Aerosolized formulations of HIV protease inhibitors for the treatment of airway reflux

Abstract

The present invention provides a method for treating airway reflux using an HIV protease inhibitor capable of binding to and inhibiting the enzymatic activity of pepsin. Compositions including aerosolized formulations of the HIV protease inhibitor are also provided.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Patent Application No. 63 / 011,039, filed April 16, 2020, the contents of which are incorporated by reference in their entirety.

[0002] [Statement Regarding Federally Funded Research] none

[0003] [Sequence table] A Sequence Listing is attached to this application and is submitted as an ASCII text file of Sequence Listing entitled "650053_00792_ST25.txt", created on April 16, 2021, and 624 bytes in size. The Sequence Listing was submitted electronically via EFS-Web along with the application and is hereby incorporated by reference in its entirety. [Background technology]

[0004] [Introduction] Laryngopharyngeal reflux (LPR), the reflux of gastric contents into the larynopharynx, is a significant health problem. LPR affects children and adults alike, and the clinical spectrum of this disease is broad (1-3). Unlike patients with gastroesophageal reflux disease (GERD), which is limited to the esophagus and causes heartburn, LPR patients experience symptoms of chronic laryngeal irritation and inflammation, including chronic cough, throat clearing, postnasal drip, hoarseness or dysphonia, globus sensation, dysphagia, and dyspnea (1-3). Furthermore, there is significant evidence that chronic LPR contributes to life-threatening diseases such as laryngeal cancer (4-11). It is estimated that LPR affects more than 20% of the US population and is present in up to 10% of patients visiting an otolaryngologist (otolaryngology) (12-14). The economic burden of LPR exceeds $52 billion annually, which is 5.6 times greater than the cost of GERD, and 52% of this cost is attributable to the use of proton pump inhibitors (PPIs) ( 15 – 16 ).

[0005] PPI therapy is the mainstay of treatment for gastroesophageal reflux disease (GERD), but its effectiveness for treating LPR is low (18, 57). In clinical practice, it was previously thought that LPR patients simply required higher doses and longer PPI trials than GERD patients. This thinking was based on the assumption that the upper airway is more sensitive to acid reflux than the esophagus (1, 58, 59). However, placebo-controlled trials have not demonstrated a therapeutic benefit of PPIs for the treatment of LPR (60-65). Nevertheless, PPIs are still commonly prescribed for the treatment of LPR due to the lack of alternatives (23, 72). Given the potential risks of long-term PPI therapy, its associated costs, and the high proportion of LPR patients for whom PPI therapy has been shown to be ineffective, alternative treatments for LPR are desperately needed (1, 18, 31, 57, 73, 74). Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides methods for treating reflux in a subject in need thereof. In one aspect, the method comprises administering to the subject a therapeutically effective amount of an HIV protease inhibitor to treat reflux. Preferably, the HIV protease inhibitor is capable of binding to pepsin and inhibiting the enzymatic activity of pepsin. In some aspects, the subject has airway reflux, and preferably, the airway reflux condition is selected from laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), and esophagopharyngeal reflux (EPR). In another aspect, the subject has gastroesophageal reflux disease (GERD), preferably GERD that is refractory to proton pump inhibition.

[0007] In another aspect, the present disclosure provides a composition comprising an aerosolized formulation of an HIV protease inhibitor capable of inhibiting pepsin and a pharmaceutically acceptable carrier.

[0008] In a further aspect, the present disclosure provides for the use of a composition described herein to treat reflux in a subject in need thereof, wherein the subject has a condition selected from the group consisting of laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), esophagopharyngeal reflux (EPR), or GERD refractory to protein pump inhibition. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows a schematic of the assays used to screen for compounds that inhibit pepsin. Assay 1 (top) is a binding assay that measures the extent to which compounds compete with fluorescently labeled pepstatin for binding sites on pepsin. Assay 2 (bottom) is a pepsin activity assay that utilizes fluorescently labeled casein as the enzyme substrate. [Figure 2] 1 shows the percent inhibition of pepsin produced by a library of pharmacologically active compounds screened using the binding assay. [Figure 3A] Binding (A) curves of pepsin in the presence of the indicated HIV protease inhibitors are shown. [Figure 3B] Activity curves of pepsin in the presence of the indicated HIV protease inhibitors (B) are shown. [Figure 4A] The crystal structure of amprenavir bound to pepsin is shown. (A) The active site of porcine pepsin with amprenavir bound. The 2Fo-Fc electron density map contoured at 1.0σ is shown in magenta mesh, and the 2Fo-Fc simulated annealing composite omit map contoured at 1.0σ is shown in green mesh. Generated using POVSCRIPT and POV-Ray. [Figure 4B] (B) Schematic of the active site with bound amprenavir, showing potential hydrogen-bonding interactions with dashed green lines. Created using a combination of MarvinSketch (www.ChemAxon.com) and Adobe Illustrator CC 2020. [Figure 4C] Crystal structure of amprenavir bound to pepsin. Structure and enzyme-inhibitor interactions at the active site of pepsin with bound darunavir. Generated using POVSCRIPT and POV-Ray. [Figure 4D] This shows the crystal structure of amprenavir bound to pepsin. It also shows the structure of darunavir bound to pepsin and enzyme-inhibitor interactions at the active site. Created using a combination of MarvinSketch (www.ChemAxon.com) and Adobe Illustrator CC 2020. [Figure 5A-D] In vivo pepsin-induced respiratory epithelial damage is shown. Representative animals are treated with various protocols: pH 7 (Panel A), pH 4 (Panel B), 0.3 mg / ml pepsin at pH 7 (Panel C), and 0.3 mg / ml pepsin at pH 4 (Panel D). Panels A–D are at 20x magnification. (A) Normal respiratory epithelium (arrow) is approximately one cell layer thick with basal polarization of the nuclei and apical ciliary surfaces. (B) Reactive respiratory epithelium characterized by hyperplasia (thick arrow) and focal squamous metaplasia (long arrow) with loss of cilia. Other areas show relative thickening of the mucosa with a moderately increased nuclear-to-cytoplasmic (N:C) ratio and irregular, condensed chromatin. (C) Thickened respiratory epithelium with pseudostratification of epithelial cells. Keratinization (arrows) is present in multiple foci. A significant increase in the N:C ratio, accompanied by loss of nuclear polarization and a reduction in apical cilia, is evident in several areas of this treatment group. (D) The respiratory epithelium becomes necrotic (arrows) and is replaced by inflammatory exudate. An active acute inflammatory infiltrate infiltrates the submucosal region. [Figure 5E-H]In vivo pepsin-induced respiratory epithelial damage is shown. Representative animals are treated with various protocols: pH 7 (Panel E), pH 4 (Panel F), 0.3 mg / ml pepsin at pH 7 (Panel G), and 0.3 mg / ml pepsin at pH 4 (Panel H). Panels E–H are at 50x magnification. (E) Normal respiratory epithelium (arrow) is approximately one cell layer thick with basal polarization of the nuclei and apical surfaces of the cilia. (F) Reactive respiratory epithelium characterized by hyperplasia (thick arrow) and focal squamous metaplasia (long arrow) with loss of cilia. Other areas show relative thickening of the mucosa with a moderately increased nuclear-to-cytoplasmic (N:C) ratio and irregular, condensed chromatin. (G) Thickened respiratory epithelium with pseudostratification of epithelial cells. Keratinization (arrows) is present in multiple foci. A significant increase in the N:C ratio, accompanied by loss of nuclear polarization and a reduction in apical cilia, is evident in several areas of this treatment group. (H) The respiratory epithelium becomes necrotic (arrows) and is replaced by inflammatory exudate. An active acute inflammatory infiltrate infiltrates the submucosal region. [Figure 6A-B] Oral administration of Lexiva® prevents pepsin-mediated airway epithelial damage in vivo. Representative animals from various treatment protocols are shown in each panel: pH7 (Panel A), Lexiva (Panel B). Magnification: 20x. (A, B) Normal-appearing respiratory epithelium consisting of a single layer of ciliated columnar epithelium with basal polarization of the nuclei and apical ciliary surfaces. [Figure 6C-D] Oral administration of Lexiva has been shown to prevent pepsin-mediated airway epithelial damage in vivo. Representative animals for various treatment protocols are shown in each panel: 0.3 mg / ml pepsin at pH 7 (Panel C), 0.3 mg / ml pepsin pH 7 + Lexiva (Panel D). Magnification 20x. (D) Normal-appearing respiratory epithelium consisting of a single layer of ciliated columnar epithelium with basal polarization of the nucleus and apical cilia. (C) Reactive multilayered epithelium with an increased nucleus-to-cytoplasm (N:C) ratio and loss of cilia. [Figure 7A-Bii]Inhaled administration of fosamprenavir and darunavir prevents pepsin-mediated laryngeal injury. Representative animals from different groups: A) Vehicle control: a single layer of respiratory epithelium with no reactive changes. B) Pepsin control: multilayered epithelium and individual cell apoptosis (arrows): i) High magnification of multilayered epithelium, and ii) high magnification of cell apoptosis. A and B: 20x magnification with scale bar = 50 μm. Bi and Bii: 40x magnification with scale bar = 20 μm. [Figure 7C-F] Inhaled administration of fosamprenavir and darunavir prevents pepsin-mediated laryngeal damage. Representative animals from different groups: C) Fosamprenavir gavage: vehicle group, histologically normal. [This tissue section is more proximal than the other sections, almost in the oropharynx, so some transitional epithelium is visible.] D) Fosamprenavir gavage: pepsin group, histologically normal. E) Fosamprenavir aerosol: vehicle group, histologically normal. F) Fosamprenavir aerosol: pepsin group, histologically normal. C-F: Scale bar = 50 μm, 20x magnification. [Figure 7G-J] Inhaled administration of fosamprenavir and darunavir prevents pepsin-mediated laryngeal damage. Representative animals from different groups: G) Darunavir gavage: mildly reactive epithelium in the vehicle group, but less than in the pepsin control. H) Darunavir gavage: mildly reactive epithelium in the pepsin group, but less than in the pepsin control. I) Darunavir aerosol: histologically normal in the vehicle group. J) Darunavir aerosol: histologically normal in the pepsin group. G-J: Scale bar = 50 μm, 20x magnification. [Figure 8A] Figure 1 is a schematic diagram of a 12-week, randomized, double-blind, placebo-controlled clinical trial designed to test the efficacy of the HIV protease inhibitor Lexiva for the treatment of LPR. [Figure 8B] Figure 1 is a schematic diagram of a 12-week, randomized, double-blind, placebo-controlled clinical trial designed to test the efficacy of the HIV protease inhibitor Lexiva for the treatment of LPR. [Figure 9A]The crystal structure of ritonavir bound to pepsin is shown. (A) The active site of porcine pepsin with ritonavir bound. The 2Fo-Fc electron density map contoured at 1.0σ is shown in magenta mesh, and the 2Fo-Fc simulated annealing composite omit map contoured at 1.0σ is shown in green mesh. [Figure 9B] The crystal structure of ritonavir bound to pepsin is shown. (B) Schematic of the active site with ritonavir bound, showing potential hydrogen-bonding interactions with dashed green lines. [Figure 10A] The crystal structure of saquinavir bound to pepsin is shown. (A) The active site of porcine pepsin with saquinavir bound. The 2Fo-Fc electron density map contoured at 1.0σ is shown in magenta mesh, and the 2Fo-Fc simulated annealing composite omit map contoured at 1.0σ is shown in green mesh. [Figure 10B] The crystal structure of saquinavir bound to pepsin is shown. (B) Schematic of the active site with saquinavir bound, showing potential hydrogen-bonding interactions with dashed green lines. DETAILED DESCRIPTION OF THE INVENTION

[0010] In this application, we disclose a novel means for treating reflux conditions, including airway reflux, such as laryngopharyngeal reflux (LPR). The deleterious laryngopharyngeal changes observed in LPR occur after direct contact of the mucosa with refluxed gastric contents, consisting of acid, pepsin, bile, and pancreatic enzymes. Recent research has led to a shift in understanding of the underlying causes of LPR, and it is now understood that the non-acidic components of gastric reflux contribute significantly to the disease. Studies using combined multichannel intraluminal impedance and pH (MII-pH) monitoring have shown that many episodes of LPR are non-acidic, and that weakly acidic and non-acidic reflux is associated with persistent symptoms in acid-suppressed patients (39-42). Pepsin, the main digestive enzyme in the stomach, has increasingly been implicated as a contributor to the damage and inflammation associated with LPR (17-23). Importantly, the stomach and esophagus have internal defense mechanisms against pepsin, such as mucus, peristalsis, and bicarbonate secretion, but laryngeal tissue does not (26). In the airway, where pH is neutral (<8), pepsin is enzymatically inactive but stable. However, once pepsin is taken up by laryngeal and hypopharyngeal cells via receptor-mediated endocytosis, it is retained in intracellular vesicles at low pH, where it is presumably reactivated and causes damage (20, 32, 33, 49, 52). Although many episodes of LPR are weakly acidic or non-acidic, pepsin is present in all reflux fluids (24) and is frequently detected in airway tissues and secretions from LPR patients. For example, we have demonstrated that endocytosed non-acidic pepsin induces the expression of proinflammatory cytokine genes in hypopharyngeal cells. This response is similar to that occurring in reflux esophagitis and contributes to the severity of disease in GERD patients (21, 31). Importantly, inhibition of pepsin's proteolytic activity (i.e., using pepstatin, curcumin, ecabet sodium, anthocyanins, or by preincubation at pH 8.0 before lowering the pH to 7.0) has been shown to abrogate this damage and inflammation (5, 7, 22, 33, 52, 54-56), making pepsin a promising therapeutic target for the treatment of airway reflux.

[0011] The inventors believe that LPR depends more on pepsin-mediated damage than acid-mediated damage, and that drugs that specifically target pepsin may be effective in patients with non-acid reflux. These drugs may ultimately provide a treatment option for patients resistant to proton pump inhibitors (PPIs). Pepsin can be inhibited by two mechanisms: (1) irreversible inactivation, which prevents pepsin from being reactivated in lower pH intracellular compartments, and (2) receptor antagonists, which prevent pepsin uptake by receptor-mediated endocytosis. The pepsin inhibitor pepstatin is already commercially available, but it has poor water solubility and pharmacokinetic properties. Therefore, new pepsin inhibitor compounds with high bioavailability are needed.

[0012] In this application, we screened therapeutic compounds for their ability to bind to pepsin and inhibit its enzymatic activity and identified specific HIV protease inhibitors that possess these abilities (see Example 1). Several HIV protease inhibitors have already been approved by the U.S. Food and Drug Administration (FDA) for the treatment of HIV, making these drugs ideal candidates for testing the efficacy of pepsin inhibition for the treatment of LPR. Using epidemiological data, we demonstrated that patients taking HIV protease inhibitors have a significantly lower incidence of airway reflux (0.2%) compared to the general population (10-34.4%), supporting the idea that these HIV medications could be repurposed to treat LPR. Of 10 commercially available HIV protease inhibitors, we determined that four (i.e., amprenavir, darunavir, ritonavir, and saquinavir) possess the ability to bind to pepsin and inhibit its enzymatic activity in vitro (Figure 3). To test these drug candidates in vivo, we established a novel mouse model of LPR (Figure 5A-H). Using these mice, we tested the ability of HIV protease inhibitors to ameliorate pepsin-mediated laryngeal mucosal damage and inflammation. Mice were administered HIV protease inhibitors by both oral gavage and aerosolized delivery, comparing the results of systemic and local delivery, respectively. Based on the results of these animal studies, we will test the efficacy of promising HIV protease inhibitors in a 12-week, randomized, double-blind, placebo-controlled clinical trial (see Example 2).

[0013] [method:] The present invention provides a method for treating reflux, preferably airway reflux, in a subject in need thereof. The method comprises administering a therapeutically effective amount of an HIV protease inhibitor to the subject to treat reflux. As used herein, the term "airway reflux" refers to inflammation of the upper and lower airways caused by the reflux of gastric contents. The term airway reflux is used interchangeably with the alternative terms "supraesophageal reflux" and "extraesophageal reflux." These broad terms encompass several related reflux conditions, including gastropharyngeal reflux (GPR; reflux of gastric contents up to the esophagus), laryngopharyngeal reflux (LPR; reflux of gastric contents beyond the esophagus into the laryngopharynx), and esophagopharyngeal reflux (EPR; a condition similar to LPR characterized by esophageal abnormalities). Reflux also includes gastroesophageal reflux disease (GERD), which refers to inflammation of the esophagus caused by the reflux of gastric contents back up into the esophagus. The reflux treated herein is preferably a GERD patient who is resistant to protein pump inhibitor (PPI) therapy.

[0014] As used herein, the term "HIV protease inhibitor" refers to any antiviral drug that inhibits one or more HIV proteases. HIV protease inhibitors selectively bind to HIV protease and prevent viral replication by blocking the proteolytic cleavage of protein precursors required for the production of infectious viral particles. Suitable HIV protease inhibitors include those already approved by the U.S. Food and Drug Administration (FDA) for the treatment of HIV, such as amprenavir (IUPAC: [(3S)-oxolan-3-yl]N-[(2S,3R)-4-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-1-phenylbutan-2-yl]carbamate), ritonavir (IUPAC: 1,3-thiazol-5-ylmethyl N-[(2S,3R)-4-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-1-phenylbutan-2-yl]carbamate), and ritonavir (IUPAC: 1,3-thiazol-5-ylmethyl N-[(2S,3R)-4-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-1-phenylbutan-2-yl]carbamate). S,5S)-3-hydroxy-5-[[(2S)-3-methyl-2-[[methyl-[(2-propan-2-yl-1,3-thiazol-4-yl)methyl]carbamoyl]amino]butanoyl]amino]-1,6-diphenylhexan-2-yl]carbamate), lopinavir (IUPAC: (2S)-N-[(2S,4S,5S)-5-[[2-(2,6-dimethylphenoxy)acetyl]amino]-4-hydroxy-1,6-diphenylhexan-2-yl ]-3-methyl-2-(2-oxo-1,3-diazinan-1-yl)butanamide), saquinavir (IUPAC: (2S)-N-[(2S,3R)-4-[(3S,4aS,8aS)-3-(tert-butylcarbamoyl)-3,4,4a,5,6,7,8,8a-octahydro-1H-isoquinolin-2-yl]-3-hydroxy-1-phenylbutan-2-yl]-2-(quinoline-2-carbonylamino)butanediamide), nelfinavir (IUPAC: AC: (3S,4aS,8aS)-N-tert-butyl-2-[(2R,3R)-2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-phenylsulfanylbutyl]-3,4,4a,5,6,7,8,8a-octahydro-1H-isoquinoline-3-carboxamide), darunavir (IUPAC: [(3aS,4R,6aR)-2,3,3a,4,5,6a-hexahydrofuro-2,3-b]furan-4-yl]N-[(2S,3R)-4-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-1-phenylbutan-2-yl]carbamate), indinavir ((2S)-1-[(2S,4R)-4-benzyl-2-hydroxy-5-[[(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]amino]-5-oxopentyl]-N-tert-butyl-4-(pyridin-3-ylmethyl)piperazine-2-carboxamide), atazanavir (IUPAC: methyl N-[(2S)-1-[2-[(2S,3S)-2-hydroxy]-3-[[(2S)-2-(methoxycarbonylamino)-3,3-dimethylbutanoyl]amino]-4-phenylbutyl]-2-[(4-pyridin-2-yl

[0033] Examples of thiazol-5-ylmethyl N-[(2R,5R)-5-[[(2S)-2-[[methyl-[(2-propan-2-yl-1,3-thiazol-4-yl)methyl]carbamoyl]amino]-4-morpholin-4-ylbutanoyl]amino]-1,6-diphenylhexan-2-yl]carbamate). The HIV protease inhibitors used in the present invention must be capable of binding to pepsin and inhibiting the enzymatic activity of pepsin. Thus, in some embodiments, the HIV protease inhibitor is amprenavir, darunavir, ritonavir, or saquinavir, which were shown in Example 1 to bind to and inhibit pepsin. In some embodiments, the HIV protease inhibitor is amprenavir (IUPAC: [(3S)-oxolan-3-yl]N-[(2S,3R)-4-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino])-3-hydroxy-1-phenylbutan-2-yl]carbamate) or its prodrug, fosamprenavir (IUPAC: [(3S)-oxolan-3-yl]N-[(2S,3R)-4-[(4-aminophenyl)]sulfonyl-(2-methylpropyl)amino]-1-phenyl-3-phosphonooxybutan-2-yl]carbamate). HIV protease inhibitors are known in the art and are commercially available.

[0015] Fosamprenavir is a prodrug of amprenavir sold by ViiV Healthcare as a calcium salt under the trade names Lexiva (USA) and Telzir (Europe). The body must metabolize fosamprenavir to form the active form of the drug, amprenavir. Thus, administering amprenavir as a prodrug acts like a sustained-release formulation, extending its availability in the body. Furthermore, because fosamprenavir has demonstrated excellent pharmacokinetics in mice and is already FDA-approved, fosamprenavir may rapidly progress into pilot clinical trials. In some embodiments, HIV protease inhibitors for use in the compositions and methods described herein have an IC value in the micromolar range (μm). 50 In some preferred embodiments, HIV protease inhibitors for use in the compositions and methods described herein have IC in the nanomolar (nm) range. 50 It has.

[0016] In the present methods, the HIV protease inhibitor can be administered using any route effective for treating reflux, preferably airway reflux. As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, administration by inhalation, intranasal administration, nebulizer administration, or other routes of administration that reach the upper respiratory tract. Administration can be continuous or intermittent.

[0017] In some embodiments, the HIV protease inhibitor is administered orally for the treatment of reflux. For example, in some embodiments, the HIV protease inhibitor is administered at approximately 0.7 to 1.4 g twice daily (i.e., an FDA-approved and therefore safe dose for the treatment of HIV). However, the inventors hypothesize that administering the drug in aerosolized form increases local delivery and improves efficacy. (See the section entitled "Compositions" for a more detailed discussion of aerosolized formulations.) Thus, in some embodiments, the HIV protease inhibitor is administered as an aerosol. For example, in certain embodiments, the HIV protease inhibitor is administered as a nasal spray, and in other embodiments, via an inhaler. In some embodiments, the HIV protease inhibitor is administered at a dose of less than 1.4 g twice daily, including less than 0.7 g twice daily, and in some embodiments, may include microgram amounts twice daily.

[0018] Oral administration of fosamprenavir at 20 mg / kg / day (as Lexiva), a dose equivalent to that used to treat HIV in humans, prevented pepsin-mediated laryngeal damage (defined as multilayered reactive epithelium and cell apoptosis) in our in vivo mouse model (Figure 7A-J). In contrast, oral administration of darunavir (Prezista 8.6 mg / kg / day), also at a human-equivalent dose, did not prevent pepsin-mediated laryngeal damage (Figure 7H). Fosamprenavir administered by inhalation at a dose of 1 mg / kg / day prevented pepsin-mediated laryngeal damage (Figure 7F). Importantly, darunavir administered as an aerosol at a dose of 12 mg / kg / day (Figure 7J) was also effective in preventing pepsin-mediated injury. The inhaled dose was calculated from the measured exposure conditions and implicitly assumed that all drug was deposited. Actual deposition is likely closer to 10%.

[0019] The methods of the present invention are used to treat reflux in a subject in need thereof. In some embodiments, the reflux may be airway reflux. In other embodiments, the reflux may be GERD, preferably GERD in a subject resistant to proton pump inhibition. As used herein, the term "subject in need thereof" or "patient" refers to any human or animal suffering from reflux. In some embodiments, the subject has airway reflux. In some embodiments, the airway reflux condition is selected from laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), and esophagopharyngeal reflux (EPR). In some embodiments, the subject is a subject having reflux episodes caused by weakly acidic or non-acidic reflux. In another embodiment, the subject is a subject resistant to proton pump inhibitor (PPI) therapy.

[0020] As used herein, the terms "treat," "treating," or "treatment" describe the management and care of a subject for the purpose of combating a disease, condition, or disorder. "Treating" includes administering a protease inhibitor or composition of the invention to prevent the onset of symptoms or complications, alleviate symptoms or complications, or eliminate a disease, condition, or disorder. In preferred embodiments, the methods and compositions of the invention reduce mucosal damage and inflammation in the airways of a subject. "Treatment" also includes reducing one or more symptoms of airway reflux, preferably LPR, GPR, or ERP, such as chronic cough, throat clearing, postnasal drip, hoarseness or dysphonia, globus sensation, dysphagia, dyspnea, or a combination thereof. Treatment also includes reducing chronic laryngeal irritation and inflammation. In one embodiment, "treatment" also includes alleviating one or more symptoms of PPI-resistant GERD, such as one or more of the following symptoms, particularly a burning sensation in the chest (heartburn), which may be worse at night, usually after meals; chest pain; difficulty swallowing; regurgitation of food or sour liquids; and a feeling of a lump in the throat.

[0021] The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological or clinical result. This result may be the reduction, alleviation, suppression, or prevention of one or more symptoms of a disease or condition, the reduction, suppression, or prevention of laryngeal irritation, the reduction, suppression, or prevention of laryngeal irritation or mucosal damage, or the reduction, alleviation, suppression, or prevention of one or more symptoms of airway reflux, or any other desired alteration of a biological system. In some embodiments, an effective amount is an amount suitable to provide a desired effect, e.g., reduce mucosal damage and inflammation in the airways. Response to treatment of airway reflux can be assessed using any standard clinical method, including, but not limited to, visual examination of the larynx (e.g., fiberoptic laryngoscopy), reflux symptom index (RSI), reflux finding score (RFS) (e.g., a physician-reported score based on visual examination of the larynx), combined esophageal multichannel intraluminal impedance and pH monitoring (MII-pH), reflux symptom score (RSS), reflux symptom assessment (RSA), or pepsin activity in saliva. Alternatively, the response to airway reflux treatment can be assessed by diagnosing inflammation in tissue samples taken from the subject's airways, for example, by hematoxylin and eosin (H&E) staining or by detecting the presence of neutrophil infiltration, keratinization, and necrosis. Another suitable method is to measure pepsin activity before and after 12 weeks of treatment. While HIV inhibitors are not expected to prevent reflux or affect pepsin protein levels, they do inactivate the pepsin enzyme, so measuring pepsin activity in saliva after treatment confirms that the treatment is inactivating pepsin in the airways. This is currently a research tool for assessing in vivo efficacy.

[0022] Patients with reflux episodes caused by weakly acidic or non-acidic reflux are largely resistant to proton pump inhibitor (PPI) therapy, which inhibits acid production but does not affect pepsin activity. The methods of the present invention are particularly beneficial for this group of refractory patients who are in dire need of an alternative to PPIs. As used herein, the phrase "refractory to treatment" refers to a condition that does not respond to treatment. For example, a patient's reflux may be considered resistant to PPI therapy if three months of twice-daily treatment with a PPI does not significantly improve the condition. Response to reflux treatment can be assessed using any standard means known in the art, including, but not limited to, the Reflux Symptom Index (RSI), the Reflux Finding Score (RFS), combined esophageal multichannel intraluminal impedance and pH monitoring (MII-pH), the Reflux Symptom Score (RSS), or the Reflux Sign Assessment (RSA). See the Examples section for a detailed description of these measurements. For example, an effective treatment would reduce the RSI and / or RFS to normal values, eg, RSI≦13, RFS≦7, or a combination thereof.

[0023] [Composition:] The present invention also provides compositions comprising an aerosolized formulation of an HIV protease inhibitor and a pharmaceutically acceptable carrier. Commercially available HIV protease inhibitors are typically formulated as tablets or oral suspensions for systemic drug delivery. However, the inventors contend that the HIV protease inhibitors used herein that can inhibit pepsin may be more effective in treating airway reflux when delivered locally. Accordingly, the present invention provides reformulated HIV protease inhibitors in aerosolized form for delivery by inhalation. For example, in some embodiments, the aerosol is formulated for oral or nasal administration. In one example, the aerosol can be formulated for inhalation. The present invention also encompasses the use of these aerosolized formulations of HIV protease inhibitors for the treatment of acid reflux in a subject in need thereof.

[0024] As used herein, the term "aerosolization" refers to the process by which a substance is converted into a fine spray or colloidal suspension in the air. Aerosolization is typically achieved using an aerosol generator, such as an aerosol drug delivery device. Aerosol drug delivery devices suitable for use in the present invention include, but are not limited to, pressurized metered-dose inhalers, dry powder inhalers, spacers and holding chambers, and nebulizers. As will be appreciated by those skilled in the art, each of these devices may require a different drug formulation. For example, for use in pressurized metered-dose inhalers, the drug is mixed with a propellant (e.g., a halogenated fluoroalkane such as HFA227 or HFA134A). For use in dry powder inhalers, the drug is formulated as a powder and is often mixed with other powders (e.g., lactose) to reduce agglomeration. Nebulizers vary significantly in concept and can be designed for use with a wide range of liquid formulations. Thus, in some embodiments, the composition is a liquid or a suspension. In other embodiments, the composition is a lyophilized or otherwise dried formulation.

[0025] Drug delivery via inhalation requires a formulation that can be successfully aerosolized. Mouse inhalation studies have involved optimized delivery in mouse models by administering pure drug in relatively small aerosol particles (i.e., 1 μm). For translation from in vivo to clinical applications, drugs can be formulated to optimize inhalation delivery to humans. Here, larger particle sizes are generated to take advantage of the fact that humans can be instructed to perform controlled, forceful inhalations to maximize particle deposition. Computational fluid dynamics analysis of particle deposition in the larynx revealed that the optimal particle size for larynx deposition is 9–12 microns (unpublished data), which is in good agreement with other studies (Perkins et al., 2018). Specifically, to produce a therapeutic effect, aerosolized drug particles or droplets must be of sufficient size and mass to be transported to the portion of the airway requiring treatment (i.e., the pharynx). When aerosol particles are inhaled orally or nasally, larger particles (>10 μm) are filtered by the nose and / or throat (mostly by inertial impaction), while 5-10 μm particles generally reach the proximal lower respiratory tract and 1-5 μm particles reach the periphery of the lungs. Thus, in some embodiments, the HIV protease inhibitor particles in the aerosol average 1-15 μm in size. However, with increased deposition in the larynx, the HIV protease inhibitor particles in the aerosol average 5-15 μm in size. In preferred embodiments, the HIV protease inhibitor particles in the aerosol average 8-12 μm, 9-12 μm, or 8-10 μm, or other average sizes in between, e.g., 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 10.5 μm, 11 μm, or 12 μm. For example, for use in the present invention, HIV protease inhibitors can be aerosolized using an ultrasonic atomizer, as described in detail in the Materials and Methods section of Example 1.

[0026] The compositions of the present invention may contain any pharmaceutically acceptable carrier that allows aerosol delivery. "Pharmaceutically acceptable carriers" are known in the art and include, but are not limited to, suitable diluents, preservatives, solubilizers, emulsifiers, liposomes, nanoparticles, and adjuvants. Pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include isotonic solutions, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media.

[0027] The compositions of the present invention may further comprise additional components that affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate of the HIV protease inhibitor. Suitable components include, but are not limited to, buffers (e.g., Tris-HCl, acetate, phosphate), additives such as albumin or gelatin to prevent absorption to surfaces, surfactants (e.g., Tween 20, Tween 80, Pluronic F68, bile salts), solubilizers (e.g., glycerol, polyethyleneglycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking agents, and tonicity adjusters (e.g., lactose, mannitol). In addition, the compositions may be formulated for controlled or sustained release of the HIV protease inhibitor, for example, through formulation in a lipophilic depot (e.g., fatty acids, waxes, oils).

[0028] The compositions may be prepared in unit dosage form for administration to a subject, with the amount and timing of administration being at the discretion of the treating clinician to achieve the desired outcome.

[0029] The HIV protease inhibitor included in the composition of the present invention can be any HIV protease inhibitor suitable for treating airway reflux, as discussed above. In some embodiments, the HIV protease inhibitor included in the composition is amprenavir, darunavir, ritonavir, saquinavir, or a derivative thereof. In a preferred embodiment, the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir. In another embodiment, the HIV protease inhibitor is darunavir.

[0030] The invention has been described in terms of one or more preferred embodiments, and it is to be understood that, except as expressly stated, many equivalents, alternatives, variations, and modifications are possible and within the scope of the invention.

[0031] It should be apparent to those skilled in the art that many additional modifications beyond those already described are possible without departing from the concept of the present invention. In interpreting this disclosure, all terms should be interpreted in the broadest possible sense consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive sense, such that a recited element, component, or step can be combined with other elements, components, or steps not expressly recited. Embodiments referred to as "comprising" particular elements are also contemplated as "consisting essentially of" and "consisting of" those elements. The terms "consisting essentially of" and "consisting of" should be interpreted consistent with the interpretations of the MPEP and the relevant Federal Circuit Court. The transitional phrase "consisting essentially of" limits a claim to the specific materials or steps of the claimed invention and to the extent that "does not materially affect the basic and novel characteristics." "Consisting of" is a closed term excluding elements, steps, or ingredients not specified in the claim. For example, reference to a sequence "consisting of" refers to the sequence recited in a SEQ ID NO, as well as to a larger sequence that may include the SEQ ID NO as a portion thereof.

[0032] All references cited herein are expressly incorporated by reference in their entirety.

[0033] The present invention will be more fully understood by consideration of the following non-limiting examples. [Example]

[0034] Example 1: In vivo and animal evaluation of HIV protease inhibitors for the treatment of airway reflux. In the following examples, we used binding and enzymatic assays to evaluate the ability of 10 commercially available, FDA-approved HIV protease inhibitors to bind to and inhibit pepsin. We then used co-crystallization of these inhibitors with pepsin to help identify the best drug candidates for testing in subsequent studies. Finally, we established a mouse model of laryngopharyngeal reflux (LPR). We used these mice to test the ability of candidate drugs to abrogate pepsin-mediated laryngeal damage both when administered by oral gavage and when administered by aerosolized delivery.

[0035] [Background technology] The adverse laryngopharyngeal changes observed in LPR occur after direct contact of the mucosa with refluxed gastric contents, consisting of acid, pepsin, bile, and pancreatic enzymes. Because the acidity of reflux alone could cause tissue damage in the upper airway, LPR was once considered an acid-mediated disease. However, recent research has changed our understanding of the underlying causes of LPR, and it is now understood that the nonacidic components of gastric reflux contribute significantly to the disease. While in vitro studies have reported mucosal damage caused by bile at nonacidic pH, there is no evidence that the same process occurs in the human larynx. This is likely because the concentration of bile reaching the proximal laryngopharynx is insufficient to cause cell membrane damage (19). Studies using combined multichannel intraluminal impedance and pH (MII-pH) monitoring have shown that many episodes of LPR are nonacidic, and that weakly acidic and nonacidic reflux are associated with persistent symptoms in acid-suppressed patients (39-42).

[0036] Pepsin, the main digestive enzyme in the stomach, has increasingly been implicated as a contributor to the damage and inflammation associated with LPR (17-23). ​​Pepsin is a proteolytic enzyme that is initially synthesized and secreted as the zymogen pepsinogen by chief cells in the gastric fundus, which then undergoes autocatalytic cleavage to generate the mature form of pepsin upon introduction into the acidic environment of the gastric lumen. Importantly, the stomach and esophagus possess internal defense mechanisms against pepsin, such as mucus, peristalsis, and bicarbonate secretion, but laryngeal tissue does not (26). Pepsin exhibits maximal activity at pH 2 and remains active down to pH 6.5. In fact, the enzyme remains stable up to pH 8, above which the molecule loses its secondary structure and the enzyme is irreversibly inactivated (20, 25). In the airway, at a neutral pH (<8), pepsin is enzymatically inactive but stable. However, once pepsin is taken up into laryngeal and hypopharyngeal cells via receptor-mediated endocytosis, it is retained in low-pH intracellular vesicles, where it is presumed to be reactivated and cause damage (20, 32, 33, 49, 52). Although many episodes of LPR are weakly acidic or non-acidic, pepsin is present in all reflux fluids (24) and is frequently detected in airway tissues and secretions from LPR patients. Furthermore, studies have demonstrated that endocytosed pepsin causes mitochondrial damage and upregulates the expression of several genes involved in stress and toxicity (53). For example, we have demonstrated that endocytosed non-acidic pepsin induces the expression of proinflammatory cytokine genes in hypopharyngeal cells. This response is similar to that occurring in reflux esophagitis and contributes to the severity of disease in GERD patients (21, 31). Importantly, inhibition of pepsin's proteolytic activity (i.e., using pepstatin, curcumin, ecabet sodium, anthocyanins, or by preincubation at pH 8.0 before lowering the pH to 7.0) has been shown to abrogate this damage and inflammation (5, 7, 22, 33, 52, 54-56), making pepsin a promising therapeutic target for the treatment of airway reflux.

[0037] Materials and Methods: Competitive Binding and Activity Assays A fluorescence polarization assay was developed to test compounds for binding and inhibition of pepsin (Figure 1). A competitive binding assay was developed using pepstatin, a known inhibitor of pepsin with subnanomolar affinity. 91 (Roberts 2003). This binding assay measures relative binding affinity but not enzyme inhibition by compounds. Therefore, another pepsin activity assay was developed using fluorescently labeled alpha-casein as a substrate. Both assays are based on the principle of fluorescence polarization (FP), which means that as the molecular size of a fluorescent species changes due to dissociation / decomposition or association / binding events, the depolarization of plane-polarized light changes accordingly, directly affecting the FP value. 92 (Lea2011). In the assay used herein, millipolarization (mP) decreases as casein-Alexa647 is degraded by pepsin and increases as pepsin binds to pepstatin-Alexa647.

[0038] Casein is a soluble fiber found in the soluble fiber of the soluble fiber. 93The samples were labeled using a 2.5 μg / mg label-to-protein ratio as described in [1]. Specifically, 200 μl (10 mg / ml) bovine alpha-casein (C6780 Sigma-Aldrich, St. Louis, MO) in 0.1 M sodium bicarbonate was incubated with 5 μl (1 mg / ml) Alexa Fluor 647 carboxylic acid, succinimidyl ester (in DMSO, A-20106 ThermoFisher Scientific, Waltham, MA) for 15 minutes at room temperature. The mixture was applied to a Sephadex G-25 column (90 x 5 mm) in a glass Pasteur pipette. Elution was performed using D-PBS, pH 7.4, containing 0.1% sodium azide (ThermoFisher Scientific). The fast-moving band containing the casein-bound fluorophore was collected in a volume of approximately 0.4 ml. The concentration of the resulting probe (casein-Alexa647 in PBS-azide) was estimated spectrophotometrically using Beer's law (Implen Nanophotometer, Implen, Inc., Westlake Village, CA). The probe was aliquoted and stored at -20°C until use.

[0039] Assays were performed in 20 μl volumes in 384-well black optical plates (Nunc, Roskilde, DK). A pepstatin dose-response curve was used to optimize pepsin / inhibitor concentrations and assay incubation times. Dose responses were performed using 0.3–1000 μM unlabeled pepstatin with 100–500 nM pepstatin-Alexa647 probe, 0.003–3 U / μl porcine pepsin (Worthington, Lakewood, NJ), and 0.1 M HCl, pH 1 + 0.01% Tween-20 containing 10–37% dimethyl sulfoxide (HIV protease inhibitor diluent). Reagents were mixed at 50% of the final reaction volume (pepsin was added last) and immediately read for 5 minutes in at least triplicate using a BioTek Cytation 5 (BioTek Instruments, Winooski, VT) equipped with a far-red FP filter cube at 620 / 680 nM excitation / emission. Mean polarization values ​​of replicate reads were plotted against probe or labeled pepstatin concentration in GraphPad Prism 8 (La Jolla, CA). The reagent concentration resulting in the largest dynamic range was selected for the assay. Pepsin activity assays were similarly optimized using a casein-Alexa647 probe in 0.1 M HCl, pH 1 + 0.01% Tween-20. Reagents were mixed (pepsin was added last) and immediately read at minimal intervals (<2 min intervals) over 30 minutes. Assays were performed in triplicate, and mean polarization values ​​were plotted over time.

[0040] HIV protease inhibitors were dissolved in DMSO. Amprenavir, ritonavir, lopinavir, saquinavir mesylate, nelfinavir mesylate hydrate, darunavir ethanol, and indinavir sulfate hydrate were obtained from Sigma-Aldrich. Compounds were tested in triplicate over a 3 log(concentration) range under optimized assay conditions and plotted using GraphPad Prism 8. Half-maximal inhibitory concentrations (IC 50 ) is an online tool (icekat.herokuapp.com / icekat, Olp2019 95) Percent binding and percent activity were calculated by normalization to polarization values ​​measured in the absence of HIV protease inhibitor compounds.

[0041] [crystal] Lyophilized porcine pepsin A (Worthington Biochemical Corporation) was dissolved in water at 200 mg / mL. All ligand stock solutions, except saquinavir mesylate, were prepared as saturated solutions in DMSO to maximize ligand dissolution. The solution was centrifuged, and the appropriate volume of supernatant was added to the pepsin solution so that the final mixture contained 1.6% of the ligand stock by volume. The mixture was incubated at room temperature for 1 hour, centrifuged at 31,000 rcf for 10 minutes, and the supernatant was used for crystallization experiments. Due to the low solubility of saquinavir, this compound was not present in initial attempts to determine the structure of the pepsin-saquinavir complex. To maximize saquinavir solubility in the crystallization solution, solutions from a CryoSol screen (Molecular Dimensions) (PMID 28626721) were tested. SM2, a mixture consisting of 37.5% v / v dioxane, 25% v / v DMSO, 12.5% ​​v / v ethylene glycol, 12.5% ​​v / v 1,2-propanediol, and 12.5% ​​v / v glycerol, allowed for an increase in the amount of ligand solution in the cocrystallization mixture without damaging the protein. A saturated solution of saquinavir in SM2 was treated in the same manner as the solutions of the other ligands, except that its amount in the solution containing pepsin was increased to 5% by volume.

[0042] Initial crystallization conditions were identified by screening 200 mg / mL of ligand-free porcine pepsin against the SaltRX screen (Hampton Research). After 1 week of incubation at room temperature, small bipyramidal crystals were obtained in 3.5 M ammonium chloride and 0.1 M sodium acetate trihydrate (pH 4.6). These crystals were used to prepare microseed stocks as described by Luft and DeTitta (PMID 10216295), which were then used to cocrystallize pepsin with amprenavir, ritonavir, and darunavir. Diffraction-quality crystals formed after 2–7 days from hanging drops containing 2 μL of pepsin at concentrations of 180–210 mg / mL and 1 μL of serially diluted 10–100-fold microseed solution on top of a well solution containing 3–4 M ammonium chloride and 0.1 M sodium acetate trihydrate (pH 4.6). Crystals formed as triangular bipyramids with dimensions of approximately 200 × 100 × 100 μm and were cryoprotected by immersion in 30% w / v glucose, 5 M ammonium chloride, and 0.1 M sodium acetate trihydrate (pH 4.6) followed by immersion in liquid nitrogen.

[0043] The saquinavir complex was crystallized in the same manner, except that 0.1 M acetic acid was used instead of 0.1 M sodium acetate trihydrate pH 4.6. This change allowed for the growth of large crystals without the need for a microseed solution. Cryoprotection was achieved using 30% w / v glucose, 3 M ammonium chloride, and 0.1 M acetic acid, followed by immersion in liquid nitrogen. Crystallographic data collection and model refinement statistics are shown in Tables 1-1 and 1-2.

[0044] [Table 1-1]

[0045] [Table 1-2]

[0046] [Data Collection] The pepsin·APV:A 1.9Å diffraction dataset was collected at the Advanced Photon Source (APS) Life Sciences Collaborative Access Team beamline (LS-CAT) 21-ID-F equipped with a MAR300 CCD detector, using a 50 × 50 μm beam at a wavelength of 0.97872 Å. A total of 262 frames were collected from φ = 0 to 130.5° with an oscillation range of 0.5° and a detector distance of 250 mm. Diffraction data were indexed, integrated, and scaled using MOSFLM (PMID 21460445).

[0047] The pepsin·RTV:A 2.1Å diffraction dataset was collected at LS-CAT beamline 21-ID-D equipped with a Dectris Eiger 9M detector using a 50 × 50 μm beam at a wavelength of 1.12721 Å. A total of 900 "frames" were collected from φ = 0–180°, oscillating at a rate of 1° / sec and slicing 5 images / °. The crystal-to-detector distance was 160 mm. Diffraction data were indexed, integrated, and scaled using MOSFLM (PMID 21460445).

[0048] The pepsin·DRV:A 1.9 Å diffraction data set was collected at LS-CAT beamline 21-ID-G equipped with a MAR300 CCD detector using a 50 × 50 μm beam at 0.97856 Å wavelength. A total of 900 frames were collected from φ = 0–180° with an oscillation range of 0.2° and a detector distance of 260 mm. The exposure time was 0.3 seconds. Diffraction data were indexed, integrated, and scaled using HKL2000 (PMID 27754618).

[0049] The pepsin·SQV:A 1.9 Å diffraction data set was collected at LS-CAT beamline 21-ID-F equipped with a MAR300 CCD detector using a 50 × 50 μm beam at a wavelength of 0.97872 Å. A total of 400 frames were collected from φ = 20–100° with an oscillation range of 0.2° and a detector distance of 200 mm. The exposure time was 0.5 seconds. Diffraction data were indexed, integrated, and scaled using MOSFLM (PMID 21460445).

[0050] [Model refinement] The initial phase was obtained by molecular replacement in PHASER (PMID 19461840). The B-factor was reset to 20.00 Å, and the unliganded structure of porcine pepsin (PDB ID 4PEP) with solvent molecules removed was used as the search model. Model refinement was performed using the phenix.refine tool in the PHENIX (PMID 20124702, PMID 21041930) suite and COOT (PMID 20383002, PMID 15572765). Geometric restraints for the compound were obtained from the CCP4 monomer library (PMID 28177307). The model was validated using the MolProbity (PMID 20057044) tool implemented in the PHENIX suite. The models of the ritonavir and saquinavir complexes were further optimized using the PDB-REDO server (PMID 25075342) before deposition.

[0051] [In vivo mouse model] An in vivo mouse model of direct application of pepsin to mimic laryngeal exposure during LPR and treatment with selected HIV protease inhibitors via oral gavage (oral systemic delivery) or aerosolized delivery (inhalation local delivery) was used. Commercial formulations of Lexiva and Prezista were used in the oral / systemic treatment groups, and equivalent amounts of the pure active drugs fosamprenavir (Anant Pharmaceuticals Pvt. Ltd., Ambernath, Maharashtra, India) and darunavir (Ambeed, Inc., Arlington Heights, IL) were aerosolized for inhalation treatment; both are referred to by their generic names throughout. Each group contained three mice. Six-week-old female Jackson A / J mice (Jackson Laboratory, Bar Harbor, ME) were weighed 1 day after arrival and every Monday thereafter. There were no significant weight differences between the test groups. Mice were housed three per cage and fed D-62 powdered Wattenberg chow at 2 g / mouse / day upon arrival until termination ( Caicedo-Granado et al., 2014 ).

[0052] In the first two experiments examining the effects of 1) pH 7 and pH 4 + / - pepsin and 2) oral fosamprenavir treatment, mice were anesthetized with Avertin (2,2,2-tribromoethanol) via intraperitoneal injection at 225–240 mg / kg. Avertin was the preferred anesthetic for the short duration required, as it has a rapid induction, produces short surgical anesthesia, and has a rapid recovery. In the third experiment comparing oral and inhaled treatment with fosamprenavir and darunavir, the IACUC requested a change in anesthetic because mice required anesthesia for several consecutive days. In this experiment, isoflurane inhalation was used. The self-scavenging isoflurane device was operated at 3% isoflurane with 2.5 liters of air per minute (LPM). The induction chamber was charged for 3–5 minutes before anesthetizing the mice. Mice were left in the induction chamber for 3–5 minutes before any wounding or administration. Anesthesia records were kept in accordance with IACUC policy.

[0053] Anesthetized mice were placed on an inclined tilting board under a surgical microscope (Caicedo-Granado et al., 2014). The animals were immobilized with copper wire attached to the board, from which the mice were suspended by their upper teeth. This allowed the mandible to open naturally, exposing the larynx, which could be visualized under a Zeiss microscope at 6x magnification. The wounding procedure was performed using a blunt needle bent at 135°. Under direct visualization, the subglottic, glottic, and supraglottic regions were superficially scratched medially from distal to proximal as the needle was gently withdrawn, creating a mild abrasion. This procedure was performed twice, with a 1-week interval between wounds.

[0054] To examine the effects of pepsin at acidic and non-acidic pH, animals were treated with laryngeal instillation of pepsin (20 μL at 0.3 mg / ml, pH 7.0 or pH 4.0) for 2 weeks at 24, 48, and 72 hours after the wound procedure. During weeks 3 and 4, animals were treated with laryngeal instillation 3 days per week. Mice were sacrificed on Friday of week 4, and samples were collected.

[0055] Groups used to examine the efficacy of HIV protease inhibitors and compare oral and inhaled treatments were as follows: control / vehicle, control / pepsin, Lexiva gavage / vehicle, Lexiva gavage / pepsin, Prezista gavage / vehicle, Prezista gavage / pepsin, darunavir aerosol / vehicle, darunavir aerosol / pepsin, fosamprenavir aerosol / vehicle, and fosamprenavir aerosol / pepsin. Twelve mice in the aerosol group were conditioned in a nose-only exposure chamber (Intox) by 10-minute exposure to dry air for three separate days. Starting the following Monday (Day 1), mice received aerosol and gavage treatments Monday through Friday (five exposures). Aerosol and gavage treatments continued Monday through Friday for three additional weeks, with the final week receiving four exposure days (Monday through Thursday) for a total of 19 days. On Friday, mice were sacrificed and tissues were collected. Each mouse received two wounds and 12 doses of vehicle or pepsin. Wounding and intratracheal administration were performed simultaneously with aerosol and oral gavage treatments. The first wound was performed on day 2, followed by vehicle or pepsin administration on days 3, 4, and 5. The second wound was performed on day 8, followed by vehicle or pepsin administration on days 9, 10, and 11. During weeks 3 and 4, mice received vehicle or pepsin on days 16, 17, 18, 23, 24, and 25. Day 26 was the day for sacrifice and tissue collection.

[0056] The aerosol generation procedure was similar to that previously described (Xie et al., 2010). A 10 mL suspension of drug in ethanol was placed in a baffle so that the concentration remained constant at equilibrium solubility. Ethanol droplets containing dissolved drug were generated by an airborne ultrasonic atomizer (nominal frequency 1.7 MHz) at a flow rate of 0.5 LPM equipped with a custom-made glass baffle (University of Minnesota, Department of Chemistry Glass Shop). The aerosol cloud then passed through a cylindrical drying column containing an annular ring of charcoal. The ethanol was removed, and the released dry aerosol particles of pure drug were directed into an exposure chamber.

[0057] The mass deposited on the filter at a fixed collection time was determined gravimetrically, allowing the calculation of the total output (mg / min). The aerosol concentration (mass / volume of air) was calculated by dividing the total output by the air flow rate (0.5 LPM).

[0058] The inhaled mass of drug (Minh) for each mouse was calculated as follows: Minh=[aerosol]*RMV*t where [aerosol] is the aerosol concentration of the drug, RMV is the minute ventilation of the mouse (0.025 L / min), and t is the time of aerosol exposure. The actual mass deposited in the mouse was not determined, but it is expected to be only 10% of the inhaled mass, which is the deposition rate in mice for aerosol particles 1 μm in size.

[0059] In particular, for human translation, it is reformulated to a larger particle size (e.g., approximately 8 µm to 12 µm) and utilized for human inhalation to maximize particle deposition.

[0060] [Aerosolization of HIV protease inhibitors] Aerosol generation is performed as previously described (Xie et al. J Pharm Sci. (2010) 99(11):4658-68). Briefly, droplets are generated by an ultrasonic atomizer (nominal frequency 1.7 MHz, purchased from MainlandMart.com) and airborne through a custom-made glass baffle. To achieve the desired particle size distribution and mass deposition, the concentration of the target compound in the baffle ranges from 0.75 to 20 mg / ml (i.e., baffle concentration). The baffle volume is 10 ml, and the run time is short to minimize changes in the baffle solution concentration assayed before and after each experiment. The aerosol cloud is then transported through a drying column, and the resulting dry particles are directed into the exposure chamber.

[0061] To determine aerosol concentration, the mass deposited on the filter by the extracted filter absorption is measured. The total output is calculated from the total output (filter) and collection time. The aerosol concentration (mass / volume of air) is calculated by dividing the total output by the air flow rate, and the liquid output is calculated by dividing the aerosol concentration by the iron concentration in the baffle. The size distribution of aerosol particles is determined using a low-flow cascade impactor (Model 1403, In-Tox Products, Moriarty, New Mexico). Collection is performed immediately prior to the exposure chamber. The mass median aerodynamic diameter (MMAD) and associated geometric standard deviation (GSD) are calculated from linear regression of an XY probability plot of the cumulative undersize mass as a function of the logarithm of the cutoff diameter using Kaleida-Graph (Synergy Software, Reading, PA). Values ​​are based on a pool of three independent measurements.

[0062] The mass of inhaled fluorescent aerosol (Minh) for each mouse is calculated as follows: Minh=[aerosol]*RMV*t where [aerosol] is the aerosol concentration of fluorescein, RMV is the minute ventilation of the mouse (0.024 L / min, based on Guyton's equation), and t is the time of aerosol exposure. The deposition rate of fluorescent aerosol in the airways is calculated as the ratio of the assayed tissue mass to the inhaled mass.

[0063] In particular, for human translation, it is reformulated to a larger particle size (e.g., approximately 8 µm to 12 µm) and utilized for human inhalation to maximize particle deposition.

[0064] [result:] (Identification of pepsin inhibitor candidates) Our team developed and optimized two high-throughput screening (HTS) assays to identify compounds that are specific and sensitive inhibitors of pepsin (Figure 1). The first assay is a binding assay that measures the ability of compounds to compete with fluorescently labeled pepstatin for binding sites on pepsin. The second assay is a pepsin activity assay that utilizes fluorescently labeled casein as the enzyme substrate. The specificity of these assays was confirmed using three protease enzymes similar to pepsin: cathepsin D, trypsin, and renin. The optimal assay conditions for the competitive binding assay (i.e., those that yield the largest dynamic range in the dose-response curve) were as follows: 100 nM pepstatin-Alexa647, 0.03 U / µL pepsin, 37.5% DMSO, 20 µL reaction. The optimal assay conditions for the pepsin activity assay were 200 nM casein-Alexa647, 0.01 U / µL pepsin, 5% DMSO, 20 µL reaction.

[0065] An example of one of the libraries screened in the binding assay is shown in Figure 2. This library contains over 1300 compounds, of which only two showed significant binding, highlighting the specificity of our HTS assay system for determining pepsin inhibition.

[0066] Ten commercially available HIV protease inhibitors were tested in binding and enzyme assays, and four of them, namely, amprenavir, darunavir, ritonavir, and saquinavir, demonstrated pepsin inhibition (Figure 3A, Figure 3B). Under optimized assay conditions, four of the seven HIV protease inhibitors demonstrated both competitive binding to pepsin (Figure 3A) and inhibition of pepsin activity (Figure 3B). These were amprenavir, darunavir, ritonavir, and saquinavir. The IC for binding was 50 The values ​​are shown in Figures 3A and 3B. The in vitro activity of these four HIV protease inhibitors against pepsin provides fundamental support for considering their use in the treatment of LPR.

[0067] For in vivo testing, we selected the most promising of these pepsin inhibitors. Both fosamprenavir and darunavir are considered good candidates for clinical trials, but other pepsin inhibitors are being considered. Saquinavir was also eliminated from consideration because it is expensive and may cause QT prolongation, heart block, high blood lipids, and liver problems. Amprenavir and ritonavir are inexpensive and cause minimal side effects (e.g., diarrhea, nausea, and vomiting). Amprenavir and its prodrug, fosamprenavir, are considered promising candidates because they have excellent pharmacokinetics and have already been tested in mice. Furthermore, a prodrug of amprenavir (fosamprenavir) is commercially available.

[0068] Ritonavir, saquinavir, and darunavir have also been found to bind to the active site of pepsin through cocrystallization studies. Amprenavir, ritonavir, and darunavir have few side effects, such as diarrhea, nausea, and vomiting. Amprenavir and ritonavir are less expensive than darunavir. Fosamprenavir, a prodrug of amprenavir, has improved oral bioavailability and a favorable tolerability profile, making it the best candidate for in vivo testing of its potential efficacy in preventing pepsin-mediated laryngeal injury / inflammation.

[0069] [crystal] To provide structural data for interpreting inhibition studies, commercially available porcine pepsin (EC 3.4.23.1) was used for crystallization experiments. Repeated attempts to crystallize human pepsin collected from volunteers failed, likely due to inherent sample heterogeneity. In assessing the quality of the porcine pepsin crystals, a dataset was collected to determine a unique pepsin structure in the absence of inhibitors (data not shown). This structure is nearly identical to that previously determined by Cooper et al. (PDB ID 5PEP [2115088]) based on a small root-mean-square deviation (RMSD) of 0.51 Å for all Cα atoms. Considering the high degree of sequence identity (86%), the structure of porcine pepsin is also nearly identical to that of the human enzyme (PDB ID 1PSN [PMID 7663352]), with an RMSD value of 0.50 Å for all Cα atoms. Most of the minor differences in tertiary structure are localized to the loop containing residues 277–282, with RMSD values ​​ranging from 1.6 to 3.6 Å. This loop is not part of the binding groove but is involved in crystal contacts in both human and porcine pepsin. Residues lining the active site cleft are highly conserved between the human and porcine enzymes. Of the 17 residues that make direct contact with the inhibitor investigated here, only two differ between human and porcine pepsin: T12 and V291, which are methionine and leucine residues in the human form, respectively. Therefore, the porcine enzyme was deemed an acceptable surrogate for human pepsin to evaluate the structural biology described below.

[0070] Using the same crystallization conditions that produced crystals of unbound porcine pepsin, we crystallized complexes of the enzyme with each of four inhibitors: ritonavir (pPEP·RTV; PDB ID 6XCY), amprenavir (pPEP·APV; PDB ID 6XCT), saquinavir (pPEP·SQV; PDB ID 6XCZ), and darunavir (pPEP·DRV; PDB ID 6XD2). All are peptidomimetics containing chemical moieties to exploit specific subsites of the protease. In all four complexes, the alcohol of the central phenylalaninol residue, which mimics the transition state for peptide bond cleavage, is bound between the catalytic aspartic acid residues D32 and D215. Furthermore, the amino group of the phenylalaninol residue is on the prime side of the binding site, and the directionality of the bond is the same in each case. This is the same directionality observed in the binding of pepstatin to human pepsin [PMID 7663352]. The binding of these compounds is dominated by van der Waals contacts between the inhibitor side chains and the side chains of residues lining the binding site, involving only a few hydrogen bonds. For example, in the pPEP·RTV complex (Figure 9A-B), the β-homophenylalanine side chain binds to the P1 subsite, making van der Waals contacts with F111, F117, and I120. The phenylalaninol side chain binds to the P1 subsite, making van der Waals contacts with I213, M289, V291, and I300. The thiazole and isopropyl-thiazole groups of RTV lack stabilizing interactions with the active site. The electron density of these groups is correspondingly poorly defined, and the B-factor, which reflects the accuracy of the atomic positioning in these parts of the molecule, is very high. The structure of the pPEP·SQV complex (Figure 10A–B) is similar in that the side chain of the phenylalaninol residue interacts with the P1′ subsite, but the two termini of the molecule, the quinoline and decahydroisoquinoline moieties, also have low density and high B factors.

[0071] The structures of amprenavir (Figure 4A-D) and darunavir follow the same pattern. The phenylalaninol residue in both inhibitors occupies the P1' site and interacts with I213, M289, V291, and I300. The isobutyl group, mimicking a leucine residue, occupies the P1 site and interacts with F111, F117, and I120. In both amprenavir and darunavir, one of the oxygen atoms of the sulfonamide moiety hydrogen bonds with the main-chain amide of T77. The aminophenyl group does not make polar contacts with the active site. At opposite ends of the two molecules, the tetrahydrofuran group of amprenavir forms a hydrogen bond with the phenolic oxygen of Y189. However, the bis-tetrahydrofuran group of darunavir cannot make this interaction with the active site and is limited to van der Waals contacts with I73, T74, I128, and Y189. It is interesting to note that both amprenavir and darunavir bind to the pepsin active site in an orientation opposite to that seen in their complex with HIV protease [PMID:20695887, PMID:24785545].

[0072] [Epidemiological support for the use of HIV protease inhibitors to treat LPR] Clinical records were retrieved using the Cohort Discovery Tool (CDT) of the Medical College of Wisconsin Clinical Data Warehouse (MCW CDW; PRO#13874). The MCW CDW provided fully de-identified clinical information for each subject, including patient demographics, ICD-coded diagnoses, ICD- and CPT-coded procedures, laboratory test results, inpatient pharmacy orders, and text containing clinical documentation, including pathology reports. Using this database, we found that of 2,062 HIV patients taking HIV protease inhibitors, only 5 (0.2%) had LPR. Using this data, we found that patients taking HIV protease inhibitors had a significantly lower incidence of airway reflux (0.2%) compared with the general population (10–34.4%) (76, 77). However, this query included all HIV protease inhibitors, some of which do not inhibit pepsin. Therefore, there were insufficient patient numbers to query each HIV protease inhibitor individually.

[0073] [Generation of a mouse model of LPR] A new mouse model was established to test the efficacy of HIV protease inhibitors for the treatment of pepsin-mediated laryngeal mucosal damage and inflammation. Currently, there is no complete animal model of LPR, and proximal reflux into the laryngopharynx is inconsistent in surgical models. Therefore, an established mouse model (78) was modified using a wound procedure for use in this study. Briefly, after anesthesia, the larynx was exposed in a suspension apparatus and visualized under a Zeiss microscope at 6x magnification. Anesthetized animals were placed in suspension, and pepsin was applied topically in a volume of 20 μl. These mice exhibited pepsin-mediated airway epithelial damage at both pH 4 and pH 7 (Figure 5A–H), demonstrating the ability of this treatment to mimic the effects of LPR. Figure 5A–H further demonstrates that, like non-acidic laryngopharyngeal reflux, pepsin causes laryngeal epithelial damage at pH 7 in vivo.

[0074] This mouse model was the first to test the ability of the HIV protease inhibitors fosamprenavir (trade name: Lexiva) and darunavir (trade name: Prezista) to alleviate laryngeal injury. Oral administration of fosamprenavir at 20 mg / kg / day (Lexiva), equivalent to the dose used to treat HIV in humans, prevented pepsin-mediated laryngeal injury (defined as multilayered reactive epithelium and cellular apoptosis) in this in vivo mouse model (Figure 7A-J). In contrast, oral administration of darunavir (8.6 mg / kg / day (Prezista)), also at a human-equivalent dose, did not abrogate pepsin-mediated laryngeal injury (Figure 7H). Fosamprenavir administered by inhalation at a dose of 1 mg / kg / day prevented pepsin-mediated laryngeal injury (Figure 7F). Importantly, darunavir administered as an aerosol at a dose of 12 mg / kg / day (Figure 7J) was also effective in preventing pepsin-mediated injury. HIV protease inhibitors were aerosolized (see Materials and Methods above), and a second group of mice received the HIV protease inhibitor via inhalation to determine whether local delivery was more effective. Specifically, mice undergoing the wound procedure and pepsin treatment were assigned to one of four groups: (1) ritonavir by gavage, (2) ritonavir by aerosol, (3) fosamprenavir by gavage, or (4) fosamprenavir by aerosol, compared with mice undergoing the wound procedure and not receiving pepsin treatment. Inflammation was assessed in laryngeal tissue sections stained with hematoxylin and eosin (H&E). Specifically, epithelial thickness (i.e., 0, 1-2, 3-4, 5+ cells and thickness in microns) and the presence of neutrophil infiltration, keratinization, and necrosis were recorded.

[0075] Specifically, slides were sectioned at 4 microns in thickness using a microtome and stained with hematoxylin and eosin (H&E) using an automated stainer. Slides were reviewed by a board-certified pathologist. Upper respiratory epithelium was identified based on histological morphology. The effects of pepsin-mediated injury were determined by cytological changes, including increased nucleus to cytoplasm ratio, multilayered epithelium, loss of cilia, individual cell apoptosis, and inflammatory infiltrates.

[0076] Figures 6A-D further demonstrate that Lexiva treatment, equivalent to current FDA-approved HIV doses and formulations, prevents pepsin-mediated laryngeal damage in our in vivo mouse model. Thus, Lexiva may be used to treat laryngopharyngeal reflux.

[0077] This study was approved by the University of Minnesota's Institutional Care and Use Committee. Thirty 6-week-old female A / J mice were purchased from Jackson Labs, Bar Harbor, ME for use in this study. Mice were housed three per cage under standard conditions. Mice had free access to water and D-62 modified pellets (standard rodent chow purchased from Research Diets, New Brunswick, NJ). Mice were weighed the day after arrival and weekly thereafter. One week after arrival, mice were randomized by weight and assigned to experimental groups. There were no significant weight differences between test groups.

[0078] Each group contained three mice and was designated as follows: control / vehicle, control / pepsin, Lexiva gavage / vehicle, Lexiva gavage / pepsin, Prezista gavage / vehicle, Prezista gavage / pepsin, darunavir aerosol / vehicle, darunavir aerosol / pepsin, fosamprenavir aerosol / vehicle, and fosamprenavir aerosol / pepsin. During the same week, 12 mice designated as aerosol were trained in the aerosol exposure machine for three days, 10 minutes each time. Starting on Monday of the following week (Day 1), mice received aerosol and gavage treatments Monday through Friday (five exposures). Aerosol and gavage treatments continued Monday through Friday for three more weeks, with the final week consisting of four exposure days (19 total days) from Monday through Thursday. On Friday, mice were sacrificed and tissues were harvested. Each mouse received two wounds and 12 vehicle or pepsin treatments. Wounding and intratracheal administration were performed simultaneously with aerosol and oral gavage treatments. The first wounding was performed on day 2, followed by vehicle or pepsin administration on days 3, 4, and 5. The second wounding was performed on day 8, followed by vehicle or pepsin administration on days 9, 10, and 11. In weeks 3 and 4, mice received vehicle or pepsin on days 16, 17, and 18 and days 23, 24, and 25. Day 26 was the day for sacrifice and tissue collection.

[0079] All aerosol training or experimental exposures lasted 10 minutes per mouse. Training, identical to the experimental exposures, was completed using air only. When aerosol exposure began, darunavir or fosamprenavir was sprayed into the airflow. In this experiment, each aerosol set had its own dedicated nebulizer and mouse exposure tube. On day 1, darunavir aerosol was administered first, followed by fosamprenavir. On day 2, fosamprenavir was administered first, followed by darunavir. This cycle continued throughout the exposure period, with daily changes. The nose cone of the exposure chamber was wiped clean with 70% ethanol and allowed to dry daily between exposures. The dedicated exposure tube was replaced to prevent cross-contamination. Mice completed daily aerosol and oral gavage before wounding or administration of vehicle or pepsin. The vehicle was PBS, pH 7. Pepsin was diluted in PBS, pH 7.0. The pepsin concentration was 0.3 mg / mL. Vehicle and pepsin doses were 30 ul and were delivered with a Hamilton syringe and custom-made dispensing needle for intratracheal administration.

[0080] This study was conducted identically to our previously published study using this intratracheal procedure, with the exception of the anesthetic used. Previously, we used injectable Avertin anesthetic, but the IACUC requested a change in anesthetic because the mice required anesthesia for several consecutive days. In this study, mice were administered isoflurane inhalation. The isoflurane machine was a self-scavenging device borrowed from University Research Animal Resources. The isoflurane machine was operated at 3% isoflurane and 2.5 liters per minute (LPM) of air. The induction chamber was charged for 3–5 minutes before anesthetizing the mice. Mice remained in the induction chamber for 3–5 minutes before any wounding or administration. Anesthesia records were kept in accordance with IACUC policy.

[0081] All gavage administrations began on day 1. The Lexiva gavage dose was 0.2 mL. The Prezista gavage dose was 0.5 mL. Each oral medication had its own gavage tip. Each oral medication was shaken for 1 minute each day before removing the dose from the bottle. Each dose was checked for accuracy and air bubbles before administration. Each gavage-administered mouse received a total of 19 doses.

[0082] Both Lexiva and Prezista, when administered by aerosol, prevent pepsin-mediated laryngeal injury (Figure 7A-J). A) Vehicle control: A single layer of respiratory epithelium with no reactive changes. B) Pepsin control: Multilayered epithelium and individual cell apoptosis (arrows): i) High magnification of multilayered epithelium, and ii) High magnification of cell apoptosis. C) Fosamprenavir gavage: Histologically normal in the vehicle group. [This tissue section is more proximal than the other sections, approximately in the oropharynx, so some transitional epithelium is visible.] D) Fosamprenavir gavage: Histologically normal in the pepsin group. E) Fosamprenavir aerosol: Histologically normal in the vehicle group. F) Fosamprenavir aerosol: Histologically normal in the pepsin group. G) Darunavir gavage: Mildly reactive epithelium is seen in the vehicle group, but less than in the pepsin control. H) Darunavir gavage: Mildly reactive epithelium is seen in the pepsin group, but less than in the pepsin control. I) Darunavir aerosol: vehicle group, histologically normal. J) Darunavir aerosol: pepsin group, histologically normal. A-J, scale bar = 50 μm, 20x magnification. Bi and Bii, scale bar = 20 μm, 40x magnification.

[0083] Figures 7A-J suggest that oral delivery of Lexiva, but not Prezista, prevents pepsin-mediated laryngeal damage at equivalent FDA-approved HIV doses. Inhaled delivery of both Lexiva and Prezista prevents pepsin-mediated laryngeal damage in vivo. Inhaled treatment better maintained normal laryngeal histology than oral treatment during pepsin challenge experiments. Local inhaled delivery, in contrast to oral delivery, which requires high doses to produce therapeutically effective drug levels in the laryngopharynx, can reduce the amount of drug administered to patients, limiting side effects. This may prove particularly beneficial for these medications, as the primary side effects are gastrointestinal-related. Therefore, local inhalation (nose / mouth spray) is more effective and potent for treating laryngopharyngeal reflux.

[0084] [Consideration] For the past two decades, treatment of LPR has focused on suppressing gastric acid production. With the introduction of MII-pH technology, it is now known that LPR is generally nonacidic, and nonacidic proximal events are associated with throat symptoms and laryngeal fiberoptic examination findings (Sharma and Castell, 2009; Tamhankar et al., 2004; Tutuian et al., 2006; Tutuian et al., 2008; Iqbal et al., 2008; Mainie et al., 2006; Klimara et al., 2020; Zhang et al., 2017; Falk et al., 2017; Sidwa et al., 2017; Lechien et al., 2019; 2020). These findings have prompted investigation of other nonacidic components of gastric refluxate. In vitro studies have revealed that both pepsin and bile can cause laryngeal inflammation at nonacidic pH. In vitro studies have reported mucosal damage caused by bile at nonacidic pH, but it has been argued that "there is no evidence that the same mechanism occurs in the human larynx" (Campagnolo et al., 2014). In vitro exposure to bile acids induces cell membrane "blebbing" (Hopwood et al., 1981), but to our knowledge, this has never been reported in the laryngeal mucosa of patients with LPR. Presumably, the concentration of bile reaching the proximal laryngopharynx is insufficient to cause cell membrane damage. The concentrations of bile salts and acids used in previous in vitro studies ranged from 5 to 50 mM. The physiological bile acid / salt content of gastric reflux fluid reaching the laryngopharynx is expected to be several orders of magnitude lower, in the range of 10 to 100 μM (Ali et al., 2013). This low concentration has not been shown to cause laryngeal injury. It should also be noted that unconjugated bile acids, which cause damage at higher pH, are rarely found in gastric reflux fluid, consistent with the laryngopharyngeal environment ( Ali et al., 2013 ; Pearson et al., 2011 ).

[0085] In contrast, pepsin is present in all reflux fluids (Samuels and Johnston, 2010). Furthermore, it is frequently detected in airway tissues and secretions from patients with LPR but absent from control subjects without reflux as confirmed by MII-pH. Therefore, gastrin can predict reflux-related symptoms and disease (Klimara et al., 2019; 2020; Bardhan et al., 2012; Samuels and Johnston, 2009; 2010; Johnston et al., 2009; 2010; 2018; Zalvan et al., 2017; Lechien et al., 2020; Calvo-Henriquez et al., 2017; Weitzendorfer et al., 2019). Pepsin-mediated damage and inflammatory changes in vitro are consistent with those observed in patients with LPR (Axford et al., 2001; Johnston et al., 2003; 2004; Gill et al., 2005). Compelling evidence from multiple groups (Bardhan et al., 2012; Niu et al., 2020; Martinucci et al., 2013) highlights the key role of pepsin (independent of gastric acid) in reflux-related laryngeal symptoms and the finding that is resistant to PPI therapy. The data presented herein further support the therapeutic potential of drugs that specifically target pepsin.

[0086] It should be noted that pepstatin, a commercially available potent inhibitor of pepsin, is a poor in vivo candidate due to its poor water solubility and poor pharmacokinetic properties. Therefore, preclinical evaluation of other pepsin inhibitors to document efficacy has been undertaken.

[0087] Considering pepsin binding in the four crystal structures (Figures 9A-B, S1, and S2), it is difficult to rationalize the observed IC50 values. Aside from interactions with the alcohol group and catalytic aspartic acid residue, and the hydrogen-bonding interactions that amprenavir and saquinavir make with Y189, all polar contacts occur in the main chain. Thus, binding is primarily stabilized by van der Waals contacts. Overall, this series of crystal structures, along with biochemical data from inhibition assays, suggests that rationally designing inhibitors of pepsin will be challenging. Therefore, a strategy of testing existing inhibitors of other aspartic proteases was considered the most direct route to identifying potent inhibitors of pepsin activity for the treatment of LPR.

[0088] Selected HIV protease inhibitors were found to bind to the active site of pepsin and inhibit the enzyme. Because HIV protease inhibitors target a foreign virus, their therapeutic activity is not directly related to potential pharmacological effects in humans. Therefore, HIV protease inhibitors are ideal drugs for repurposing, as well as enabling proof-of-concept testing that pepsin inhibitors are effective in patients with LPR disease. Currently, there are 10 commercially available HIV protease inhibitors, seven of which were suitable for testing in our assay. Cocrystallization studies with pepsin, competitive binding assays with fluorescently labeled pepstatin, and enzyme assays with a labeled protein substrate (casein) revealed that amprenavir, ritonavir, saquinavir, and darunavir bind to and inhibit pepsin with IC50 values ​​in the low micromolar range. Saquinavir may be excluded from clinical trials based on its known side effects and interactions (QT prolongation, heart block, elevated blood lipids, and liver problems) and cost. Amprenavir, ritonavir, and darunavir have few side effects, such as diarrhea, nausea, and vomiting. Amprenavir and ritonavir are less expensive than darunavir, but darunavir had the lowest IC50 for pepsin. Fosamprenavir, a prodrug of amprenavir with improved oral bioavailability and a favorable tolerability profile, is commercially available. Therefore, darunavir and fosamprenavir were considered leading candidates and were subsequently tested in established in vivo mouse models to assess their efficacy in suppressing pepsin-mediated laryngopharyngeal and inflammatory processes.

[0089] Oral systemic administration of fosamprenavir, but not darunavir, prevented pepsin-mediated laryngeal injury in vivo. However, treatment with both of these HIV protease inhibitors maintained normal laryngeal histology despite pepsin exposure when administered locally by inhalation. Reformulation of these drugs for local inhalation delivery (oral or nasal) may be beneficial for the treatment of LPR. In addition to the obvious advantage of delivering the drug directly to the site of reflux-induced damage, inhalation delivery can also reduce the amount of drug administered to patients, as opposed to oral delivery. The latter approach overcomes the apparent low oral bioavailability, requiring high doses to achieve effective drug concentrations in laryngeal tissue, which can cause dose-limiting side effects. This latter aspect may prove particularly beneficial for the drugs used here, as the primary side effects are gastrointestinal-related.

[0090] In the current inhalation study, optimized delivery was performed in a mouse model administering pure drug in relatively small aerosol particles (i.e., 1 μm). To translate from in vivo to clinical applications, drugs can be formulated to optimize inhalation delivery to humans. Here, larger particle sizes are generated to take advantage of the fact that humans can be instructed to perform controlled, forceful inhalations to maximize particle deposition. Computational fluid dynamics analysis of particle deposition in the larynx revealed that the optimal particle size for laryngeal deposition is 9–12 microns (unpublished data), which is in good agreement with other studies (Perkins et al., 2018).

[0091] Pilot epidemiological data on the incidence of LPR in patients taking HIV protease inhibitors support our hypothesis, with only 5 / 2062 (0.2%) patients taking HIV protease inhibitors reporting LPR in their medical records. This includes all HIV protease inhibitors, some of which do not inhibit pepsin. Unfortunately, there were insufficient patient numbers to interrogate each HIV protease inhibitor individually. By comparison, the reported incidence of LPR ranges from 10% to 34.4% in the general population (Kamani et al., 2012; Lowden et al., 2009), similar to the incidence of LPR in matched populations of MCWCDWs. Although limited, this epidemiological data supports our hypothesis. Given these aggregate data, a 12-week, randomized, double-blind, placebo-controlled clinical trial will be conducted to evaluate the efficacy of Lexiva or Prezista for LPR.

[0092] [Conclusion:] Compelling evidence highlights the key role of pepsin (independent of gastric acid) in reflux-induced laryngeal symptoms and endoscopic findings refractory to PPI therapy. As shown in this example, fosamprenavir and darunavir, FDA-approved retroviral therapies for HIV / AIDS, bind to and inhibit pepsin, abrogating pepsin-mediated laryngeal inflammation and mucosal damage in vivo. Because these drugs target a foreign virus, they are ideal for repurposing, allowing clinical trials to assess the efficacy of much-needed treatment for patients more quickly than can be achieved with novel compounds. If FDA-approved oral formulations resolve the symptoms and endoscopic findings of LPR in clinical trials, reformulation for local inhaled delivery may further improve outcomes and limit side effects.

[0093] Example 2: Clinical Trial In Example 1, the inventors identified an FDA-approved HIV protease inhibitor (fosamprenavir, trade name: Lexiva) that binds to and inhibits pepsin. The inventors aim to enter clinical trials after confirming that this drug suppresses pepsin-mediated laryngeal inflammation and mucosal damage in a mouse model of LPR.

[0094] Reformulating HIV protease inhibitors that bind to and inhibit pepsin for local inhalation delivery (oral or nasal) could be beneficial for the treatment of LPR. In addition to the obvious logic of applying local delivery, inhalation delivery can also reduce the amount of drug given to the patient, limiting side effects, as opposed to oral delivery, which requires high doses to get sufficient drug from the gastrointestinal tract to the laryngeal tissues via the bloodstream.

[0095] To translate the in vivo data from mice to humans, we reformulated the formulation to larger particle sizes and maximized particle deposition using human inhalation. Computational fluid dynamics analysis of particle deposition in the larynx revealed that the optimal particle size for deposition in the larynx was 9–12 microns (unpublished data), which is consistent with other studies (Perkins EL, Basu S, Garcia GJM, Buckmire RA, Shah RN, Kimbell JS). Ideal particle size for inhaled steroids targeting vocal granulomas: a preliminary study using computational fluid dynamics. Otolaryngol Head Neck Surg. 2018;158(3):511–9. Epub 2017 / 11 / 22. doi:10.1177 / 0194599817742126. PubMed PMID: 29160160; PMCID: PMC5832637).

[0096] The following example describes a proposed 12-week, randomized, double-blind, placebo-controlled clinical trial designed by the inventors to test the ability of the HIV protease inhibitor Lexiva to resolve the symptoms and endoscopic findings of LPR. Additionally, the study is designed to evaluate the predictive value of pepsin protein and its activity in saliva on the efficacy of Lexiva treatment. If the results of this study suggest that Lexiva can inactivate pepsin in saliva, the drug formulation will be optimized for local delivery via inhaler or nasal spray.

[0097] Materials and Methods: (Research Design) A prospective, placebo-controlled clinical trial of Lexiva will be conducted for 12 weeks in medically refractory patients with clinically diagnosed moderate / severe LPR (Reflux Symptom Index (RSI) ≥ 20, Reflux Finding Score (RFS) ≥ 11, and a laryngeal reflux event confirmed by combined esophageal multichannel intraluminal impedance and pH monitoring (MII-pH)). Twelve weeks is the conventional time for determining the effectiveness of LPR treatment (1). Routine clinical outcome measures of LPR (RSI and RFS) will be documented before and after treatment with Lexiva or placebo. Saliva will be collected before and after treatment and subjected to both pepsin protein analysis and kinetic activity assays to compare pre- and post-treatment RSI, RFS, and MII-pH data. The study schema is shown in Figures 8A-B.

[0098] Patients aged 18 years or older with a clinical diagnosis of moderate / severe HIV who are medically refractory to proton pump inhibitor (PPI) treatment for at least 3 months will be recruited for this study. An experienced clinical research coordinator will obtain informed consent. Patients will be randomly assigned by the Froedtert Hospital Pharmacy Investigational Drug Service to receive either Lexiva, administered at the same dose and frequency as FDA-approved HIV treatment, or placebo for 12 weeks. Patients will provide saliva samples for pepsin concentration (ELISA) and activity (kinetic) analysis before starting Lexiva / placebo treatment. A $200 study incentive (paid at the end of the study) will be offered to encourage patients to follow up after the 12-week treatment period.

[0099] The Sandhill Scientific ZepHr Sleuth MII device (Z07-2000-B) and Sandhill Scientific ComforTec Z / pH disposable impedance / pH probe (ZAI-BL-56) were used in this clinical trial to monitor total reflux, esophageal reflux, and LPR. A proximal reflux event was defined as an episode reaching both impedance sensors in the hypopharynx.

[0100] In addition to RFS, RSI, and MII-pH, measurements routinely used in clinical practice to assess LPR, the reflux symptom score (RSS) and reflux symptom assessment (RSA), were recently developed to assess LPR at diagnosis and throughout treatment (81-84) and were also obtained for this study. The RSS, like the RSI, is a patient-administered questionnaire. An RSS >13 is considered suggestive of LPR. A 20-39.9% decrease in RSS is defined as a mild response, 40-59.9% as a moderate response, 60-79.9% as a good response, and an RSS of ≥80% or ≤13 after treatment is defined as a complete treatment response (83). RSA, like RFS, is used to assess physical findings of LPR. An RSA >14 suggests LPR. Notably, both RSS and RSA assessments have demonstrated high intra- and inter-rater reliability and responsiveness to change (82-84).

[0101] [Rationale for selection of clinical and research methods] Patients with confirmed moderate / severe LPR who were resistant to acid suppression therapy and therefore likely to benefit from pepsin inhibition were selected for this study. Patients were selected based on the following: confirmation of proximal laryngopharyngeal reflux by MII-pH testing, RSI ≥ 20, RFS ≥ 11, and failure of 3 months of BID PPI therapy. RSI ≥ 13 and RFS ≥ 7 were used to diagnose LPR, and higher values ​​were selected for inclusion in this study to increase the likelihood of a response after the 12-week treatment period. In 2001, Belafsky et al. reported an RFS of 11.5 (+ / - 5.2 SD) at enrollment, which improved to 9.3 (+ / - 4.7 SD) at 2 months and 7.3 (+ / - 5.5 SD) at 4 months (79). In 2002, the same group reported an RSI of 21.2 (+ / - 10.7 SD) at enrollment, which improved to 12.8 (+ / - 10 SD) after 6 months (80). Therefore, patients with an RSI ≥ 20 and an RFS ≥ 11 would be expected to decrease to near-normal values ​​after 1 week of treatment with Lexiva if the pepsin inhibitor was effective. "Responders" are defined as patients with a ≥ 6-point reduction in RSI score after treatment, based on U.S. Food and Drug Administration guidelines published in 2015 (85). Changes in each item on the RSI questionnaire compared with the chief complaint at the initial clinic visit are also assessed.

[0102] Selected patients will undergo MII-pH testing and fiberoptic laryngeal examination according to routine clinical care, avoiding additional costly and invasive testing and examinations for this research study. Based on our experience, patients with medically refractory LPR desperately seek options to resolve their symptoms and improve their quality of life. Furthermore, Lexiva is an FDA-approved medication with minimal side effects. Therefore, we do not anticipate any issues with enrollment.

[0103] The presence and levels of pepsin protein in saliva will be measured by ELISA, and pepsin proteolytic activity will be measured by a kinetic assay, as described below. Pepsin is a sensitive and specific biomarker of reflux, and its presence in saliva indicates proximal airway reflux (30, 34, 86, 88). We hypothesize that its presence in saliva preparations will predict the effectiveness of Lexiva in reducing RFS and RSI. Treatment with Lexiva is expected to have no effect on pepsin protein levels but to inactivate the enzyme, preventing pepsin-mediated mucosal damage and inflammation. Loss of pepsin activity is expected to correlate with reduced RFS and RSI after treatment. We have previously shown that salivary pepsin correlates with RSI and proximal reflux events, as measured by MII-pH (86). Therefore, patients with a higher number of proximal reflux events and a higher percentage of proximal reflux time (as measured by MII-pH) during the study period are expected to show the greatest improvement after treatment with Lexiva.

[0104] For saliva sample collection, patients are asked to cough, clear their throat, and spit into an empty tube during their appointment to demonstrate collection technique. They then place the tube in a cold bag to take home. They are asked to spit into the tube if they experience symptoms (cough, throat clearing, and postnasal drip, up to three times), and the following morning, upon standing, before eating, drinking, or brushing their teeth. Each patient is asked to cough and spit into the tube a minimum of two times and a maximum of five times if they experience symptoms within 24 hours of their appointment. We have shown that repeated collection of saliva into a single vial improves sensitivity without affecting the sensitivity of pepsin detection by ELISA (unpublished data). As in other studies, patients are given a pre-addressed and stamped box to send their samples to the laboratory via FedEx Priority. While some groups collect saliva with citric acid as a preservative, we have shown significant agreement between saliva samples collected with and without citric acid (87). We have also shown that pepsin is stable at room temperature without degradation, and have previously reported pepsin concentration and activity analysis by ELISA and kinetic assays ( 20 , 24 , 30 , 38 , 88 , 89 ).

[0105] [Pepsin ELISA] The presence and concentration of pepsin in saliva specimens was determined by noncompetitive indirect sandwich ELISA, as previously described (75, 90). Nunc-ImmunoMaxisorp 96-well flat-bottom microtiter plates (Thermo Fisher Scientific, Inc.) were coated with affinity-purified rabbit anti-Hu3 antibody (3 μg / mL) raised against the N-terminus of mature human pepsin in 100 μL of 0.2 M sodium carbonate buffer (pH 9.6) per well and incubated for 18–20 h at room temperature. The plates were then washed three times with PBS, pH 7.4, containing 0.1% Tween-20 (PBS-T), for 2 min per wash, and incubated for 90 min at 37°C and 200 rpm on an orbital shaker in blocking buffer (SuperBlock PBS Blocking Buffer; Thermo Fisher Scientific, Inc.). The plate is rinsed three times with shaking in PBS, pH 7.4, for 2 minutes per wash. Saliva specimens and pepsin standards are prepared in sample diluent (1% bovine serum albumin [BSA] [Sigma-Aldrich] in PBS). Saliva specimens are prepared at a 1:25 ratio with sample diluent. Sample diluent alone is used as a blank control. After applying the specimens and standards to the microtiter plate in triplicate at 100 μL per well, the plate is incubated at 37°C and 200 rpm for 1 hour. The samples are removed from the plate by vacuum aspiration, and the wells are washed three times with PBS-Tween (PBS-T) for 2 minutes per wash with shaking. Captured pepsin is detected by incubating with goat anti-human pepsin antibody diluted to 26 μg / mL in 1% BSA / PBS-T for 40 minutes at 37°C and 200 rpm. Wash the plate three times with PBS-T for 2 min each wash and incubate for 40 min at 37 °C and 200 rpm with peroxidase-conjugated mouse anti-goat immunoglobulin G (Sigma-Aldrich) diluted 1:30,000 in 1% BSA / PBS-T.Enzymatic color development was performed using the 1-Step Ultra TMB ELISA (ThermoFisher Scientific). Color development was stopped by adding an equal volume of 1N sulfuric acid, and wells were read at 450 nm on a microplate reader (SpectraMax Plus; Molecular Devices, Sunnyvale, CA). Triplicate measurements for each standard, sample, and blank were averaged, and the average absorbance at 450 nm (A450) of the blank sample was subtracted from the average absorbance of the sample and standard. A standard curve was plotted using the blank-subtracted average absorbance and the concentration of the pepsin standard dilution. The pepsin concentration (ng / ml) in the saliva sample was calculated using a best-fit curve model. Samples with an absorbance A450 less than twice that of the blank were considered pepsin-negative.

[0106] [Pepsin activity assay] A synthetic peptide substrate for pepsin (Lys-Pro-Ala-Glu-Phe-PNP-Arg-Leu-COOH (SEQ ID NO: 1), molecular weight = 1,052.18; PNP = paranitrophenylalanine) is specifically cleaved by pepsin between Phe-5 and PNP-6. Cleavage is monitored by the decrease in absorbance at 300 nm (20, 24). The rate of hydrolysis of the synthetic peptide substrate (70 μmol / L) by saliva samples is measured by a kinetic assay at 300 nm. The rate of the first 10% hydrolysis is calculated for each reaction, and a graph of the mean (±SEM) rate (nmol / L / min, n = 3) is plotted for each sample. Activity (U / ml, 1 U = pepsin producing a change in A300 of 0.001 per second) is calculated.

[0107] [Inclusion criteria] Clinical diagnosis of LPR Age ≥ 18 RSI ≥ 20 RFS≧11 Documentation of LPR (>1 proximal event) by MII-pH testing Failure of 3-month bid PPI therapy Attending a laryngology clinic and undergoing flexible laryngoscopy and MII-pH testing as part of routine clinical care, with at least 3 months between clinic visits (standard practice) Patients must be deemed able to comply with saliva sample collection, treatment plan, and follow-up schedule. Patients must provide study-specific informed consent before participating in a study Exclusion criteria Lexiva is not recommended for the elderly, pregnant women (or those planning to become pregnant), or nursing mothers. - Taking HIV inhibitors -History of liver problems Sulfa allergy ·hemophilia Currently undergoing treatment with another investigational medical device and / or drug - History of stomach or esophageal surgery Gastrointestinal diseases that may interfere with symptom questionnaires, e.g., IBD History of laryngeal or neck surgery, including thyroidectomy and microlaryngoscopy Suspected esophageal cancer Nasopharyngeal cancer - Previously had anti-reflux surgery Polypharmacy (using five or more medications due to comorbid conditions) Potential or known incompatibilities with Lexiva Poor understanding or compliance with the clinical trial protocol is expected

[0108] [Data / Efficacy Analysis] The data obtained in this study were recorded and analyzed using REDCap, a secure web platform for online databases and surveys. REDCap provides automated export procedures for seamless data downloads to Excel and common statistical packages (SPSS, SAS, Stata, R), as well as advanced features such as an integrated project calendar, scheduling module, ad hoc reporting tools, and branching logic, file uploads, and calculated fields. REDCap electronically permits the recording of signed informed consent. Two questionnaires (RSI and RSS) managed by patients for this study were electronically completed by patients on the clinic tablets, and the data were automatically saved to REDCap.

[0109] [Data Recorded in REDCap] · Age, gender, race · Smoking history (yes / no), current smoker (yes / no) · Alcohol intake (> 1 drink / day) · Hypertension (yes / no) · Diabetes (yes / no) · Mental illness (yes / no) · Duration of symptoms (≥ 6 months) · Antireflux medications (proton pump inhibitors, H2 receptor antagonists, alginates) before and during the trial · MII-pH data: total number of proximal reflux events, percentage of time of proximal events, percentage of time of proximal events < pH 4.0, total number of distal reflux events, percentage of time of distal events, percentage of time of distal events < 4.0, DeMeester score · Reflux Symptom Index (RSI) · Reflux Finding Score (RFS) · Pepsin protein in saliva - yes / no · Pepsin protein concentration (ng / ml) · Pepsin activity - yes / no · Pepsin activity (U / ml) · Reflux Symptom Score (RSS) · Reflux Sign Assessment (RSA)

[0110] Power Analysis To determine the sample size required for the proposed study, we used the means and standard deviations (SDs) of RFS and RSI from previous reports (79, 80) and assumed a mean difference of 4.2 (6.5) and SD of 6 (10) for the change in RFS (RSI) after 12 weeks of treatment with Lexiva. Based on these assumptions and a test for significance of the change in RFS / RSI between the two arms using a two-tailed, two-sample, equal-variance t-test, a sample size of 44 and 44 for the Lexiva and placebo groups would achieve 80% power to detect a minimum effect size of 0.6 at a significance level of 0.05. A 15% dropout rate is expected over the 3-month treatment course. Therefore, we plan to recruit a total of 104 patients.

[0111] A sample size of 44 in the Lexiva group would achieve 80% power to detect a minimum correlation of 0.41 between pepsin levels at baseline and change in RFS and RSI after 12 weeks of treatment with Lexiva, at a significance level of 0.05, using two-sided hypothesis testing.

[0112] [Data Management] The accuracy of data collected and entered into REDCap is verified at the time of entry. Data is obtained from patients, EPIC, VPS, PC4, and billing offices (via CDR requests). All data is stored securely with password-protected access to REDCap (www.project-redcap.org / ).

[0113] [Missing data] Every effort is made to avoid missing data using all available sources. Logistic regression (LR) is used to investigate the assumption that data are missing at random. When data appear to be missing at random (MAR), multiple imputation of items is used. Sensitivity analyses are employed to impute values ​​using clinical information and various assumptions.

[0114] [Statistical analysis] The distribution of variables and relationships between variables are examined with statistical summaries such as mean, median, standard deviation (SD), range, and correlations with plots such as boxplots, scatterplots, and local regression smooth curves. When parametric assumptions are necessary, appropriate transformations are employed, along with justification for their use. To test the efficacy of Lexiva treatment, two-sample t-tests are performed by comparing the mean changes in RFS and RSI between the treatment and placebo groups. Pearson correlation analysis or linear regression is used to assess the association between the presence and level of pepsin in saliva before treatment and the changes in RFS and RSI after treatment. Nonparametric tests, such as Wilcoxon and Mann-Whitney tests or Spearman correlation, are used as appropriate, especially in the presence of outliers.

[0115] [Risks and protection against risks] Lexiva oral suspension (50 mg / ml, a prodrug of amprenavir, generic name: fosamprenavir) is currently FDA-approved for the treatment of HIV-1 infection. For adults, the oral suspension should be taken without food. Treatment-naive patients should take 1.4 g twice daily. This clinical trial will use the same route of administration and dosage levels as Lexiva to evaluate its efficacy against laryngopharyngeal reflux (LPR).

[0116] There is a dire need for medical treatment for patients with LPR. Patients with LPR experience symptoms that substantially impact their quality of life, including chronic cough, throat clearing, postnasal drip, hoarseness or dysphonia, globus sensation, difficulty swallowing, and difficulty breathing. Furthermore, chronic LPR contributes to life-threatening conditions such as laryngeal cancer. It is estimated that LPR affects over 20% of the U.S. population and occurs in up to 10% of patients visiting an otolaryngologist. Given that HIV protease inhibitors, such as Lexiva, target a foreign virus, investigation of Lexiva in patients with LPR should not result in significant side effects or reduce the risk of this disease.

[0117] The procedures utilized in this study pose minimal risk to patients. MII-pH testing and flexible laryngoscopy (to obtain RFS and RSI) are routine clinical care for this patient population. Study-specific tests include saliva collection for pepsin analysis, RSS, and RSA. The RSS is a patient-administered questionnaire that will be obtained at the same time as the clinical RSI. The RSA will be obtained when the investigator obtains the clinical RFS during fiberoptic laryngoscopy.

[0118] [References] 1.Ford CN.Evaluation and management of laryngopharyngeal reflux.JAMA.2005;294(12):1534-40.Epub 2005 / 09 / 29.doi:10.1001 / jama.294.12.1534.PubMed PMID:16189367. 2.Koufman JA.The otolaryngologic manifestations of gastroesophageal reflux disease(GERD):a clinical investigation of 225 patients using ambulatory 24-hour pH monitoring and an experimental investigation of the role of acid and pepsin in the development of laryngeal injury.Laryngoscope.1991;101(4 Pt 2 Suppl 53):1-78.Epub 1991 / 04 / 01.doi:10.1002 / lary.1991.101.s53.1.PubMed PMID:1895864. 3.Vaezi MF.Extraesophageal manifestations of gastroesophageal reflux disease.Clin Cornerstone.2003;5(4):32-8;discussion 9-40.Epub 2004 / 04 / 23.doi:10.1016 / s1098-3597(03)90097-4.PubMed PMID:15101493. 4.Gabriel CE,Jones DG.The importance of chronic laryngitis.J Laryngol Otol.1960;74:349-57.Epub 1960 / 06 / 01.doi:10.1017 / s0022215100056693.PubMed PMID:13825824. 5.Johnston N,Yan JC,Hoekzema CR,Samuels TL,Stoner GD,Blumin JH,Bock JM.Pepsin promotes proliferation of laryngeal and pharyngeal epithelial cells.Laryngoscope.2012;122(6):1317-25.Epub 2012 / 05 / 10.doi:10.1002 / lary.23307.PubMed PMID:22570308;PMCID:PMC3816638. 6.Kelly EA, Samuels TL,Johnston N. Chronic pepsin exposure promotes anchorage-independent growth and migration of a hypopharyngeal squamous cell line.Otolaryngol Head Neck Surg.2014;150(4):618-24.Epub 2014 / 01 / 01.doi:10.1177 / 0194599813517862.PubMed PMID: 24376122;PMCID: PMC4423599. 7.Kim SY,Park B,Lim H,Kim M,Kong IG,Choi HG.Increased risk of larynx cancer in patients with gastroesophageal reflux disease from a national sample cohort.Clin Otolaryngol.2019;44(4):534-40.Epub 2019 / 03 / 19.doi: 10.1111 / coa.13328.PubMed PMID:30884136. 8.Parsel SM,Wu EL,Riley CA,McCoul ED.Gastroesophageal and Laryngopharyngeal Reflux Associated With Laryngeal Malignancy:A Systematic Review and Meta-analysis.Clin Gastroenterol Hepatol.2019;17(7):1253-64 e5.Epub 2018 / 10 / 27.doi:10.1016 / j.cgh.2018.10.028.PubMed PMID:30366155. 9.Riley CA,Wu EL,Hsieh MC,Marino MJ,Wu XC, McCoul ED.Association of Gastroesophageal Reflux With Malignancy of the Upper Aerodigestive Tract in Elderly Patients.JAMA Otolaryngol Head Neck Surg.2018;144(2):140-8.Epub 2017 / 12 / 23.doi:10.1001 / jamaoto.2017.2561.PubMed PMID:29270624;PMCID:PMC5839296. 10.Tae K,Jin BJ,Ji YB,Jeong JH,Cho SH,Lee SH.The role of laryngopharyngeal reflux as a risk factor in laryngeal cancer:a preliminary report.Clin Exp Otorhinolaryngol.2011;4(2):101-4.Epub 2011 / 07 / 01.doi:10.3342 / ceo.2011.4.2.101.PubMed PMID:21716948;PMCID:PMC3109325. 11.Wight R,Paleri VArullendran P.Current theories for the development of nonsmoking and nondrinking laryngeal carcinoma.Curr Opin Otolaryngol Head Neck Surg.2003;11(2):73-7.Epub 2003 / 09 / 30. doi: 10.1097 / 00020840-200304000-00002.PubMed PMID:14515082. 12.Altman KW,Stephens RM,Lyttle CS,Weiss KB.Changing impact of gastroesophageal reflux in medical and otolaryngology practice.Laryngoscope.2005;115(7):1145-53.Epub 2005 / 07 / 05.doi:10.1097 / 01.MLG.0000165464.75164.E5.PubMed PMID:15995499. 13.Koufman JA,Amin MR,Panetti M.Prevalence of reflux in 113 consecutive patients with laryngeal and voice disorders.Otolaryngol Head Neck Surg.2000;123(4):385-8.Epub 2000 / 10 / 06.doi:10.1067 / mhn.2000.109935.PubMed PMID:11020172. 14.Reulbach TR,Belafsky PC,Blalock PD,Koufman JA,Postma GN.Occult laryngeal pathology in a community-based cohort.Otolaryngol Head Neck Surg.2001;124(4):448-50.Epub 2001 / 04 / 03.doi:10.1067 / mhn.2001.114256.PubMed PMID:11283505. 15.Francis DO,Rymer JA,Slaughter JC,Choksi Y,Jiramongkolchai P,Ogbeide E,Tran C,Goutte M,Garrett CG,Hagaman D,Vaezi MF.High economic burden of caring for patients with suspected extraesophageal reflux.Am J Gastroenterol.2013;108(6):905-11.Epub 2013 / 04 / 03.doi:10.1038 / ajg.2013.69.PubMed PMID:23545710. 16.Gelardi M,Ciprandi G.Focus on gastroesophageal reflux(GER)and laryngopharyngeal reflux(LPR):new pragmatic insights in clinical practice.J Biol Regul Homeost Agents.2018;32(1 Suppl.2):41-7.Epub 2018 / 02 / 13.PubMed PMID:29436209. 17.Ali MS,Parikh S,Chater P,Pearson JP.Bile acids in laryngopharyngeal refluxate:will they enhance or attenuate the action of pepsin?Laryngoscope.2013;123(2):434-9.Epub 2012 / 10 / 17.doi:10.1002 / lary.23619.PubMed PMID:23070961. 18.Bardhan KD,Strugala V,Dettmar PW.Reflux revisited:advancing the role of pepsin.Int J Otolaryngol.2012;2012:646901.Epub 2012 / 01 / 14. doi:10.1155 / 2012 / 646901.PubMed PMID:22242022;PMCID:PMC3216344. 19.Campagnolo AM,Priston J,Thoen RH,Medeiros T,Assuncao AR.Laryngopharyngeal reflux:diagnosis, treatment,and latest research.Int Arch Otorhinolaryngol.2014;18(2):184-91.Epub 2015 / 05 / 21. doi:10.1055 / s-0033-1352504.PubMed PMID:25992088;PMCID:PMC4297018. 20.Johnston N,Dettmar PW,Bishwokarma B,Lively MO,Koufman JA.Activity / stability of human pepsin:implications for reflux attributed laryngeal disease.Laryngoscope.2007;117(6):1036-9.Epub 2007 / 04 / 10.doi:10.1097 / MLG.0b013e31804154c3.PubMed PMID:17417109. 21.Samuels TL,Johnston N.Pepsin as a causal agent of inflammation during nonacidic reflux.Otolaryngol Head Neck Surg.2009;141(5):559-63.Epub 2009 / 10 / 29.doi:10.1016 / j.otohns.2009.08.022.PubMed PMID:19861190. 22.Tan JJ,Wang L,Mo TT,Wang J,Wang MG,Li XP.Pepsin promotes IL-8 signaling-induced epithelial-mesenchymal transition in laryngeal carcinoma.Cancer Cell Int.2019;19:64.Epub 2019 / 04 / 03.doi:10.1186 / s12935-019-0772-7.PubMed PMID:30936780;PMCID:PMC6425698. 23.Lechien JR,Bock JM,Carroll TL,Akst LM.Is empirical treatment a reasonable strategy for laryngopharyngeal reflux?A contemporary review.Clin Otolaryngol.2020.Epub 2020 / 02 / 26.doi:10.1111 / coa.13518.PubMed PMID:32097534. 24.Samuels TL,Johnston N.Pepsin as a marker of extraesophageal reflux.Ann Otol Rhinol Laryngol.2010;119(3):203-8.Epub 2010 / 04 / 16.doi:10.1177 / 000348941011900310.PubMed PMID:20392035. 25.Piper DW,Fenton BH.pH stability and activity curves of pepsin with special reference to their clinical importance.Gut.1965;6(5):506-8.Epub 1965 / 10 / 01.doi:10.1136 / gut.6.5.506.PubMed PMID:4158734;PMCID:PMC1552331. 26.Axford SE,Sharp N,Ross PE,Pearson JP,Dettmar PW,Panetti M,Koufman JA.Cell biology of laryngeal epithelial defenses in health and disease:preliminary studies.Ann Otol Rhinol Laryngol.2001;110(12):1099-108.Epub 2002 / 01 / 05.doi:10.1177 / 000348940111001203.PubMed PMID:11768697. 27.Hopwood D,Bateson MC,Milne G,Bouchier IA.Effects of bile acids and hydrogen ion on the fine structure of oesophageal epithelium.Gut.1981;22(4):306-11.Epub 1981 / 04 / 01.doi:10.1136 / gut.22.4.306.PubMed PMID:7239322;PMCID:PMC1419165. 28.Eto T,Tompkins RK.Further studies on the inhibition of pepsin by bile salts.Ann Surg.1986;203(1):8-12.Epub 1986 / 01 / 01.doi:10.1097 / 00000658-198601000-00002.PubMed PMID:3079997;PMCID:PMC1251031. 29.Pearson JP,Parikh S,Orlando RC,Johnston N,Allen J,Tinling SP,Johnston N,Belafsky P,Arevalo LF,Sharma N,Castell DO,Fox M,Harding SM,Morice AH,Watson MG,Shields MD,Bateman N,McCallion WA,van Wijk MP,Wenzl TG,Karkos PD,Belafsky PC.Review article:reflux and its consequences--the laryngeal,pulmonary and oesophageal manifestations.Conference held in conjunction with the 9th International Symposium on Human Pepsin(ISHP)Kingston-upon-Hull,UK,21-23 April 2010.Aliment Pharmacol Ther.2011;33 Suppl 1:1-71.Epub 2011 / 03 / 05.doi:10.1111 / j.1365-2036.2011.04581.x.PubMed PMID:21366630. 30.Calvo-Henriquez C,Ruano-Ravina A,Vaamonde P,Martinez-Capoccioni G,Martin-Martin C.Is Pepsin a Reliable Marker of Laryngopharyngeal Reflux?A Systematic Review.Otolaryngol Head Neck Surg.2017;157(3):385-91.Epub 2017 / 06 / 07.doi: 10.1177 / 0194599817709430.PubMed PMID:28585488. 31.Johnston N,Dettmar PW,Ondrey FG,Nanchal R,Lee SH,Bock JM.Pepsin:biomarker,mediator,and therapeutic target for reflux and aspiration.Ann N Y Acad Sci.2018;1434(1):282-9.Epub 2018 / 05 / 19.doi:10.1111 / nyas.13729.PubMed PMID:29774546. 32.Johnston N,Wells CW,Samuels TL,Blumin JH.Pepsin in nonacidic refluxate can damage hypopharyngeal epithelial cells.Ann Otol Rhinol Laryngol.2009;118(9):677-85.Epub 2009 / 10 / 09.doi:10.1177 / 000348940911800913.PubMed PMID:19810610. 33.Johnston N,Wells CW,Samuels TL,Blumin JH.Rationale for targeting pepsin in the treatment of reflux disease.Ann Otol Rhinol Laryngol.2010;119(8):547-58.Epub 2010 / 09 / 24.doi:10.1177 / 000348941011900808.PubMed PMID:20860281. 34.Weitzendorfer M,Antoniou SA,Schredl P,Witzel K,Weitzendorfer IC,Majerus A,Emmanuel K,Koch OO.Pepsin and oropharyngeal pH monitoring to diagnose patients with laryngopharyngeal reflux.Laryngoscope.2019.Epub 2019 / 10 / 12.doi:10.1002 / lary.28320.PubMed PMID:31603541. 35.Zalvan CH,Hu S,Greenberg B,Geliebter J.A Comparison of Alkaline Water and Mediterranean Diet vs Proton Pump Inhibition for Treatment of Laryngopharyngeal Reflux.JAMA Otolaryngol Head Neck Surg.2017;143(10):1023-9.Epub 2017 / 09 / 08.doi:10.1001 / jamaoto.2017.1454.PubMed PMID:28880991;PMCID:PMC5710251. 36.Gill GA,Johnston N,Buda A,Pignatelli M,Pearson J,Dettmar PW,Koufman J.Laryngeal epithelial defenses against laryngopharyngeal reflux:investigations of E-cadherin,carbonic anhydrase isoenzyme III,and pepsin.Ann Otol Rhinol Laryngol.2005;114(12):913-21.Epub 2006 / 01 / 24.doi:10.1177 / 000348940511401204.PubMed PMID:16425556. 37.Johnston N,Bulmer D,Gill GA,Panetti M,Ross PE,Pearson JP,Pignatelli M,Axford SE,Dettmar PW,Koufman JA.Cell biology of laryngeal epithelial defenses in health and disease:further studies.Ann Otol Rhinol Laryngol.2003;112(6):481-91.Epub 2003 / 07 / 02.doi:10.1177 / 000348940311200601.PubMed PMID:12834114. 38.Johnston N,Knight J,Dettmar PW,Lively MO,Koufman J.Pepsin and carbonic anhydrase isoenzyme III as diagnostic markers for laryngopharyngeal reflux disease.Laryngoscope.2004;114(12):2129-34.Epub 2004 / 11 / 27.doi:10.1097 / 01.mlg.0000149445.07146.03.PubMed PMID:15564833. 39.Agrawal A,Roberts J,Sharma N,Tutuian R,Vela M,Castell DO.Symptoms with acid and nonacid reflux may be produced by different mechanisms.Dis Esophagus.2009;22(5):467-70.Epub 2009 / 02 / 19.doi:10.1111 / j.1442-2050.2009.00940.x.PubMed PMID:19222535. 40.Tamhankar AP,Peters JH,Portale G,Hsieh CC,Hagen JA,Bremner CG, DeMeester TR.Omeprazole does not reduce gastroesophageal reflux:new insights using multichannel intraluminal impedance technology.J Gastrointest Surg.2004;8(7):890-7;discussion 7-8.Epub 2004 / 11 / 09.doi:10.1016 / j.gassur.2004.08.001.PubMed PMID:15531244. 41.Tutuian R,Mainie I,Agrawal A,Adams D,Castell DO.Nonacid reflux in patients with chronic cough on acid-suppressive therapy.Chest.2006;130(2):386-91.Epub 2006 / 08 / 11.doi:10.1378 / chest.130.2.386.PubMed PMID:16899836. 42.Tutuian R,Vela MF,Hill EG,Mainie I,Agrawal A,Castell DO.Characteristics of symptomatic reflux episodes on Acid suppressive therapy.Am J Gastroenterol.2008;103(5):1090-6.Epub 2008 / 05 / 01.doi: 10.1111 / j.1572-0241.2008.01791.x.PubMed PMID:18445095. 43.Falk GL,Van der Wall H,Burton L,Falk MG,O’Donnell H,Vivian SJ.Fundoplication for laryngopharyngeal reflux despite preoperative dysphagia.Ann R Coll Surg Engl.2017;99(3):224-7.Epub 2017 / 03 / 03.doi:10.1308 / rcsann.2016.0330.PubMed PMID:28252352;PMCID:PMC5450280. 44.Iqbal M,Batch AJ,Moorthy K,Cooper BT,Spychal RT.Outcome of surgical fundoplication for extra-oesophageal symptoms of reflux.Surg Endosc.2009;23(3):557-61.Epub 2008 / 03 / 28.doi: 10.1007 / s00464-008-9861-8.PubMed PMID:18365279. 45.Lechien JR,Dapri G,Dequanter D,Rodriguez Ruiz A,Marechal MT,De Marrez LG,Saussez S,Fisichella PM.Surgical Treatment for Laryngopharyngeal Reflux Disease:A Systematic Review.JAMA Otolaryngol Head Neck Surg.2019;145(7):655-66.Epub 2019 / 05 / 03.doi:10.1001 / jamaoto.2019.0315.PubMed PMID:31046069. 46.Mainie I,Tutuian R,Shay S,Vela M,Zhang X,Sifrim D,Castell DO.Acid and non-acid reflux in patients with persistent symptoms despite acid suppressive therapy:a multicentre study using combined ambulatory impedance-pH monitoring.Gut.2006;55(10):1398-402.Epub 2006 / 03 / 25.doi:10.1136 / gut.2005.087668.PubMed PMID:16556669;PMCID:PMC1856433. 47.Sidwa F,Moore AL,Alligood E,Fisichella PM.Surgical Treatment of Extraesophageal Manifestations of Gastroesophageal Reflux Disease.World J Surg.2017;41(10):2566-71.Epub 2017 / 05 / 17.doi:10.1007 / s00268-017-4058-8.PubMed PMID:28508234. 48.Zhang C,Hu ZW,Yan C,Wu Q,Wu JM,Du X,Liu DG,Luo T,Li F,Wang ZG.Nissen fundoplication vs proton pump inhibitors for laryngopharyngeal reflux based on pH-monitoring and symptom-scale.World J Gastroenterol.2017;23(19):3546-55.Epub 2017 / 06 / 10.doi:10.3748 / wjg.v23.i19.3546.PubMed PMID:28596691;PMCID:PMC5442091. 49.Johnston N,Wells CW,Blumin JH,Toohill RJ,Merati AL.Receptor-mediated uptake of pepsin by laryngeal epithelial cells.Ann Otol Rhinol Laryngol.2007;116(12):934-8.Epub 2008 / 01 / 26.doi:10.1177 / 000348940711601211.PubMed PMID:18217514. 50.Rees LE,Pazmany L,Gutowska-Owsiak D,Inman CF,Phillips A,Stokes CR,Johnston N,Koufman JA,Postma G,Bailey M,Birchall MA.The mucosal immune response to laryngopharyngeal reflux.Am J Respir Crit Care Med.2008;177(11):1187-93.Epub 2008 / 03 / 08.doi:10.1164 / rccm.200706-895OC.PubMed PMID:18323539;PMCID:PMC2643204. 51.Samuels TL,Altman KW,Gould JC,Kindel T,Bosler M, MacKinnon A,Hagen CE,Johnston N.Esophageal pepsin and proton pump synthesis in barrett’s esophagus and esophageal adenocarcinoma.Laryngoscope.2019;129(12):2687-95.Epub 2019 / 05 / 03.doi:10.1002 / lary.28051.PubMed PMID:31046139. 52.Niu K,Guo C,Teng S,Zhou D,Yu S,Yin W,Wang P,Zhu W,Duan M.Pepsin promotes laryngopharyngeal neoplasia by modulating signaling pathways to induce cell proliferation.PLoS One.2020;15(1):e0227408.Epub 2020 / 01 / 16. doi:10.1371 / journal.pone.0227408.PubMed PMID 31940393;PMCID:PMC6961942. 53.Johnston N,Dettmar PW,Lively MO,Postma GN,Belafsky PC,Birchall M,Koufman JA.Effect of pepsin on laryngeal stress protein(Sep70,Sep53,and Hsp70)response:role in laryngopharyngeal reflux disease.Ann Otol Rhinol Laryngol.2006;115(1):47-58.Epub 2006 / 02 / 10.doi:10.1177 / 000348940611500108.PubMed PMID:16466100. 54.Hurley BP,Jugo RH,Snow RF,Samuels TL,Yonker LM,Mou H,Johnston N,Rosen R.Pepsin Triggers Neutrophil Migration Across Acid Damaged Lung Epithelium.Sci Rep.2019;9(1):13778.Epub 2019 / 09 / 26.doi:10.1038 / s41598-019-50360-4.PubMed PMID:31551494;PMCID:PMC6760148. 55.Nagahama K,Yamato M,Nishio H,Takeuchi K.Essential role of pepsin in pathogenesis of acid reflux esophagitis in rats.Dig Dis Sci.2006;51(2):303-9.Epub 2006 / 03 / 15.doi:10.1007 / s10620-006-3129-8.PubMed PMID:16534673. 56.Samuels TL,Pearson AC,Wells CW,Stoner GD,Johnston N.Curcumin and anthocyanin inhibit pepsin-mediated cell damage and carcinogenic changes in airway epithelial cells.Ann Otol Rhinol Laryngol.2013;122(10):632-41.Epub 2013 / 12 / 04.PubMed PMID:24294686. 57.Martinucci I,de Bortoli N,Savarino E,Nacci A,Romeo SO,Bellini M,Savarino V,Fattori B,Marchi S.Optimal treatment of laryngopharyngeal reflux disease.Ther Adv Chronic Dis.2013;4(6):287-301.Epub 2013 / 11 / 02.doi:10.1177 / 2040622313503485.PubMed PMID:24179671;PMCID:PMC3807765. 58.Koufman JA,Aviv JE,Casiano RR,Shaw GY.Laryngopharyngeal reflux:position statement of the committee on speech,voice, and swallowing disorders of the American Academy of Otolaryngology-Head and Neck Surgery.Otolaryngol Head Neck Surg.2002;127(1):32-5.Epub 2002 / 08 / 06.doi:10.1067 / mhn.2002.125760.PubMed PMID:12161727. 59.Park W,Hicks DM,Khandwala F,Richter JE,Abelson TI,Milstein C,Vaezi MF.Laryngopharyngeal reflux:prospective cohort study evaluating optimal dose of proton-pump inhibitor therapy and pretherapy predictors of response.Laryngoscope.2005;115(7):1230-8.Epub 2005 / 07 / 05.doi:10.1097 / 01.MLG.0000163746.81766.45.PubMed PMID:15995512. 60.Eherer AJ,Habermann W,Hammer HF,Kiesler K,Friedrich G,Krejs GJ.Effect of pantoprazole on the course of reflux-associated laryngitis:a placebo-controlled double-blind crossover study.Scand J Gastroenterol.2003;38(5):462-7.Epub 2003 / 06 / 11.doi:10.1080 / 00365520310001860.PubMed PMID:12795454. 61.El-Serag HB,Lee P,Buchner A,Inadomi JM,Gavin M,McCarthy DM.Lansoprazole treatment of patients with chronic idiopathic laryngitis:a placebo-controlled trial.Am J Gastroenterol.2001;96(4):979-83.Epub 2001 / 04 / 24.doi:10.1111 / j.1572-0241.2001.03681.x.PubMed PMID:11316215. 62.Noordzij JP,Khidr A,Evans BA,Desper E,Mittal RK,Reibel JF,Levine PA.Evaluation of omeprazole in the treatment of reflux laryngitis:a prospective,placebo-controlled,randomized,double-blind study.Laryngoscope.2001;111(12):2147-51.Epub 2002 / 01 / 22.doi:10.1097 / 00005537-200112000-00013.PubMed PMID:11802014. 63.Steward DL,Wilson KM,Kelly DH,Patil MS,Schwartzbauer HR,Long JD,Welge JA.Proton pump inhibitor therapy for chronic laryngo-pharyngitis: a randomized placebo-control trial.Otolaryngol Head Neck Surg.2004;131(4):342-50.Epub 2004 / 10 / 07.doi:10.1016 / j.otohns.2004.03.037.PubMed PMID:15467597. 64.Vaezi MF,Richter JE,Stasney CR,Spiegel JR,Iannuzzi RA,Crawley JA,Hwang C,Sostek MB,Shaker R.Treatment of chronic posterior laryngitis with esomeprazole.Laryngoscope.2006;116(2):254-60.Epub 2006 / 02 / 10.doi:10.1097 / 01.mlg.0000192173.00498.ba.PubMed PMID:16467715. 65.Wo JM,Koopman J,Harrell SP,Parker K,Winstead W,Lentsch E.Double-blind,placebo-controlled trial with single-dose pantoprazole for laryngopharyngeal reflux.Am J Gastroenterol.2006;101(9):1972-8;quiz 2169.Epub 2006 / 09 / 14.doi:10.1111 / j.1572-0241.2006.00693.x.PubMed PMID:16968502. 66.Lam PK,Ng ML,Cheung TK,Wong BY,Tan VP,Fong DY,Wei WI,Wong BC.Rabeprazole is effective in treating laryngopharyngeal reflux in a randomized placebo-controlled trial.Clin Gastroenterol Hepatol.2010;8(9):770-6.Epub 2010 / 03 / 23.doi:10.1016 / j.cgh.2010.03.009.PubMed PMID:20303417. 67.Reichel O,Dressel H,Wiederanders K,Issing WJ.Double-blind, placebo-controlled trial with esomeprazole for symptoms and signs associated with laryngopharyngeal reflux.Otolaryngol Head Neck Surg.2008;139(3):414-20.Epub 2008 / 08 / 30.doi:10.1016 / j.otohns.2008.06.003.PubMed PMID:18722223. 68.Vaezi MF.Gastroesophageal reflux-related chronic laryngitis:con.Arch Otolaryngol Head Neck Surg.2010;136(9):908-9.Epub 2010 / 09 / 22. doi:10.1001 / archoto.2010.149.PubMed PMID:20855684. 69.Lien HC,Wang CC,Liang WM,Sung FC,Hsu JY,Yeh HZ,Chong K,Chang CS.Composite pH predicts esomeprazole response in laryngopharyngeal reflux without typical reflux syndrome.Laryngoscope.2013;123(6):1483-9.Epub 2013 / 04 / 05.doi:10.1002 / lary.23780.PubMed PMID:23553459. 70.Masaany M,Marina MB,Sharifa Ezat WP,Sani A.Empirical treatment with pantoprazole as a diagnostic tool for symptomatic adult laryngopharyngeal reflux.J Laryngol Otol.2011;125(5):502-8.Epub 2011 / 03 / 02.doi:10.1017 / S0022215111000120.PubMed PMID:21356141. 71.Kahrilas PJ.When proton pump inhibitors fail.Clin Gastroenterol Hepatol.2008;6(5):482-3.Epub 2008 / 04 / 02.doi:10.1016 / j.cgh.2008.02.010.PubMed PMID:18378500;PMCID:PMC2474735. 72.Barry DW,Vaezi MF.Laryngopharyngeal reflux:More questions than answers.Cleve Clin J Med.2010;77(5):327-34.Epub 2010 / 05 / 05.doi:10.3949 / ccjm.77a.09121.PubMed PMID:20439565. 73.Belafsky PC.PRO:Empiric treatment with PPIs is not appropriate without testing.Am J Gastroenterol.2006;101(1):6-8.Epub 2006 / 01 / 13.doi:10.1111 / j.1572-0241.2006.00448_2.x.PubMed PMID:16405525. 74.Hvid-Jensen F,Pedersen L,Funch-Jensen P,Drewes AM.Proton pump inhibitor use may not prevent high-grade dysplasia and oesophageal adenocarcinoma in Barrett’s oesophagus:a nationwide study of 9883 patients.Aliment Pharmacol Ther.2014;39(9):984-91.Epub 2014 / 03 / 13. doi:10.1111 / apt.12693.PubMed PMID:24617286. 75.Luebke K,Samuels TL,Chelius TH,Sulman CG,McCormick ME,Kerschner JE,Johnston N,Chun RH.Pepsin as a biomarker for laryngopharyngeal reflux in children with laryngomalacia.Laryngoscope.2017;127(10):2413-7.Epub 2017 / 02 / 23.doi:10.1002 / lary.26537.PubMed PMID:28224634. 76.Kamani T,Penney S,Mitra I,Pothula V.The prevalence of laryngopharyngeal reflux in the English population.Eur Arch Otorhinolaryngol.2012;269(10):2219-25.Epub 2012 / 05 / 12.doi:10.1007 / s00405-012-2028-1.PubMed PMID:22576243. 77.Lowden M,McGlashan JA,Steel A,Strugala V,Dettmar PW.Prevalence of symptoms suggestive of extra-oesophageal reflux in a general practice population in the UK.Logoped Phoniatr Vocol.2009;34(1):32-5.Epub 2009 / 02 / 14.doi:10.1080 / 14015430902735847.PubMed PMID:19214865. 78.Caicedo-Granados E,Galbraith AR,Schachern MG,Hartle DE,Wattenberg LW,Wuertz BR,Keel S,Yueh B,Ondrey FG.N-methylnitrosourea-induced carcinoma as a model for laryngeal carcinogenesis.Head Neck.2014;36(12):1802-6.Epub 2014 / 12 / 31.doi:10.1002 / hed.23536.PubMed PMID:25548813. 79.Belafsky PC,Postma GN,Koufman JA.The validity and reliability of the reflux finding score(RFS).Laryngoscope.2001;111(8):1313-7.Epub 2001 / 09 / 25.doi:10.1097 / 00005537-200108000-00001.PubMed PMID:11568561. 80.Belafsky PC,Postma GN,Koufman JA.Validity and reliability of the reflux symptom index(RSI).J Voice.2002;16(2):274-7.Epub 2002 / 08 / 02.doi:10.1016 / s0892-1997(02)00097-8.PubMed PMID:12150380. 81.Lechien JR,Akst LM,Hamdan AL,Schindler A,Karkos PD,Barillari MR,Calvo-Henriquez C,Crevier-Buchman L,Finck C,Eun YG,Saussez S,Vaezi MF.Evaluation and Management of Laryngopharyngeal Reflux Disease:State of the Art Review.Otolaryngol Head Neck Surg.2019;160(5):762-82.Epub 2019 / 02 / 13.doi:10.1177 / 0194599819827488.PubMed PMID:30744489. 82.Lechien JRBobin F,Mouawad F,Zelenik K,Calvo-Henriquez C,Chiesa-Estomba CM,Enver N,Nacci A,Barillari MR,Schindler A,Crevier-Buchman L,Hans S,Simeone V,Wlodarczyk E,Harmegnies B,Remacle M,Rodriguez A,Dequanter D,Eisendrath P,Dapri G,Finck C,Karkos P,Pendleton H,Ayad T,Muls V,Saussez S.Development of scores assessing the refluxogenic potential of diet of patients with laryngopharyngeal reflux.Eur Arch Otorhinolaryngol.2019;276(12):3389-404.Epub 2019 / 09 / 14.doi:10.1007 / s00405-019-05631-1.PubMed PMID:31515662. 83.Lechien JR,Bobin F,Muls V,Thill MP,Horoi M,Ostermann K,Huet K, Harmegnies B,Dequanter D,Dapri G,Marechal MT,Finck C,Rodriguez Ruiz A,Saussez S.Validity and reliability of the reflux symptom score.Laryngoscope.2020;130(3):E98-E107.Epub 2019 / 04 / 16.doi:10.1002 / lary.28017.PubMed PMID:30983002. 84.Lechien JR,Rodriguez Ruiz A,Dequanter D,Bobin F,Mouawad F,Muls V,Huet K,Harmegnies B,Remacle S,Finck C, Saussez S.Validity and Reliability of the Reflux Sign Assessment.Ann Otol Rhinol Laryngol.2020;129(4):313-25.Epub 2019 / 11 / 16.doi:10.1177 / 0003489419888947.PubMed PMID:31729247. 85.Lien HC,Wang CC,Lee SW,Hsu JY,Yeh HZ,Ko CW,Chang CS,Liang WM.Responder Definition of a Patient-Reported Outcome Instrument for Laryngopharyngeal Reflux Based on the US FDA Guidance.Value Health.2015;18(4):396-403.Epub 2015 / 06 / 21.doi:10.1016 / j.jval.2015.01.001.PubMed PMID:26091593. 86.Klimara MJ,Johnston N,Samuels TL,Visotcky AM,Poetker DM,Loehrl TA,Blumin JH,Bock JM.Correlation of salivary and nasal lavage pepsin with MII-pH testing.Laryngoscope.2020;130(4):961-6.Epub 2019 / 07 / 23.doi:10.1002 / lary.28182.PubMed PMID:31329290. 87.Marshall S,McCann AJ,Samuels TL,Blair A,Bonne V,Johnston N,Koufman J.Detection of pepsin and IL-8 in saliva of adult asthmatic patients.J Asthma Allergy.2019;12:155-61.Epub 2019 / 06 / 20.doi:10.2147 / JAA.S205482.PubMed PMID:31213853;PMCID:PMC6549784. 88.Potluri S,Friedenberg F,Parkman HP,Chang A,MacNeal R,Manus C,Bromer MQ,Malik A,Fisher RS,Nugent T,Thangada VK,Kueppers F,Miller LS.Comparison of a salivary / sputum pepsin assay with 24-hour esophageal pH monitoring for detection of gastric reflux into the proximal esophagus,oropharynx,and lung.Dig Dis Sci.2003;48(9):1813-7.Epub 2003 / 10 / 17.doi:10.1023 / a:1025467600662.PubMed PMID:14561007. 89.Crapko M,Kerschner JE,Syring M,Johnston N.Role of extra-esophageal reflux in chronic otitis media with effusion.Laryngoscope.2007;117(8):1419-23.Epub 2007 / 06 / 23.doi:10.1097 / MLG.0b013e318064f177.PubMed PMID:17585281. 90.Knight J,Lively MO,Johnston N,Dettmar PW,Koufman JA.Sensitive pepsin immunoassay for detection of laryngopharyngeal reflux.Laryngoscope.2005;115(8):1473-8.Epub 2005 / 08 / 12.doi:10.1097 / 01.mlg.0000172043.51871.d9.PubMed PMID:16094128. 91.Roberts NB,Taylor WH.Comparative Pepstatin Inhibition Studies on Individual Human Pepsins and Pepsinogens 1,3 and 5(gastricsin)and Pig Pepsin A.J Enzyme Inhib Med Chem.2003;18(3):209-217. 92.Lea WA,Simeonov A.Fluorescence polarization assays in small molecule screening.Expert Opin Drug Discov.2011;6(1):17-32. 93.Jolley ME.Fluorescence polarization assays for the detection of proteases and their inhibitors.J.Biomol.Screen.1996;1(1):33-8,https: / / doi.org / 10.1177 / 108705719600100112 94.Schade SZ, Jolley ME, Sarauer BJ, Simonson LG.BODIPY-alpha-casein, a pH-independent protein substrate for protease assays using fluorescence polarization.Anal Biochem.1996 Dec 1;243(1):1-7 95.Olp MD,Kalous KS,Smith BC.An online tool for calculating initial rates from continuous enzyme kinetic traces.bioRxiv 700138;Jul 14 2019.doi:https: / / doi.org / 10.1101 / 700138

[0119] [array] (SEQ ID NO: 1 Synthetic peptide substrate for pepsin) Lys-Pro-Ala-Glu-Phe-PNP-Arg-Leu (PNP = paranitrophenylalanine)

Claims

1. 1. A pharmaceutical composition for use in treating reflux in a subject in need thereof, comprising a therapeutically effective amount of an HIV protease inhibitor, wherein the HIV protease inhibitor is fosamprenavir or darunavir; The pharmaceutical composition, wherein the reflux is reflux of gastric contents.

2. 10. The composition for use of claim 1, wherein the HIV protease inhibitor is formulated for oral administration.

3. 3. The composition for use according to claim 1 or 2, wherein the HIV protease inhibitor is formulated for administration as an aerosol.

4. 4. The composition for use of claim 3, wherein the HIV protease inhibitor is formulated for administration as a nasal spray.

5. 4. The composition for use of claim 3, wherein the HIV protease inhibitor is formulated for administration via an inhaler or nebulizer.

6. 6. A composition for use according to any one of claims 1 to 5, wherein the HIV protease inhibitor is formulated for administration twice daily at a dosage of 1.4g or less.

7. 7. The composition for use according to any one of claims 1 to 6, wherein the subject has an airway reflux condition selected from laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), and esophagopharyngeal reflux (EPR).

8. 8. The composition for use according to claim 7, wherein the subject's condition is refractory to treatment with a proton pump inhibitor (PPI).

9. A composition for use according to any one of claims 1 to 8, wherein administration of said composition reduces damage and inflammation of the laryngeal mucosa.

10. The composition for use according to any one of claims 1 to 6, wherein the subject has gastroesophageal reflux disease (GERD).

11. The composition for use according to claim 10, wherein the GERD is proton pump inhibition resistant GERD.

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