, every 27 days, every 28 days, every 29 days, every 30 days, every 31 days, every 32 days, every 33 days, every 34 days, every 35 days, every 36 days, every 37 days, every 38 days, every 39 days, every 40 days, every 41 days, every 42 days, every 43 days, every 44 days, every 45 days, every 46 days, every 47 days, every 48 days, every 49 days, every 50 days, every 51 days, every 52 days, every 53 days, every 54 days, every 55 days, every 56 days, every 57 days, every 58 days, every 59 days, every 60 days, every 61 days, every 62 days, every 63 days, every 64 days, every 65 days, every 66 days, every 67 days, every 68 days, every 69 days, every 70 days, every 71 days, every 72 days, every 73 days, every 74 days, every 75 days, every 76 days, every 77 days, every 78 days, every 79 days, every 80 days, every 81 days, every 82 days, every 83 days, every 84 days, every 85 days, every 86 days, every 87 days, every 88 days, every 89 days, every 90 days, every 91 days, every 92 days, every 93 days, every 94 days, every 95 days, every 96 days, every 97 days, every 98 days, every 99 days, every 100 days, every 101 days, every 102 days, every 103 days, every 104 days, every 105 days, every 106 days, every 107 days, every 108 days, every 109 days, every

[0089] 110 days, every 111 days, every 112 days, every 113 days, every 114 days, every 115 days, every

[0090] 116 days, every 117 days, every 118 days, every 119 days, or every 120 days.

[0091] In some embodiments, the treatment produces a therapeutic effect may include improved respiratory function or improved survival. In some embodiments, the treatment results in at least 20% improvement respiratory function as compared to an untreated subject.

[0092] The term “therapeutic effect” is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance. The phrase “therapeutically effective amount” means the amount of such a substance that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The therapeutically effective amount of such substance will vary depending upon the subject and disease or condition being treated, the weight and age of the subject, the severity of the disease or condition, the manner of administration, and the like, which can readily be determined by one of ordinary skill in the art. For example, certain compositions described herein may be administered in a sufficient amount to produce a desired effect at a reasonable benefit / risk ratio applicable to such treatment. Docket No.: 6-25007 / 161118.08401

[0093] In another aspect, this disclosure provides use of an agent that inhibits a GADD34 in the manufacture of a medicament for treating a fibrotic airways disease in a subject in need thereof.

[0094] In yet another aspect, this disclosure provides an agent that inhibits a GADD34 for use in treatment of a fibrotic airways disease in a subject in need thereof.

[0095] Pharmaceutical Compositions and Kits

[0096] Also provided in this disclosure is a pharmaceutical composition comprising (i) an agent (e.g., a GADD34 inhibitor) described above, a stereoisomer thereof, a derivative / analog thereof, a prodrug thereof, a metabolite thereof, or a pharmaceutically acceptable salt thereof, and (ii) optionally a pharmaceutically acceptable carrier.

[0097] In some embodiments, pharmaceutical compositions described herein, e.g, for treating a myotonic disorder in a subject in need thereof, comprise: a GADD34 inhibitor such as Sephinl, a stereoisomer thereof, a derivative thereof or a pharmaceutically acceptable salt thereof, or a combination thereof.

[0098] In some embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of a compound. In other embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.

[0099] As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one component useful within the invention with other components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of one or more components of the invention to an organism.

[0100] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the composition, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0101] The term “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, Docket No.: 6-25007 / 161118.08401 excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.

[0102] As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof.

[0103] The compounds that can form salts are also within the scope of this disclosure. Unless otherwise indicated, reference to an inventive compound is understood to include reference to one or more salts thereof. The term “salt(s)” denotes acidic and / or basic salts formed with inorganic and / or organic acids and bases. In addition, the term “salt(s) may include zwitterions (inner salts), e.g., when a compound contains both a basic moiety, such as an amine or a pyridine or imidazole ring, and an acidic moiety, such as a carboxylic acid. Pharmaceutically acceptable (z.e., non-toxic, Docket No.: 6-25007 / 161118.08401 physiologically acceptable) salts are preferred, such as acceptable metal and amine salts in which the cation does not contribute significantly to the toxicity or biological activity of the salt. However, other salts may be useful, e.g., in isolation or purification steps which may be employed during preparation, and thus, are contemplated within the scope of the disclosure. Salts of the compounds may be formed, for example, by reacting a compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

[0104] Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bi sulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), hydroiodides, maleates (formed with maleic acid), 2-hydroxyethanesulfonates, lactates, methanesulfonates (formed with methanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3 -phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates (such as those mentioned herein), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.

[0105] Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; barium, zinc, and aluminum salts; salts with organic bases (for example, organic amines) such as trialkylamines such as tri ethylamine, procaine, dibenzylamine, N-benzyl-P-phenethylamine, 1 -ephenamine, N,N’-dibenzylethylene-diamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, dicyclohexylamine or similar pharmaceutically acceptable amines and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g, methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long-chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides and iodides), aralkyl halides ( ?.g., benzyl and phenethyl bromides), and others. In some embodiments, examples of salts include monohydrochloride, hydrogensulfate, methanesulfonate, phosphate or nitrate salts. Docket No.: 6-25007 / 161118.08401

[0106] Various forms of prodrugs are well known in the art and are described in: (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., Ch 31, (Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds. Ch 5, pgs 113-191 (Harwood Academic Publishers, 1991); and (d) Hydrolysis in Drug and Prodrug Metabolism, Bernard Testa and Joachim M. Mayer, (Wiley-VCH, 2003).

[0107] In addition, the compounds, subsequent to their preparation, can be isolated and purified to obtain a composition containing an amount by weight equal to or greater than 99% of a compound (“substantially pure”), which is then used or formulated as described herein. Such “substantially pure” compounds are also contemplated herein as part of the present disclosure.

[0108] “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. The present disclosure is intended to embody stable compounds.

[0109] The compounds of the present disclosure are intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium (D) and tritium (T). Isotopes of carbon include13C and14C. Isotopically labeled compounds of the disclosure can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically labeled reagent in place of the non-labeled reagent otherwise employed. For example, methyl ( — CH3) also includes deuterated methyl groups such as — CD3.

[0110] Compounds and / or pharmaceutically acceptable salts thereof can be administered by any means suitable for the condition to be treated, which can depend on the need for site-specific treatment or quantity of the compound to be delivered. Also embraced within this disclosure is a class of pharmaceutical compositions comprising a compound and / or pharmaceutically acceptable salts thereof; and one or more non-toxic, pharmaceutically acceptable carriers and / or diluents and / or adjuvants (collectively referred to herein as “carrier” materials) and, if desired, other active ingredients. The compounds may be administered by any suitable route, e.g., in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment Docket No.: 6-25007 / 161118.08401 intended. The compounds and compositions of the present disclosure may, for example, be administered orally, mucosally, rectally, or parentally including intravascularly, intravenously, intraperitoneally, subcutaneously, intramuscularly, and intrastemally in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. For example, the pharmaceutical carrier may contain a mixture of mannitol or lactose and microcrystalline cellulose. The mixture may contain additional components such as a lubricating agent, e.g., magnesium stearate, and a disintegrating agent such as crospovidone. The carrier mixture may be filled into a gelatin capsule or compressed as a tablet. The pharmaceutical composition may be administered as an oral dosage form or an infusion, for example.

[0111] Techniques and formulations generally may be found in Remmington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the agents can be formulated in liquid solutions, e.g., in physiologically compatible buffers such as Hank’s solution or Ringer’s solution. In addition, the agents may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

[0112] For oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, liquid capsule, suspension, or liquid. The pharmaceutical composition is, e.g., made in the form of a dosage unit containing a particular amount of the active ingredient. For example, the pharmaceutical composition may be provided as a tablet or capsule comprising an amount of active ingredient in the range of from about 0.1 to 1000 mg, e.g., from about 0.25 to 250 mg, and e.g., from about 0.5 to 100 mg. A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but can be determined using routine methods.

[0113] Any pharmaceutical composition contemplated herein can, for example, be delivered orally via any acceptable and suitable oral preparations. Exemplary oral preparations include, but are not limited to, for example, tablets, troches, lozenges, aqueous and oily suspensions, dispersible powders or granules, emulsions, hard and soft capsules, liquid capsules, syrups, and elixirs. Pharmaceutical compositions intended for oral administration can be prepared according to any methods known in the art for manufacturing pharmaceutical compositions intended for oral administration. In order to provide pharmaceutically palatable preparations, a pharmaceutical Docket No.: 6-25007 / 161118.08401 composition in accordance with the disclosure can contain at least one agent selected from sweetening agents, flavoring agents, coloring agents, demulcents, antioxidants, and preserving agents.

[0114] A tablet can, for example, be prepared by admixing at least one compound and / or at least one pharmaceutically acceptable salt thereof with at least one non-toxic pharmaceutically acceptable excipient suitable for the manufacture of tablets. Exemplary excipients include, but are not limited to, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, and sodium phosphate; granulating and disintegrating agents, such as microcrystalline cellulose, sodium croscarmellose, corn starch, and alginic acid; binding agents, such as starch, gelatin, polyvinylpyrrolidone, and acacia; and lubricating agents, such as magnesium stearate, stearic acid, and talc. Additionally, a tablet can either be uncoated or coated by known techniques to either mask the bad taste of an unpleasant tasting drug or delay disintegration and absorption of the active ingredient in the gastrointestinal tract, thereby sustaining the effects of the active ingredient for a longer period. Exemplary water-soluble tastemasking materials include, but are not limited to, hydroxypropyl-methylcellulose and hydroxypropyl-cellulose. Exemplary time delay materials include, but are not limited to, ethylcellulose and cellulose acetate butyrate.

[0115] Hard gelatin capsules can, for example, be prepared by mixing at least one compound and / or at least one salt thereof with at least one inert solid diluent, such as calcium carbonate; calcium phosphate; and kaolin.

[0116] Soft gelatin capsules can, for example, be prepared by mixing at least one compound and / or at least one pharmaceutically acceptable salt thereof with at least one water-soluble carrier, such as polyethylene glycol; and at least one oil medium, such as peanut oil, liquid paraffin, and olive oil.

[0117] An aqueous suspension can be prepared, for example, by admixing at least one compound and / or at least one pharmaceutically acceptable salt thereof with at least one excipient suitable for the manufacture of an aqueous suspension. Exemplary excipients suitable for the manufacture of an aqueous suspension, include, but are not limited to, for example, suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, alginic acid, polyvinyl-pyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents, Docket No.: 6-25007 / 161118.08401 such as a naturally-occurring phosphatide, e.g., lecithin; condensation products of alkylene oxide with fatty acids, such as polyoxyethylene stearate; condensation products of ethylene oxide with long-chain aliphatic alcohols, such as heptadecaethylene-oxycetanol; condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate; and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan monooleate. An aqueous suspension can also contain at least one preservative, such as ethyl and n-propyl p- hydroxybenzoate; at least one coloring agent; at least one flavoring agent; and / or at least one sweetening agent, including but not limited to, for example, sucrose, saccharin, and aspartame.

[0118] Oily suspensions can, for example, be prepared by suspending at least one compound and / or at least one pharmaceutically acceptable salt thereof in either vegetable oil, such as Arachis oil; olive oil; sesame oil; and coconut oil; or in mineral oil, such as liquid paraffin. An oily suspension can also contain at least one thickening agent, such as beeswax, hard paraffin, and cetyl alcohol. In order to provide a palatable oily suspension, at least one of the sweetening agents already described hereinabove, and / or at least one flavoring agent can be added to the oily suspension. An oily suspension can further contain at least one preservative, including, but not limited to, for example, an antioxidant, such as butylated hydroxy anisole, and alpha- tocopherol.

[0119] Dispersible powders and granules can, for example, be prepared by admixing at least one compound and / or at least one pharmaceutically acceptable salt thereof with at least one dispersing and / or wetting agent; at least one suspending agent; and / or at least one preservative. Suitable dispersing agents, wetting agents, and suspending agents are as already described above. Exemplary preservatives include, but are not limited to, for example, antioxidants, e.g., ascorbic acid. In addition, dispersible powders and granules can also contain at least one excipient, including, but not limited to, for example, sweetening agents, flavoring agents, and coloring agents.

[0120] An emulsion of at least one compound and / or at least one pharmaceutically acceptable salt thereof can, for example, be prepared as an oil-in-water emulsion. The oily phase of the emulsions comprising compounds may be constituted from known ingredients in a known manner. The oil phase can be provided by, but is not limited to, for example, a vegetable oil, such as olive oil, Arachis oil, mineral oil, such as liquid paraffin, and mixtures thereof. While the phase may comprise merely an emulsifier, it may comprise a mixture of at least one emulsifier with a fat or Docket No.: 6-25007 / 161118.08401 an oil or with both fat and oil. Suitable emulsifying agents include, but are not limited to, for example, naturally occurring phosphatides, e.g., soybean lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate; and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. In some embodiments, a hydrophilic emulsifier is included together with a lipophilic emulsifier, which acts as a stabilizer. It is also preferred to include both oil and fat. Together, the emulsifier(s) with or without stabilizer(s) make-up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. An emulsion can also contain a sweetening agent, a flavoring agent, a preservative, and / or an antioxidant. Emulsifiers and emulsion stabilizers suitable for use in the formulation of the present disclosure include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate alone or with a wax, or other materials well known in the art.

[0121] A pharmaceutical composition described herein can also be incorporated into a topical formulation containing a topical carrier that is generally suited to topical drug administration and comprising any such material known in the art. The topical carrier may be selected so as to provide the composition in the desired form, e.g., as an ointment, lotion, cream, microemulsion, gel, oil, solution, or the like, and may be comprised of a material of either naturally occurring or synthetic origin. It is preferable that the selected carrier does not adversely affect the active agent or other components of the topical formulation. Examples of suitable topical carriers for use herein include water, alcohols and other nontoxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like.

[0122] Pharmaceutical compositions may be incorporated into gel formulations, which generally are semisolid systems consisting of either suspension made up of small inorganic particles (two- phase systems) or large organic molecules distributed substantially uniformly throughout a carrier liquid (single-phase gels). Single-phase gels can be made, for example, by combining the active agent, a carrier liquid, and a suitable gelling agent, such as tragacanth (at 2 to 5%), sodium alginate (at 2-10%), gelatin (at 2-15%), methylcellulose (at 3-5%), sodium carboxymethylcellulose (at 2- 5%), carbomer (at 0.3-5%) or polyvinyl alcohol (at 10-20%) together and mixing until a characteristic semisolid product is produced. Other suitable gelling agents include methylhydroxycellulose, polyoxyethylene-polyoxypropylene, hydroxyethylcellulose, and gelatin. Docket No.: 6-25007 / 161118.08401

[0123] Although gels commonly employ aqueous carrier liquid, alcohols and oils can be used as the carrier liquid as well.

[0124] The compounds and / or at least one pharmaceutically acceptable salt thereof can, for example, also be delivered intravenously, subcutaneously, and / or intramuscularly via any pharmaceutically acceptable and suitable injectable form. Exemplary injectable forms include, but are not limited to, for example, sterile aqueous solutions comprising acceptable vehicles and solvents, such as water, Ringer’s solution, and isotonic sodium chloride solution; sterile oil-in- water microemulsions; and aqueous or oleaginous suspensions.

[0125] Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules using one or more of the carriers or diluents mentioned for use in the formulations for oral administration or by using other suitable dispersing or wetting agents and suspending agents. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth gum, and / or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art. The active ingredient may also be administered by injection as a composition with suitable carriers, including saline, dextrose, or water, or with cyclodextrin ( / .<?., Captisol), cosolvent solubilization (z.e., propylene glycol) or micellar solubilization (i.e., Tween 80).

[0126] The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid, find use in the preparation of injectables.

[0127] A sterile injectable oil-in-water microemulsion can, for example, be prepared by (1) dissolving at least one compound in an oily phase, such as a mixture of soybean oil and lecithin; (2) combining containing oil phase with a water and glycerol mixture; and (3) processing the combination to form a microemulsion. Docket No.: 6-25007 / 161118.08401

[0128] A sterile aqueous or oleaginous suspension can be prepared in accordance with methods already known in the art. For example, a sterile aqueous solution or suspension can be prepared with a non-toxic parenterally acceptable diluent or solvent, such as 1,3 -butanediol; and a sterile oleaginous suspension can be prepared with a sterile, non-toxic acceptable solvent or suspending medium, such as sterile fixed oils, e.g., synthetic mono- or diglycerides; and fatty acids, such as oleic acid.

[0129] Pharmaceutically acceptable carriers, adjuvants, and vehicles that may be used in the pharmaceutical compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as alpha-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens, polyethoxylated castor oil, such as cremophor surfactant (BASF), or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as alpha-, beta-, and gammacyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.

[0130] The pharmaceutically active compounds of this disclosure can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals. The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and / or may contain conventional adjuvants, such as additives, preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. Tablets and pills can additionally be prepared with enteric coatings. Such compositions may also comprise adjuvants, such as wetting, sweetening, flavoring, and perfuming agents.

[0131] Various additives, known to those skilled in the art, may be included in formulations, e.g., topical formulations. Examples of additives include, but are not limited to, solubilizers, skin Docket No.: 6-25007 / 161118.08401 permeation enhancers, opacifiers, preservatives (e.g, antioxidants), gelling agents, buffering agents, surfactants (particularly nonionic and amphoteric surfactants), emulsifiers, emollients, thickening agents, stabilizers, humectants, colorants, fragrance, and the like. Inclusion of solubilizers and / or skin permeation enhancers is particularly preferred, along with emulsifiers, emollients, and preservatives. An optimum topical formulation comprises approximately: 2 wt. % to 60 wt. %, e.g., 2 wt. % to 50 wt. %, solubilizer and / or skin permeation enhancer; 2 wt. % to 50 wt. %, e.g., 2 wt. % to 20 wt. %, emulsifiers; 2 wt. % to 20 wt. % emollient; and 0.01 to 0.2 wt. % preservative, with the active agent and carrier (e.g., water) making up the remainder of the formulation. A skin permeation enhancer serves to facilitate passage of therapeutic levels of active agent to pass through a reasonably sized area of unbroken skin. Suitable enhancers are well known in the art and include, for example, lower alkanols such as methanol ethanol and 2-propanol; alkyl methyl sulfoxides such as dimethyl sulfoxide (DMSO), decylmethylsulfoxide (C. sub.10 MSO) and tetradecylmethyl sulfoxide; pyrrolidones such as 2-pyrrolidone, N-methyl-2-pyrrolidone and N-(- hydroxyethyl)pyrrolidone; urea; N,N- diethyl-m-toluamide; C2.-C6 alkane diols; miscellaneous solvents such as dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and tetrahydrofurfuryl alcohol; and the 1 -substituted azacycloheptan-2-ones, particularly 1-n- dodecylcyclazacycloheptan-2-one (laurocapram; available under the trademark AzoneRTM from Whitby Research Incorporated, Richmond, Va.).

[0132] Examples of solubilizers include, but are not limited to, the following: hydrophilic ethers such as diethylene glycol monoethyl ether (ethoxydiglycol, available commercially as Transcutol™) and diethylene glycol monoethyl ether oleate (available commercially as Softcutol™); polyethylene castor oil derivatives such as poly oxy 35 castor oil, poly oxy 40 hydrogenated castor oil, etc.; polyethylene glycol, particularly lower molecular weight polyethylene glycols such as PEG 300 and PEG 400, and polyethylene glycol derivatives such as PEG-8 caprylic / capric glycerides (available commercially as Labrasol™); alkyl methyl sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone and N-methyl -2-pyrrolidone; and DMA. Many solubilizers can also act as absorption enhancers. A single solubilizer may be incorporated into the formulation, or a mixture of solubilizers may be incorporated therein.

[0133] Suitable emulsifiers and co-emulsifiers include, without limitation, those emulsifiers and co-emulsifiers described with respect to microemulsion formulations. Emollients include, for Docket No.: 6-25007 / 161118.08401 example, propylene glycol, glycerol, isopropyl myristate, polypropylene glycol-2 (PPG-2) myristyl ether propionate, and the like.

[0134] Other active agents may also be included in formulations, e.g., anti-inflammatory agents, analgesics, antimicrobial agents, antifungal agents, antibiotics, vitamins, antioxidants, and sunblock agents commonly found in sunscreen formulations including, but not limited to, anthranilates, benzophenones (particularly benzophenone-3), camphor derivatives, cinnamates (e.g., octyl methoxy cinnamate), dibenzoyl methanes (e.g., butyl methoxy dibenzoyl methane), p- aminobenzoic acid (PABA) and derivatives thereof, and salicylates (e.g., octyl salicylate). In certain topical formulations, the active agent is present in an amount in the range of approximately 0.25 wt. % to 75 wt. % of the formulation, e.g., in the range of approximately 0.25 wt. % to 30 wt. % of the formulation, e.g., in the range of approximately 0.5 wt. % to 15 wt. % of the formulation, and e.g., in the range of approximately 1.0 wt. % to 10 wt. % of the formulation. Topical skin treatment compositions can be packaged in a suitable container to suit its viscosity and intended use by the consumer. For example, a lotion or cream can be packaged in a bottle or a roll-ball applicator, or a propellant-driven aerosol device or a container fitted with a pump suitable for finger operation. When the composition is a cream, it can simply be stored in a non- deformable bottle or squeeze container, such as a tube or a lidded jar. The composition may also be included in capsules such as those described in U.S. Pat. No. 5,063,507. Accordingly, also provided are closed containers containing a cosmetically acceptable composition.

[0135] The amounts of compounds that are administered and the dosage regimen for treating a disease condition with the compounds and / or compositions of this disclosure depends on a variety of factors, including the age, weight, sex, medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. A daily dose of about 0.001 to 100 mg / kg body weight, e.g., between about 0.0025 and about 50 mg / kg body weight and, e.g., between about 0.005 to 10 mg / kg body weight, may be appropriate. The daily dose can be administered in one to four doses per day. Other dosing schedules include one dose per week and one dose per two-day cycle.

[0136] For therapeutic purposes, the active compounds of this disclosure are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered Docket No.: 6-25007 / 161118.08401 orally, the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets may contain a controlled-release formulation as may be provided in a dispersion of active compound in hydroxypropylmethylcellulose.

[0137] Pharmaceutical compositions of this disclosure comprise at least one compound and / or at least one pharmaceutically acceptable salt thereof, and optionally an additional agent selected from any pharmaceutically acceptable carrier, adjuvant, and vehicle. Alternate compositions of this disclosure comprise a compound described herein, or a prodrug thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0138] The compound, the composition or the pharmaceutical composition described herein can be provided in a kit. In one embodiment, the kit includes (a) a container that contains the composition and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and / or the use of the agents for therapeutic benefit. For example, kits may include instructions for the manufacturing, for the therapeutic regimen to be used, and periods of administration. In an embodiment, the kit includes also includes an additional therapeutic agent. The kit may comprise one or more containers, each with a different reagent. For example, the kit includes a first container that contains the composition and a second container for the additional therapeutic agent.

[0139] The containers can include a unit dosage of the pharmaceutical composition. In addition to the composition, the kit can include other ingredients, such as a solvent or buffer, an adjuvant, a stabilizer, or a preservative. The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device. The device can be provided pre- loaded with one or both of the agents or can be empty, but suitable for loading.

[0140] Combination Therapies

[0141] Active ingredients described herein can also be used in combination with one or more additional agents. Such combinations can be selected based on the condition to be treated, crossreactivities of ingredients and pharmaco-properties of the combination. Docket No.: 6-25007 / 161118.08401

[0142] In some embodiments, one or more additional agents may include another agent for treating a respiratory disease. In some embodiments, one or more additional agents may include another agent for treating fibrotic airways disease (e.g, bronchiolitis obliterans or constrictive bronchiolitis). In some embodiments, one or more additional agents may include another GADD34 inhibitor. In some embodiments, one or more additional agents may include an inhibitor capable of down-regulating the integrated stress response (ISR) pathway.

[0143] In some embodiments, one or more additional agents may include an agent disclosed in U.S. Patent No. 10,905,663 and U.S. Patent Application Publication Nos. US 2009 / 0181934 Al and US 2023 / 0082287 Al, the relevant disclosures of which are herein incorporated by reference.

[0144] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent or therapy. In some embodiments, the additional therapeutic agent is selected from ranolazine mexiletine, flecainide, tocainide, phenytoin, carbamazepine, lamotrigine, and a combination thereof. In some embodiments, the additional therapeutic agent or therapy is administered to the subject before, after, or concomitantly with the agent.

[0145] “Combination” therapy, as used herein, unless otherwise clear from the context, is meant to encompass administration of two or more therapeutic agents in a coordinated fashion and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time. See, e.g., Kohrt et al. (2011) Blood 117:2423.

[0146] As used herein, the term “co-administration” or “co-administered” refers to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co- administration of two or more agents / therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents / therapies used may vary.

[0147] It is also possible to combine a compound of the invention with one or more other active ingredients in a unitary dosage form for simultaneous or sequential administration to a patient. The Docket No.: 6-25007 / 161118.08401 combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

[0148] The combination therapy may provide synergy and be synergistic, i.e., the effect achieved when the active ingredients used together are greater than the sum of the effects that result from using the compounds separately. A synergistic effect may be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., in separate tablets, pills, or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas, in combination therapy, effective dosages of two or more active ingredients are administered together. A synergistic effect denotes an effect that is greater than the predicted purely additive effects of the individual compounds of the combination.

[0149] Combination therapy is further described by U.S. Pat. Nos. 11103514, 10702495, 9382215, and 6833373, which include additional active agents that can be combined with the compounds described herein, and additional types of ailments and other conditions that can be treated with a compound or combination of compounds described herein.

[0150] Accordingly, it is an aspect of this invention that an active agent (e.g., verapamil, ranolazine mexiletine, flecainide, tocainide, phenytoin, carbamazepine, and lamotrigine) can be used in combination with another agent or therapy method. An active agent may precede or follow treatment of the other agent by intervals ranging from minutes to weeks. In embodiments where the other agent and expression construct are applied separately to a cell, one would generally ensure that a significant period of time did not elapse between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) with the disclosed active.

[0151] In some embodiments, one or more agents may be administered within about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about Docket No.: 6-25007 / 161118.08401

[0152] 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 28 hours, about 31 hours, about 35 hours, about 38 hours, about 42 hours, about 45 hours, to about 48 hours or more prior to and / or after administering the disclosed active agent. In certain other embodiments, an agent may be administered within from about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 8 days, about 9 days, about 12 days, about 15 days, about 16 days, about 18 days, about 20 days, to about 21 days prior to and / or after administering the disclosed active. In some situations, it may be desirable to extend the time period for treatment significantly; however, where several weeks (e.g, about 1, about 2, about 3, about 4, about 6, or about 8 weeks or more) lapse between the respective administrations.

[0153] Administration of the compositions of the invention to a patient will follow general protocols for the administration of therapeutics, taking into account the toxicity, if any. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies or adjunct therapies, as well as surgical intervention, may be applied in combination with the described active agent. These therapies include but are not limited to chemotherapy, radiotherapy, immunotherapy, gene therapy and surgery.

[0154] Additional Definitions

[0155] To aid in understanding the detailed description of the compositions and methods according to the disclosure, a few express definitions are provided to facilitate an unambiguous disclosure of the various aspects of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0156] Doses are often expressed in relation to body weight. Thus, a dose which is expressed as [g, mg, or other unit] / kg (or g, mg, etc.) usually refers to [g, mg, or other unit] “per kg (or g, mg, etc.) body weight,” even if the term “bodyweight” is not explicitly mentioned.

[0157] As used herein, the term “zzz vitro” refers to events that occur in an artificial environment, c.g, in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism. Docket No.: 6-25007 / 161118.08401

[0158] As used herein, the term “zzz vivo" refers to events that occur within a multi-cellular organism, such as a non-human animal.

[0159] It is noted here that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0160] The terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0161] The phrases “In some embodiments,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

[0162] The terms “and / or” or “ / ” means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0163] The word “substantially” does not exclude “completely,” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

[0164] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless indicated otherwise herein, the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

[0165] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. Docket No.: 6-25007 / 161118.08401

[0166] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0167] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. When used in this document, the term “exemplary” is intended to mean “by way of example” and is not intended to indicate that a particular exemplary item is preferred or required.

[0168] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise.

[0169] In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0170] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure. Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0171] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0172] Examples

[0173] EXAMPLE 1 Docket No.: 6-25007 / 161118.08401

[0174] This example describes the materials and methods used in the subsequent EXAMPLES below.

[0175] Animals

[0176] Studies were approved by the Institutional Animal Care and Use Committee of the University of Rochester Medical Center (URMC) and adhered to the National Institutes of Health Guidelines. Seven-week-old outbred male Sprague-Dawley (SD) rats weighing 250 - 300 grams (Charles River Laboratory, Wilmington, MA) were maintained in an AAALAC -accredited animal care facility.

[0177] Human Donor l ung Samples

[0178] De-identified human donor lung samples were authorized by written informed consent of next of kin for donation for research through the United Network of Organ Sharing (UNOS) in coordination with National Disease Research Interchange (NDRI) and International Institute for Advancement of Medicine (IIAM). The University of Rochester IRB approved and oversees the placement and management of these samples in the Biorepository for Investigation of Diseases of the Lung (BRINDL) (RSRB00047606).

[0179] Aerosol Generation, Animal Exposures, and Post-Exposure Animal Monitoring

[0180] Diacetyl (DA; 2,3 -butanedi one; Sigma-Aldrich, St. Louis, MO) inhalation exposures were performed at the University of Rochester’s Inhalation Exposure Facility (IEF), as described previously (Wang J, et al. Arch Toxicol. 2021;95(7):2469-83). Animals were exposed to 200- parts-per-million (ppm) DA using whole-body inhalation chambers for 6 h / day for 5 days. Two- hundred ppm DA is comparable to 12 ppm in humans when corrected for nasal scrubbing due to larger surface area in rodents and DA concentration seen for occupational exposures. Immediately after the final day of DA exposure (Day 5), animals were returned to housing for weights and oxygen saturation using an animal oximetry probe (Starr Life Science Technologies, Oakmont, PA) for up to 14 days post-exposure (Day 19; ‘monitoring period’). Sample size for animals exposed to DA was estimated from previous studies (Flake GP, et al. Toxicology. 2017;388:40-7), accounting for early death due to upper respiratory tract necrosis and airway obstruction. At study’s end, euthanasia was performed via terminal pentobarbital sodium intraperitoneal (IP) Docket No.: 6-25007 / 161118.08401 injection (100 mg / kg; Abbott Laboratories, Abbott Park, IL) followed by diaphragmatic puncture and exsanguination.

[0181] GADD34 Inhibitor (Sephinl) Treatments

[0182] Rats were randomly assigned to the groups prior to inhalation exposures. Exposure groups included: (1) Air-exposed (Air) + dimethyl sulfoxide (DMSO), (2) Air + Sephinl (Icerguatstat, MedChemExpress, Monmouth Junction, NJ, USA; Cat #: HY-111022), (3) DA+DMSO, or (4) DA+Sephinl. Sephinl stock solution was prepared by dissolving 100 mg in 10.2 ml of DMSO solution (50 millimolar (mM), Sigma-Aldrich, St. Louis, MO, USA), and then stored at -20°C. Final solutions were prepared by diluting 50mM stock Sephinl solution in DMSO on the day of injection with each rat receiving 5mg / kg / day Sephinl solution or equivalent volume (approximately 130 microliters) DMSO. Treatments were administered via intraperitoneal injection on Day 5 (immediately at the end of DA exposures) and then daily until study’s end (Day 19). Dose and timing are based on prior publications of Sephinl use in rats.

[0183] Rat Nose-Only Exposure to GADD 34 Inhibitor (Sephinl) via Nebulization

[0184] Sephinl stock solution was prepared by dissolving 10 mg in 1 ml of DMSO solution (50 millimolar (mM), Sigma-Aldrich, St. Louis, MO, USA), and then stored at -20°C. Final solutions were prepared by diluting 50mM stock Sephinl solution in 10 ml’s 0.9% normal saline on the day of nebulization. Treatments were administered daily for 30 minutes. Aerosol was generated by administering filtered air at 35 PSI through the Aero-Mist nebulizer (BIS-US, Bedford, MA, USA) containing 10 ml’s of diluted Sephinl solution.

[0185] Rats were exposed to e-cigarette aerosol for 30 minutes of aerosol daily for 14 consecutive days via a nose-only exposure system (IET 200B Chambers, IES 324 Inhalation Tower; ElectroMedical Measurement System (EMMS), Bordon, Hampshire, UK). Control animals were housed in identical nose only chambers and were exposed to humidified (30-50%) filtered air only.

[0186] Immunohistochemistry and Immunofluorescence

[0187] After terminal anesthesia and exsanguination, lung tissue was cleared of blood by flushing the right ventricle with 10 ml phosphate-buffered solution (PBS). The trachea was then cannulated, left bronchus clamped, and the right lung was lavaged with two 2.5 ml washes of normal saline (0.9%) solution. The left lung was then unclamped and intratracheally fixed with 10% neutral Docket No.: 6-25007 / 161118.08401 buffered formalin at 20 cm H2O for 30 min prior to removal by gross dissection. Left lung lobes were paraffin-embedded, sectioned (5 pm) and stained for hematoxylin and eosin (H&E) or Gomori Trichrome stain, similar to previously described experiments1.

[0188] All airways were considered bronchi when the luminal diameter was greater than 250 pm nor connected directly to the alveolar space. Bronchiolitis obliterans (BO) was defined as airway obstruction due to concentric sub-epithelial collagen deposition with luminal obliteration of intrapulmonary bronchi (Morgan DL, et al. Toxicol Pathol. 2012;40(3):448-65; Morgan DL, etal. Toxicol Pathol. 2016;44(5):763-83). Two investigators blinded to exposure condition independently graded Trichrome-stained sections of the left lung lobe for: (1) the presence or absence of airway lesions in each rat, and (2) if present, the number of affected bronchial lesions per animal at 10* magnification per animal. Each fixed left lung lobe was also noted for the presence or absence of parenchymal fibrosis.

[0189] For immunofluorescence, fixed and embedded left lung sections were de-paraffinized with serial xylene incubations, followed by dehydration in graded alcohol and then heated for 15 minutes in antigen retrieval solution (Vector, Newark, CA). Sections were washed in PBS buffer and blocked with 10% bovine serum in PBS for 1 hr. at room temperature. Slides were then rinsed and incubated overnight at 4°C with respective primary antibody. Primary antibodies included: GADD34 (PPP1R15A, 1:200; ProteinTech, Rosemont, Ill., USA; Cat. #: 10449-1-AP), alphasmooth muscle actin (a-SMA, 1 : 100; Invitrogen, Waltham, MA, USA; Cat. #: 14-9760-82), acetylated alpha-tubulin (a-tubulin, 1: 1000, Millipore Sigma, St. Louis, MO, USA; Cat #: T7451). Rabbit and Mouse IgG (1: 1000, Agilent, Santa Clara, CA) were used as negative controls. After PBS rinse, the slides were counterstained with an AlexaFluor secondary immunofluorescent antibody (1 : 1000; ThermoFisher Scientific, Rockford, Illinois, USA) and mounted with DAPI Fluoromount-G (Southern Biotechnology, Birmingham, Alabama).

[0190] Human Lung Tissue Procurement & Staining

[0191] Samples of 0.5cm3from a standard region of the right middle lobe were flash frozen and stored at 80°C, as described previously. The right lower lobes of each case were inflated with 10% buffered formalin to 20 cm H2O, fixed for 24 h, sliced, blocked, and then fixed for an additional 24 h before dehydration to 70% ethanol. Tissue sections (4-5 pm) from these formalin fixed paraffin embedded (FFPE) blocks, selected to provide comparable bronchiolar and alveolar Docket No.: 6-25007 / 161118.08401 regions, were placed on 0.1% poly-L lysine-coated glass coverslips or Superfrost Plus Microscope Slides (Thermo Fisher Scientific, Waltham, MA, USA) and baked at 50°C for 30 min to enhance slide adherence. FFPE sections (5 pm) were stained by standard hematoxylin & eosin (H&E) methods. For immunofluorescent staining, similar protocol as listed for rat lung sections was used as described above. Primary antibodies included: GADD34 (PPP1R15A, 1 :200; ProteinTech, Rosemont, Ill., USA; Cat. #: 10449-1-AP) and pan-cytokeratin (pan-CK, 1 :200; Abeam, Waltham, MA, USA; Cat # ab7753). Images were from tiles captured with the Keyence BZ-X810 microscope, transferred, and visualized in Omero version 5.8.1.

[0192] Hydroxyproline Assay

[0193] Following the manufacturer’s protocol (Cayman Chemical, Ann Arbor, MI; Cat. #702440), 50 mg of rat lung tissue was homogenized in 500 pl of water, then hydrolyzed with 10 N NaOH. Samples were subsequently neutralized with 10 N HC1, oxidized and developed using the Hydroxyproline Assay Reagent in a stepwise manner. Absorbance was measured at 560 nm using a SpectraMax M5 microplate reader (Molecular Devices; San Jose, CA).

[0194] Lung Sample Preparation for Liquid Chromatography-Mass Spectrometry (LC-MS)

[0195] Frozen lung tissue samples were homogenized in 80% methanol (MeOH) using a Precellys® cold tissue homogenizer (Bertin Instruments) at a ratio of 10 mg of tissue per 1 mL of solvent. Following homogenization, the samples were stored at -80 °C for 30 minutes and then transferred to ice for an additional 30 minutes, with vortexing performed every 10 minutes. The samples were subsequently centrifuged at 17,000 * g for 10 minutes. A volume of 800 pL of the resulting supernatant was collected and evaporated as two 400 pL aliquots using a vacuum evaporator (Thermo Fisher Scientific). Dried samples were reconstituted in 80 pL of 50% acetonitrile (A955, Fisher Scientific) and transferred to glass vials for LC-MS analysis.

[0196] Plasma Sample Preparation

[0197] Plasma samples (100 pL) were extracted in a final volume of 1 mL of 80% methanol by incubation on ice for 30 minutes, with brief vortexing every 10 minutes. Samples were then centrifuged at 17,000 * g for 10 minutes. A volume of 900 pL of the resulting supernatant was collected and evaporated as two 450 pL aliquots using a vacuum evaporator (Thermo Fisher Docket No.: 6-25007 / 161118.08401

[0198] Scientific). Dried samples were reconstituted in 225 pL of 50% acetonitrile (A955, Fisher Scientific) and transferred to glass vials for LC-MS analysis.

[0199] LC-MS Analysis

[0200] Metabolite extracts were analyzed by high-resolution mass spectrometry using an Orbitrap™ Exploris™ 240 mass spectrometer (Thermo Fisher Scientific) coupled to a Vanquish™ Flex UHPLC system (Thermo Fisher Scientific). Samples were injected onto a Thermo Accucore™ column (100 mm length x 2.1 mm internal diameter, 2.6 pm particle size) maintained at 40 °C.

[0201] Mobile phase A consisted of 100% LC-MS grade water containing 10 mM ammonium formate and 0.125% formic acid. Mobile phase B consisted of 90% acetonitrile with 10 mM ammonium formate and 0.125% formic acid. The chromatographic gradient was programmed as follows: 0 minutes, 15% B; 1 minute, 15% B; 3 minutes, 25% B; 9 minutes, 65% B; 13 minutes, 100% B; 15 minutes, 100% B; 15.1 minutes, 15% B; and 20 minutes, 15% B. The flow rate was 400 pL / min, and the total run time was 20 minutes.

[0202] The H-ESI ion source was operated in positive ionization mode at a spray voltage of 3,500 V. Additional source parameters included a sheath gas flow of 50 arbitrary units (au), auxiliary gas flow of 10 au, sweep gas flow of 1 au, an ion transfer tube temperature of 325 °C, and a vaporizer temperature of 300 °C. Full MS 1 spectra were acquired at a mass resolution of 120,000 (FWHM), and data-dependent MS2 (ddMS2) spectra were acquired using normalized collision energies of 20%, 30%, and 40%, with an MS2 resolution of 15,000 (FWHM).

[0203] LC-MS data were analyzed using Maven software for peak area determination. The Sephinl peak was annotated by matching its mass-to-charge ratio (m / z), retention time, and MS2 fragmentation pattern to those of a Sephinl chemical standard. Quantification of Sephinl was performed using linear regression based on a standard curve generated by spiking known concentrations of the Sephinl chemical standard into blank (untreated) lung and plasma matrices.

[0204] Tracheal Airway Epithelial Cell Isolation and Ex Vivo Growth of 3D Organoids

[0205] After isolation of the lower lung lobes, rat trachea was carefully dissected and immediately placed into dispase solution (1.867 U / mg or 5U / ml in Hank’s Buffered Salt Solution (HBSS) for 45 minutes. Immediately following, the airway epithelial cell layer was surgically dissected from Docket No.: 6-25007 / 161118.08401 the surrounding cartilage, and collected in 8 ml in airway medium (3: 1 v / v Fl 2 Nutrient Mixture and Dulbecco’s Modified Eagle’s Medium (DMEM)) supplemented with 5% fetal bovine serum, 9.4 pg / ml hydrocortisone, 5 pg / ml insulin, 8.4 ng / ml cholera toxin, 10 ng / ml epidermal growth factor, 24 pg / ml adenine, and 5 pmol Y-27632. The cells were mixed with chilled Matrigel (1 :2) and seeded onto a 6-well culture plate with 2ml airway media. After 7 days in culture, a subfraction of cells (10 microliters) was assessed by viability by staining with trypan blue via an automated cell counter (TC20 automated cell counter, Bio-Rad, Hercules, CA, USA). Additional cells were collected and homogenized for total cell protein or imaged for viability via Live / Dead staining.

[0206] Live / Dead Staining

[0207] Following standard protocol (ThermoFisher; Cat. #R37601), calcein AM (green, ‘live’) was mixed with BOBO-3 Iodide (deep red, ‘dead’), and then equal volume added of the 2x stock to the cells in fresh media. Following 15 minutes at room temperature, the cells were imaged using a LEICA DM5500 B compound microscope with a LEICA DFC365 FX camera (Wetzlar, Germany).

[0208] Statistical Analysis

[0209] Prism 8.0 software (GraphPad Software) was used for statistical analysis with one-way ANOVA followed by Tukey’s post hoc analysis for multiple comparisons with naive controls. A log-rank (Mantel-Cox) test was performed for survival analysis. Mean values are reported with standard deviation. A p-value < 0.05 was considered significant.

[0210] EXAMPLE 2

[0211] Reproducible Inhalation Exposure Model of Chemical-Induced Airway Remodeling in Spragu -Daw ley Rats

[0212] The rat model of repetitive DA vapor exposures was used for modeling bronchiolitis obliterans (BO) (Wang J, et al. Arch Toxicol. 2021;95(7):2469-83; House EL, et al. Toxics. 2021 ;9( 12)). Sprague-Dawley rats (6-8-week-old; 180-250g; Charles River) were exposed to 200- parts-per-million (ppm) diacetyl (DA; 2,3 -butanedione; 98.5% purity; Sigma) vapors for 6 hours per day for 5 consecutive days using whole body exposure chambers. This DA concentration when corrected for nasal scrubbing in rats is comparable to 12-ppm DA in humans (Morris IB, et al. Docket No.: 6-25007 / 161118.08401

[0213] Toxicol Sci. 2009; 108(1 ): 173-83; Gloede E, et al. Toxicol Sci. 2011;123(1):231-46) - a chemical vapor concentration relevant to DA occupational exposures. Animal monitoring with daily weights and oxygen saturations occurred for 2 weeks after DA exposure cessation to model the fibroproliferation phase of airway disease induction. Three separate inhalation exposures using both male and female rats were performed.

[0214] At the study end (Day 19 or 2 weeks post-DA exposure), survival was significantly worse in DA-exposed rats compared to air-exposed rats (59% vs. 100%; Mantel-Cox test; ***p=0.0002, Figure 1A). Oxygen saturations (Figure IB) declined significantly in DA-exposed rats one week after DA exposure cessation and remained significantly lower than air controls at the study end. Rats exposed to DA lost weight during exposure as well as failed to gain weight at similar rates post-DA exposure and demonstrated significant impairment on lung function testing at 2 weeks s / p exposure versus air-matched controls (Figure 1C). Greater than 80% of DA-exposed rats demonstrated histologic evidence of BO pathology with sub-epithelial collagen deposition and concentric luminal narrowing of intrapulmonary airways (Fig 1, bottom right). The number of airway lesions per rat was significantly increased compared to air controls assessed via blinded review of rat lung histology. This rat model of repetitive DA exposures clearly emulates the key histopathologic features of humans exposed to certain environmental toxicants who develop BO.

[0215] Increased GADD34 Expression in Rat Airways After Repetitive Diacetyd Exposures

[0216] A common adaptive pathway for sustained ER stress is activation of the integrated stress response (ISR) (Pakos-Zebrucka K, et al. EMBO Rep. 2016;17(10): 1374-95). The central signaling event that results from ISR activation is phosphorylation of eukaryotic translational initiation factor 2 alpha (eIF2a). With phosphorylation, eIF2a halts global protein synthesis as a cytoprotective strategy to prevent further protein accumulation. In parallel, p-EIF2a activates the transcription factor ATF4 to promote cellular recovery. Although the ISR is primarily ‘prosurvival’, sustained ISR activation can also result in a maladaptive response. With sustained ISR activation, the cell upregulates the DNA damage-inducible transcript 3 gene (DDIT3; equivalent protein: CCAAT / enhancer-bind protein homologous protein (CHOP)) (Kaspar S, et al. Sci Adv. 2021;7(22)). Conversely, ISR termination occurs through an inducible feedback loop when ATF4 and / or CHOP upregulates growth arrest and DNA damage-inducible protein (GADD34; gene: PPP1R15A). GADD34’s primary function is to dephosphorylate p-EIF2a with protein Docket No.: 6-25007 / 161118.08401 phosphatase 1 (PPI) (Monkley S, et al. Sci Rep. 2021 ; 11 (1 ):21584; Sun Y, et al. Oxid Med Cell Longev. 2015;2015: 170309).

[0217] Next, GADD34 expression was assessed by immunofluorescent staining in rat lungs exposed to diacetyl vapors versus air control rat lungs (Figure 2). GADD34 expression in rat lungs increased immediately after DA exposure (Day 5) compared to air-exposed controls. Increased GADD34 expression primarily localized to DA-exposed airway epithelium while limited-to-no GADD34 expression was seen in air-matched controls. Looking at 2 weeks after DA exposure cessation (Day 19), GADD34 expression in DA-exposed rat airways remained elevated compared to air controls and localized primarily to DA-exposed airways with significant remodeling (Figure 3).

[0218] To localize GADD34 expression in DA-exposed airways at the cellular level (Figure 4), lungs from DA-exposed rats were sectioned and co-stained for GADD34 with alpha-smooth muscle actin (a-SMA, smooth muscle marker) or acetylated-tubulin (a-tubulin, cilia cell marker). Two weeks after DA exposure cessation (Day 19) GADD34 localized to the airway epithelia of DA-exposed rats, adjacent to increased a-SMA+ staining and below a-tubulin staining. These results indicate GADD34 expression remains increased in DA-exposed rat airways weeks after DA exposure cessation, and primarily localizes to the basolateral airway epithelia (below the more differentiated ciliated epithelia) and adjacent to sub-epithelial remodeling (marked by increased a- SMA+ staining).

[0219] Increased GADD34 Expression in Humans Lung Sections Affected by Bronchiolitis Obliterans

[0220] The gold standard for diagnosing BO remains histopathologic evaluation of human lung tissue either following lung biopsy or via autopsy. To begin to evaluate for GADD34 expression in human lung tissue with airway remodeling, human lung sections from a 6-year-old male who died from respiratory failure secondary to post-infectious bronchiolitis obliterans (aka ‘PIBO’) were stained with hematoxylin and eosin (H&E; Figure 5). H&E staining was conducted to identify those airways affected by significant remodeling. The black arrow in Figure 5 (left image) denotes one of the airways severely affected by remodeling with loss of the airway lumen, histopathology consistent with bronchiolitis obliterans. Docket No.: 6-25007 / 161118.08401

[0221] Human lung tissue from the same donor was then sectioned and co-stained for GADD34 with pan-cytokeratin (Figure 5, airway epithelial cell marker). Multiple airway epithelial cells stained positive for pan-cytokeratin and also co-stained positive for GADD34 (Figure 5 right image denoted withn a white arrow). These findings support the presence of GADD34 expression in human airway epithelial cells and in lungs affected by bronchiolitis obliterans (BO).

[0222] GADD34 Inhibition in Rats Exposed Repetitively to Diacetyl Vapors to Prevent BO Development

[0223] Sephinl (icerguastat) is a selective inhibitor of the phosphatase regulatory subunit PPP1R15A (GADD34 protein). It is a derivative of guanabenz but lacks the alpha2-adrenergic activity. This example investigated whether Sephinl given to rats exposed repetitively to DA would prevent and / or delay the onset of BO after exposure.

[0224] Prior to exposure, rats were randomly assigned to 1 of 4 groups: Diacetyl + Sephinl, Diacetyl + DMSO, Air + Sephinl, and Air + DMSO (Figure 6). Sprague-Dawley rats (6-8-week- old; 180-250g; Charles River) were either exposed to 200-parts-per-million (ppm) diacetyl (DA; 2,3-butanedione; 98.5% purity; Sigma) vapors for 6 hours per day for 5 consecutive days using whole body exposure chambers or air, as described previously1. Immediately following the last day’s exposure (Day 5), rats were either treated with Sephinl (5mg / kg) or DMSO via I.P. injection (130 pl). Treatment injections were continued daily for 2 weeks (Day 5 - Day 19). All animals were monitored for change in weight and oxygen saturations during the post-exposure monitoring period.

[0225] Similar to previous DA exposures, survival in DA-exposed rats treated with DMSO was significantly worse compared to Air-exposed control mice treated with vehicle control treatments (DMSO). In contrast, survival in DA-exposed rats treated with Sephinl was significantly improved versus DA+DMSO and not different from Air+Sephinl control rats (Figure 7) at the end of study (Day 19). The percent weight change was again lower in those rats exposed to DA compared to air-matched controls; however, in DA-exposed rats who received Sephinl percent weight change from baseline were significantly improved in the second week after DA exposure cessation (Days 13 - 19) versus DA+DMSO rats (Figure 8). At 1-week post-DA exposure, oxygen saturations historically decreased (Day 12 of study) in DA-exposed rats. Oxygen saturations were also significantly improved in those rats exposed to DA and treated with Sephinl compared to Docket No.: 6-25007 / 161118.08401

[0226] DA-exposed rats + DMSO (Figure 9). Collectively, these results support improved survival, improved percent weight change and improved oxygen saturations in those rats exposed to DA vapors treated with Sephinl compared to rats exposed to DA and treated with DMSO.

[0227] Rats Lungs Exposed to Diacetyl and Treated with Sephinl Demonstrate Reduced Collagen Content

[0228] Next, rat lungs were assessed by both blinded histopathologic evaluation as well as total lung content to semi-quantitate changes in lung fibrosis. In rats exposed to DA and treated with DMSO alone, airway remodeling was significantly increased compared to Air+DMSO alone (Figure 10A). Conversely, airway remodeling was significantly reduced in those rats exposed to DA and treated with Sephinl (DA+Sephinl) compared to rats exposed to DA and treated with DMSO (DA+DMSO) assessed by blinded histologic grading of Tri chrome-stained lung sections. To further support that Sephinl treatment reduces airway fibrosis, lung samples were semiquantitated for total collagen content via hydroxyproline assay for each exposure - treatment group (Figure 10B). Lung collagen content was significantly increased in rats exposed to DA vapors and treated with DMSO versus Air+DMSO exposed rats, while lung collagen content was significantly reduced in rats exposed to DA treated with Sephinl versus DA+DMSO rats. Collectively, these two assessments using histopathology evaluation on Trichrome-stained rat lung sections and hydroxyproline assay support Sephinl significantly reducing airway fibrosis after repetitive DA vapor exposures.

[0229] Isolated rat airway epithelial cells grown ex vivo 3D organoid cultures develop apoptosis after DA exposure with improved viability with Sephinl treatment

[0230] To better assess the cell-type specificity of Sephinl effects on rat airways after DA exposure, primary airway epithelial cells were isolated from Air- or DA-exposed rats and treated with DMSO or Sephinl. Airway epithelial cell isolation occurred 1 week post-vapor exposure and after 1 week (7 days) treatment with Sephinl (Figure 11). Cells were then grown for 7 days in 3D Matrigel culture to assess basal cell function. Following 7 days in 3D culture, organoids were assessed for cell viability by trypan and live / dead immunocytochemistry staining as well as stained for the common basal cell marker keratin 5 (Krt5). Brightfield microscopy and immunocytochemistry staining were performed on organoid cultures to assess organoid shape and keratin 5 protein expression, respectively. As shown, all cells grown in organoid culture created a Docket No.: 6-25007 / 161118.08401 spherical shape after 7 days in culture with an inner lumen. Additionally, nearly all organoids stained positive for keratin 5 (Figure 11 A). Organoids were isolated from rats exposed to DA and treated with DMSO demonstrated reduced viability after 7 days in culture (Figure 1 IB). In contrast, airway epithelial cells isolated from DA-exposed rats treated with Sephinl demonstrated improved viability compared to DA+DMSO treatment seen on Live / Dead imaging. These qualitative results were further supported by viability staining and automated cell counting of dissociated organoid cells. The number of viable cells at Day 7 in culture was significantly increased when isolated from rats exposed to DA and treated with Sephinl versus DA+DMSO. Additionally, the number of viable cells was significantly increased in DA+Sephinl rats vs. Air+Sephinl rats. Collectively, these results suggest that Sephinl treatment promotes airway epithelial cell survival and improved viability in rats exposed to DA and treated with Sephinl compared to those rats exposed to DA vapors receiving DMSO treatment.

[0231] Rat Airway Epithelial Cells Show Increased Integrated Stress Response Activation with Sephinl Treatment

[0232] For verify that Sephinl treatment works directly on rat airway epithelial cells, airway epithelial cells were isolated from rat tracheas 7 days after the end of DA vapor exposure and 7 days of consecutive treatment. Epithelial cells were dissociated by dispase digestion of rat trachea, and then homogenized for further evaluation of protein expression by western blot (Figures 12A and 12B). Relative protein expression of both phosphorylated and total eukaryotic translation initiation factor 2A (eIF2a), considering the proposed mechanism of action for GADD34 inhibition via Sephinl is through reduced de-phosphorylation of eIF2a that then potentiates the integrated stress response. In DA-exposed rat airway epithelial cells treated with DMSO, both phosphorylated and total eIF2a levels did not differ significantly from Air-exposed rats treated with DMSO. With Sephinl treatment, airway epithelial cells demonstrated increased p-eIF2a compared to both DA+DMSO as well as Air+Sephinl without significant change in total eIF2a. Thus, airway epithelial cells isolated from DA-exposed rats treated with Sephinl show enhanced activation of the integrated stress response with increased protein expression of phosphorylation of p-eIF2a that is not seen in airway epithelial cells exposed to DA and treated with DMSO alone.

[0233] Successful Characterization and Identification of Sephinl in Rat Lungs and Plasma Following Nebulization Docket No.: 6-25007 / 161118.08401

[0234] Considering that most of the damage from DA vapor exposures is local to the lung, a set of experiments was developed to nebulize Sephinl in solution. Through nebulization, the majority of drug delivery is local to the lung and without off-organ toxicity. Similar to prior experiments, rats were exposed to DA vapors for 6 hours per day for 5 consecutive days and then randomized to 1 of 4 groups. Different from intraperitoneal delivery, rats were placed into nose-only exposure chambers for 30 minutes daily for 14 days. Sephinl solution was nebulized via an Aero-Mist nebulizer (aerosol characterization - MMAD of 1.38 pm and GSD of 2.28).

[0235] After 14 days of treatment, both lung tissue and plasma were collected from each group to assess for both local and systemic delivery of Sephinl, respectively. Solutions with solvent alone (negative control), Sephinl standard solution (positive control) as well as homogenized lung tissue from rats treated with Sephinl via intraperitoneal injection (treatment controls) were also assessed for Sephinl concentration by high resolution mass spectroscopy (LC / MS). Using both high resolution mass accurate m / z (mass-to-charge ratio) measurements (sub 3ppm) and column retention (RT) time values, Sephinl was successfully identified in all experimental samples and a standardized relative Sephinl chemical standard was used to identify sephinl peaks in experimental samples (Figures 13A and 13B). Hence, Sephinl concentrations can be successfully identified within the lung and within circulation of Sprague-Dawley rats after exposure.

[0236] Nebulization of Sephinl Allows Concentrated Sephinl Specific to the Lungs Limiting Systemic A bsorption

[0237] Last, rat lungs and plasma were assessed for differences in concentration with respect to exposure and treatment (Figure 14). In the lung, Sephinl concentrations were significantly higher in those treated with nebulized Sephinl versus those rats treated with DMSO alone. The average Sephinl concentration in rat lungs exposed to air + nebulized Sephinl was 92.1 (33.4) ng / ml, while the average Sephinl concentration in rat lungs exposed to DA + nebulized was 51.5 (12.9) ng / ml and did not differ significantly from Air+Sephinl. Lung concentrations were also comparable to those rats treated with Sephinl via intraperitoneal injection (avg: 186.1 (34.1) ng / ml (positive standard)). Equally important, the concentration of Sephinl in plasma after nebulized Sephinl treatment was markedly lower in circulation in comparison to the concentrations seen in the lung. Plasma Sephinl concentration did not differ significantly in those rats exposed to Air treated with nebulized Sephinl vs. Air + nebulized DMSO (2.5 (1.4) vs. 0.02 (0.03) ng / ml, p=0.08). Plasma Docket No.: 6-25007 / 161118.08401

[0238] Sephinl concentration increased significantly in those rats exposed to DA treated with nebulized Sephinl vs. DA+ nebulized DMSO (2.4 (1.1) vs. 0.3 (0.4) ng / ml, *p=0.048), however, was nearly 25-fold lower in concentration than that of lung Sephinl concentrations. Plasma Sephinl concentration in Air+IP Sephinl was 35.4 (16.4) ng / ml (positive standard). In summary, nebulization of Sephinl allows for concentrated delivery of Sephinl via aerosolization while limiting systemic absorption and potential off-organ / target toxicity.

[0239] The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present disclosure as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present disclosure as set forth in the claims. Such variations are not regarded as a departure from the scope of the disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.

Claims

Docket No.: 6-25007 / 161118.08401CLAIMSWhat is claimed is:

1. A method of treating a fibrotic airways disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits a growth arrest and DNA-damage inducible protein 34 (GADD34) in a cell of the subject.

2. The method of claim 1, wherein the fibrotic airways disease is associated with the GADD34 pathway.

3. The method of claim 1, wherein inhibition of the GADD34 results in down-regulation of the integrated stress response (ISR) pathway.

4. The method of any one of the preceding claims, wherein the fibrotic airways disease is selected from bronchiolitis obliterans, constrictive bronchiolitis, lymphocytic bronchiolitis, tracheal stenosis, an infection, and an autoimmune disease.

5. The method of any one of the preceding claims, wherein the subject has an increased expression level of the GADD34 compared to a reference subject.

6. The method of any one of the preceding claims, comprising identifying the subject having an increased expression level of the GADD34 compared to a reference subject.

7. The method of claim 6, comprising analyzing a GADD34 gene or a GADD34 protein of a biological sample from the subject or the reference subject.

8. The method of any one of the preceding claims, wherein the reference subject does not have the fibrotic airways disease.

9. The method of any one of the preceding claims, wherein the agent is an antibody, an aptamer, a protein, a peptide, a nucleic acid, or a small molecule.Docket No.: 6-25007 / 161118.0840110. The method of any one of the preceding claims, wherein the agent comprises Sephinl , a stereoisomer thereof, a derivative thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

11. The method of claim 10, wherein the Sephin 1 has a chemical structure below:

12. The method of any one of the preceding claims, wherein the subject is a mammal.

13. The method of any one of the preceding claims, wherein the subject is a human.

14. The method of any one of the preceding claims, wherein the agent is administered to the subject at one or more doses of from about 0.01 to about 10 mg / kg of body weight of the subject.

15. The method of any one of the preceding claims, wherein the agent is administered at one or more doses of from about 0.1 to about 5 mg / kg of body weight of the subject.

16. The method of any one of the preceding claims, wherein the agent is administered at one or more doses of from about 0.1 to about 1 mg / kg of body weight of the subject.

17. The method of any one of claims 14-16, wherein the one or more doses of the agent are administered at least every 6 hours, 12 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks.

18. The method of any one of the preceding claims, wherein the agent is administered to the subject intratumorally, intravenously, subcutaneously, intraosseously, orally, transdermally, sublingually, intratracheally, by nebulization, in sustained release, in controlled release, in delayed release, or as a suppository.Docket No.: 6-25007 / 161118.0840119. The method of any one of the preceding claims, wherein the agent is administered to the subject via inhalation or via direct instillation into lower airways.

20. The method of claim 19, wherein the inhalation is carried out with a nebulizer, an inhaler, a sprayer, a powder dispenser, or a dry powder generator.

21. The method of claim 19, wherein the direct instillation is carried out by intratracheal or mucosa.

22. The method of claim 19, wherein the direct instillation is carried out with an intrapulmonary aerosolizer or a sub-miniature aerosolizer.

23. The method of any one of the preceding claims, further comprising administering to the subject an additional therapeutic agent or therapy.

24. Use of an agent that inhibits a GADD34 in the manufacture of a medicament for treating a fibrotic airways disease in a subject in need thereof.

25. The use of claim 24, wherein the fibrotic airways disease is bronchiolitis obliterans or constrictive bronchiolitis.

26. The use of claim 25, wherein the agent comprises Sephinl, a stereoisomer thereof, a derivative thereof or a pharmaceutically acceptable salt thereof, or a combination thereof.

27. An agent that inhibits a GADD34 for use in treatment of a fibrotic airways disease in a subject in need thereof.

28. The agent for use of claim 27, wherein the fibrotic airways disease is bronchiolitis obliterans or constrictive bronchiolitis.

29. The agent for use of claim 27, comprising Sephinl, a stereoisomer thereof, a derivative thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

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