Sustained-release oral fosamprenavir formulations for the treatment of reflux disease

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. Also provided are compositions comprising sustained-release formulations of HIV protease inhibitors for oral administration.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Application No. 63 / 392,929, filed July 28, 2022, the entire contents of which are incorporated herein by reference.

[0002] [Statement regarding federally sponsored research] Not applicable

[0003] [Array List] A sequence listing is attached to this application and has been filed as a 2,375 byte sequence listing XML file entitled "650053_00979_Sequence_Listing" created on July 25, 2023. The sequence listing was submitted electronically via the Patent Center 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 the disease is broad.

[0005] More than 20% of the US population suffers from laryngopharyngeal reflux. Although dietary and lifestyle modifications and alginates have been effective in some people, there is no gold standard treatment. Increasing evidence suggests that pepsin may be responsible, in part or in whole, for the damage and inflammation associated with laryngopharyngeal reflux. Therapies specifically targeting pepsin, suitable for local inhaled administration, may be effective for the endoscopic signs and symptoms associated with non-acid reflux.

[0006] Laryngopharyngeal reflux (LPR), a condition characterized by the reflux of gastric contents into the larynopharynx, is a significant health problem. LPR affects both children and adults and has a wide clinical spectrum. Unlike patients with gastroesophageal reflux (GER), which is limited to the esophagus, many LPR patients do not experience gastric hyperacidity. Instead, they present with symptoms of chronic laryngeal irritation, such as chronic cough, throat clearing, postnasal drip, dysphonia, laryngeal bulbar sounds, dysphagia, and dyspnea. Significant evidence suggests that chronic LPR contributes to serious and life-threatening conditions, including airway stenosis, reactive airway disease, and laryngeal cancer. LPR is estimated to affect more than 20% of the US population and account for 10% of otolaryngology (ENT) visits. The economic burden of LPR exceeds $52 billion annually, 5.6 times that of GER. 52% of the burden is attributable to proton pump inhibitors (PPIs).

[0007] PPI therapy is the mainstay of treatment for gastroesophageal reflux disease (GERD), but its effectiveness in LPR is low. In clinical practice, due to the assumption that the upper airway is more sensitive to acid reflux than the esophagus, it has been assumed that patients with reflux laryngitis require higher doses of PPIs and longer trials than patients with typical GERD. However, placebo-controlled trials have not demonstrated the therapeutic efficacy of PPIs. Reichel et al. and Lam et al. reported symptomatic improvement in a randomized, double-blind, placebo-controlled trial, but Vaezi argued that the improvement was related to heartburn, not throat symptoms. When laryngeal symptom improvement was reported, it was found to be proportionally higher in GERD patients than in patients without GERD. Due to a lack of data supporting acid suppression therapy for extraesophageal symptoms, the American College of Gastroenterology's GERD guidelines recommend against the use of acid suppression therapy for the acute treatment of patients with extraesophageal reflux (EER) syndromes (laryngitis, chronic cough) who may not have typical GERD symptoms. Despite this advice, empirical treatment with PPIs continues to be frequently used to treat LPR.

[0008] Although reflux acidity alone can damage the upper airway, combined multichannel intraluminal impedance pH (MII-pH) monitoring has demonstrated that many episodes of LPR are non-acidic, and both weakly acidic and non-acidic reflux are associated with persistent symptoms in acid-suppressed patients. These symptoms may be alleviated by anti-reflux surgery and improved by less invasive strategies (e.g., dietary and lifestyle modifications, over-the-counter alginate products) that limit reflux occurrence or neutralize non-acidic reflux components. Therefore, one or more non-acidic components of gastric reflux fluid must contribute to laryngeal injury. There is increasing evidence that pepsin, present in all reflux fluids, is partially, if not entirely, responsible for the damage and inflammation caused by LPR.

[0009] Pepsin is a proteolytic enzyme synthesized and secreted as the enzyme precursor pepsinogen by chief cells in the gastric fundus, which then degrades to produce pepsin upon entering the acidic gastric cavity. Pepsin is maximally active at pH 2 and remains active down to pH 6.5. It is stable at pH 8 but irreversibly inactivated at higher pH levels. While the stomach and esophagus possess inherent defense mechanisms against pepsin (mucus, peristalsis, and bicarbonate secretion), laryngeal tissue lacks these. Pepsin is thought to play an important role in mucosal injury and inflammation during non-acidic reflux. At neutral pH, pepsin is internalized by laryngeal and hypopharyngeal cells by receptor-mediated endocytosis and is retained in low-pH intracellular vesicles, where it is thought to be reactivated. This results in chronic inflammation and symptoms. Endocytosed non-acidic pepsin induces a proinflammatory cytokine gene expression profile in hypopharyngeal cells, which is similar to that contributing to disease severity during GERD. Inhibiting the proteolytic activity of pepsin reverses this damage and inflammation.

[0010] Given the compelling evidence of an association between non-acidic proximal reflux of pepsin and laryngeal and pharyngeal symptoms and endoscopic findings, the lack of a gold-standard medical therapy, the significant costs and risks of ongoing long-term PPI therapy despite its ineffectiveness, and the limitations of alternative non-surgical treatment options, such as the short-lived effects of over-the-counter products intended for temporary relief and the burden of adherence to dietary and lifestyle changes, new medical therapies specifically targeting pepsin would be of great benefit.

[0011] We and other investigators have discussed the possibility of GI activity inhibitors and / or receptor antagonists as potential new therapeutic agents for LPR. Summary of the Invention

[0012] In one aspect, the present disclosure provides an oral sustained release formulation for the treatment of reflux disease, comprising an effective amount of an HIV protease inhibitor, sodium alginate, and a pharmaceutically acceptable carrier.

[0013] In another aspect, the present disclosure provides a method of treating reflux disease in a subject in need thereof, the method comprising orally administering to the subject a formulation described herein for the treatment of reflux disease. [Brief explanation of the drawings]

[0014] [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 pepsin's binding sites. Assay 2 (bottom) is a digestion activity assay that utilizes fluorescently labeled casein as the enzyme substrate. [Figure 2] Figure 2 shows the percent inhibition of pepsin produced by a library of pharmacologically active compounds screened using the binding assay. [Figure 3A]Figures 3A-B show the co-crystal structure of amprenavir bound to pepsin. Figure 3A shows the 2|Fo|-|Fc| electron density map (green mesh) of amprenavir (purple carbons) bound to the pepsin active site (yellow carbons), and Figure 3B shows a schematic of the active site with amprenavir bound, with potential hydrogen-bonding interactions indicated by dashed green lines. [Figure 3B] Figures 3A-B show the co-crystal structure of amprenavir bound to pepsin. Figure 3A shows the 2|Fo|-|Fc| electron density map (green mesh) of amprenavir (purple carbons) bound to the pepsin active site (yellow carbons), and Figure 3B shows a schematic of the active site with amprenavir bound, with potential hydrogen-bonding interactions indicated by dashed green lines. [Figure 3C] Figures 3C and 3D show the crystal structure of the active site of pepsin with darunavir bound and the enzyme-inhibitor interactions. [Figure 3D] Figures 3C and 3D show the crystal structure of the active site of pepsin with darunavir bound and the enzyme-inhibitor interactions. [Figure 4A-B] Figures 4A-4D show that oral administration of Lexiva prevents pepsin-mediated airway epithelial damage in vivo. Representative animals from different treatment protocols are shown in each panel: pH 7 (Figure 4A), Lexiva (Figure 4B), 0.3 mg / ml pepsin at pH 7 (Figure 4C), and 0.3 mg / ml pepsin pH 7 + Lexiva (Figure 4D). 20x magnification. (Figures 4A, 4B, and 4D) Normal-appearing respiratory epithelium consisting of a single layer of ciliated columnar epithelium with basal polarization of the nuclei and apical cilia. (Figure 4C) Reactive multilayered epithelium with an increased nucleus-to-cytoplasm (N:C) ratio and loss of cilia. [Figure 4C-D]Figures 4A-4D show that oral administration of Lexiva prevents pepsin-mediated airway epithelial damage in vivo. Representative animals from different treatment protocols are shown in each panel: pH 7 (Figure 4A), Lexiva (Figure 4B), 0.3 mg / ml pepsin at pH 7 (Figure 4C), and 0.3 mg / ml pepsin pH 7 + Lexiva (Figure 4D). 20x magnification. (Figures 4A, 4B, and 4D) Normal-appearing respiratory epithelium consisting of a single layer of ciliated columnar epithelium with basal polarization of the nuclei and apical cilia. (Figure 4C) Reactive multilayered epithelium with an increased nucleus-to-cytoplasm (N:C) ratio and loss of cilia. [Figure 5A] Figures 5A and 5B are schematic diagrams of a 12-week, randomized, double-blind, placebo-controlled clinical trial designed to test the efficacy of the HIV protease inhibitor Lexiva in treating LPR. [Figure 5B] Figures 5A and 5B are schematic diagrams of a 12-week, randomized, double-blind, placebo-controlled clinical trial designed to test the efficacy of the HIV protease inhibitor Lexiva in treating LPR. [Figure 6] FIG. 6 shows the treatment scheme for the in vivo mouse study. [Figure 7A-B] 7A and 7B show the binding curve (FIG. 7A) and activity curve (FIG. 7B) of the HIV protease inhibitor with pepsin. [Figure 8A]Figures 8A–8D show structural data for pepsin and HIV protease inhibitors. The left panels of Figures 8A–8D show the active site of porcine pepsin with the bound HIV protease inhibitor. The 2Fo-Fc electron density map, contoured at 1.0σ, is shown as a magenta mesh, and the 2Fo-Fc simulated annealing composite omit map, also contoured at 1.0σ, is shown as a green mesh. The right panels of the figures show the binding curve (Figure 7A) and activity curve (Figure 7B) for the HIV protease inhibitor and pepsin. Figures 8A–8D are schematic diagrams of the active site with the bound HIV protease inhibitor, showing potential hydrogen-bonding interactions with green dashed lines. The electron density maps were generated using POVSCRIPT and POV-Ray, and the schematics were created using MarvinSketch and Adobe Illustrator. [Figure 8B] Figures 8A–8D show structural data for pepsin and HIV protease inhibitors. The left panels of Figures 8A–8D show the active site of porcine pepsin with the bound HIV protease inhibitor. The 2Fo-Fc electron density map, contoured at 1.0σ, is shown as a magenta mesh, and the 2Fo-Fc simulated annealing composite omit map, also contoured at 1.0σ, is shown as a green mesh. The right panels of the figures show the binding curve (Figure 7A) and activity curve (Figure 7B) for the HIV protease inhibitor and pepsin. Figures 8A–8D are schematic diagrams of the active site with the bound HIV protease inhibitor, showing potential hydrogen-bonding interactions with green dashed lines. The electron density maps were generated using POVSCRIPT and POV-Ray, and the schematics were created using MarvinSketch and Adobe Illustrator. [Figure 8C]Figures 8A–8D show structural data for pepsin and HIV protease inhibitors. The left panels of Figures 8A–8D show the active site of porcine pepsin with the bound HIV protease inhibitor. The 2Fo-Fc electron density map, contoured at 1.0σ, is shown as a magenta mesh, and the 2Fo-Fc simulated annealing composite omit map, also contoured at 1.0σ, is shown as a green mesh. The right panels of the figures show the binding curve (Figure 7A) and activity curve (Figure 7B) for the HIV protease inhibitor and pepsin. Figures 8A–8D are schematic diagrams of the active site with the bound HIV protease inhibitor, showing potential hydrogen-bonding interactions with green dashed lines. The electron density maps were generated using POVSCRIPT and POV-Ray, and the schematics were created using MarvinSketch and Adobe Illustrator. [Figure 8D] Figures 8A–8D show structural data for pepsin and HIV protease inhibitors. The left panels of Figures 8A–8D show the active site of porcine pepsin with the bound HIV protease inhibitor. The 2Fo-Fc electron density map, contoured at 1.0σ, is shown as a magenta mesh, and the 2Fo-Fc simulated annealing composite omit map, also contoured at 1.0σ, is shown as a green mesh. The right panels of the figures show the binding curve (Figure 7A) and activity curve (Figure 7B) for the HIV protease inhibitor and pepsin. Figures 8A–8D are schematic diagrams of the active site with the bound HIV protease inhibitor, showing potential hydrogen-bonding interactions with green dashed lines. The electron density maps were generated using POVSCRIPT and POV-Ray, and the schematics were created using MarvinSketch and Adobe Illustrator. [Figure 9A-D]Figures 9A-9H show in vivo damage to laryngeal epithelium by pepsin and acid. These are representative specimens from the treatment groups. Paired images at 40x magnification (Figures 9A-9D) and 200x magnification (Figures 9E-9H) were collected from the anterior portion of the vocal cords, representing the larynx: pH 7 (Figures 9A and 9E), pH 4 (Figures 9B and 9F), pH 7 with 0.3 mg / ml pepsin (Figures 9C and 9G), and pH 4 with 0.3 mg / ml pepsin (Figures 9D and 9H). (Figures 9A and 9E) Normal respiratory columnar epithelium approximately one cell layer thick with basal nuclear polarization and a ciliated apical surface (arrows). (Figures 9B and 9F) Reactive epithelium is characterized by thickened epithelium (thick arrows) and focal squamous epithelium (long arrows) 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, g) Thickened respiratory epithelium with pseudostratification of epithelial cells. Keratinization (arrows) is present in multiple foci. In some areas of this treatment group, a significantly increased N:C ratio is evident, accompanied by loss of nuclear polarization and a reduction in apical cilia. (d, h) The respiratory epithelium is necrotic (arrows) and replaced by inflammatory exudate. An active, acute inflammatory infiltrate infiltrates the submucosa. Scale bars are 100 μm in Figures 9A-9D and 50 μm in Figures 9E-9H. [Figure 9E-F]Figures 9A-9H show in vivo damage to laryngeal epithelium by pepsin and acid. These are representative specimens from the treatment groups. Paired images at 40x magnification (Figures 9A-9D) and 200x magnification (Figures 9E-9H) were collected from the anterior portion of the vocal cords, representing the larynx: pH 7 (Figures 9A and 9E), pH 4 (Figures 9B and 9F), pH 7 with 0.3 mg / ml pepsin (Figures 9C and 9G), and pH 4 with 0.3 mg / ml pepsin (Figures 9D and 9H). (Figures 9A and 9E) Normal respiratory columnar epithelium approximately one cell layer thick with basal nuclear polarization and a ciliated apical surface (arrows). (Figures 9B and 9F) Reactive epithelium is characterized by thickened epithelium (thick arrows) and focal squamous epithelium (long arrows) 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, g) Thickened respiratory epithelium with pseudostratification of epithelial cells. Keratinization (arrows) is present in multiple foci. In some areas of this treatment group, a significantly increased N:C ratio is evident, accompanied by loss of nuclear polarization and a reduction in apical cilia. (d, h) The respiratory epithelium is necrotic (arrows) and replaced by inflammatory exudate. An active, acute inflammatory infiltrate infiltrates the submucosa. Scale bars are 100 μm in Figures 9A-9D and 50 μm in Figures 9E-9H. [Figure 9G-H]Figures 9A-9H show in vivo damage to laryngeal epithelium by pepsin and acid. These are representative specimens from the treatment groups. Paired images at 40x magnification (Figures 9A-9D) and 200x magnification (Figures 9E-9H) were collected from the anterior portion of the vocal cords, representing the larynx: pH 7 (Figures 9A and 9E), pH 4 (Figures 9B and 9F), pH 7 with 0.3 mg / ml pepsin (Figures 9C and 9G), and pH 4 with 0.3 mg / ml pepsin (Figures 9D and 9H). (Figures 9A and 9E) Normal respiratory columnar epithelium approximately one cell layer thick with basal nuclear polarization and a ciliated apical surface (arrows). (Figures 9B and 9F) Reactive epithelium is characterized by thickened epithelium (thick arrows) and focal squamous epithelium (long arrows) 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, g) Thickened respiratory epithelium with pseudostratification of epithelial cells. Keratinization (arrows) is present in multiple foci. In some areas of this treatment group, a significantly increased N:C ratio is evident, accompanied by loss of nuclear polarization and a reduction in apical cilia. (d, h) The respiratory epithelium is necrotic (arrows) and replaced by inflammatory exudate. An active, acute inflammatory infiltrate infiltrates the submucosa. Scale bars are 100 μm in Figures 9A-9D and 50 μm in Figures 9E-9H. [Figure 10A]Figures 10A and 10B show that oral administration and aerosol of fosamprenavir and aerosol of darunavir protect against pepsin-induced laryngeal injury in vivo. Representative specimens are shown at 400x magnification. The laryngeal epithelium in the vehicle control group was characterized by a single layer of respiratory epithelium without reactive changes. Mice treated with pepsin pH 7 exhibited reactive epithelial changes and apoptotic debris in the laryngeal epithelium. Normal histological findings were observed in mice receiving oral administration or aerosol of fosamprenavir with saline (vehicle) or oral administration or aerosol of fosamprenavir with pepsin pH 7. Fosamprenavir oral administration and aerosol protected against pepsin-mediated laryngeal injury. Oral darunavir induced a mild reaction (rare appearance of intraepithelial lymphocytes) in the saline-treated group. The same was true for the oral darunavir with pepsin pH 7 group. Darunavir aerosol provided mild protection against pepsin-mediated injury. Although epithelial damage was still present (a mild increase in intraepithelial inflammatory cells and reactive epithelial cells), no apoptosis was observed. Scale bar = 200 μm. [Figure 10B] Figures 10A and 10B show that oral administration and aerosol of fosamprenavir and aerosol of darunavir protect against pepsin-induced laryngeal injury in vivo. Representative specimens are shown at 400x magnification. The laryngeal epithelium in the vehicle control group was characterized by a single layer of respiratory epithelium without reactive changes. Mice treated with pepsin pH 7 exhibited reactive epithelial changes and apoptotic debris in the laryngeal epithelium. Normal histological findings were observed in mice receiving oral administration or aerosol of fosamprenavir with saline (vehicle) or oral administration or aerosol of fosamprenavir with pepsin pH 7. Fosamprenavir oral administration and aerosol protected against pepsin-mediated laryngeal injury. Oral darunavir induced a mild reaction (rare appearance of intraepithelial lymphocytes) in the saline-treated group. The same was true for the oral darunavir with pepsin pH 7 group. Darunavir aerosol provided mild protection against pepsin-mediated injury. Although epithelial damage was still present (a mild increase in intraepithelial inflammatory cells and reactive epithelial cells), no apoptosis was observed. Scale bar = 200 μm. [Figure 11] Figure 11 is the certificate of analysis for 1 kg. [Figure 12] Figure 12 is the Certificate of Analysis for 250g. DETAILED DESCRIPTION OF THE INVENTION

[0015] [Detailed explanation] In this application, the inventors disclose a novel means for treating reflux conditions, including airway reflux diseases such as GERD and laryngopharyngeal reflux disease (LPR). The deleterious pharyngeal changes observed in LPR occur after direct contact of the mucosa with refluxed gastric contents consisting of acid, pepsin, bile, and pancreatic enzymes.

[0016] This application provides an oral alginate formulation that provides sustained release and relieves one or more symptoms of reflux in a subject. This novel approach is suitable for localized treatment of easily accessible airways affected by LPR, allowing for reduced dosages. The benefits are clear: targeted delivery improves efficacy and limits systemic side effects.

[0017] Here, therapeutic compounds were screened for pepsin binding and inhibition. Specific HIV protease inhibitors that inhibit pepsin were administered orally and by inhalation to a mouse model of LPR to evaluate their potential for treating LPR. An oral alginate formulation was developed that provided sustained release for optimal delivery.

[0018] Local esophageal treatment of GERD has been an area of interest for decades, but drug delivery to the esophagus is challenging due to the extremely short esophageal transit time of orally administered drugs (less than 16 seconds, even in the supine position). To overcome this limitation, thickeners and mucoadhesive materials that extend the esophageal residence and contact time of liquid suspensions have been investigated. Previous efforts have primarily focused on materials that coat the esophagus and provide local protection from refluxed acid, a concept Potts et al. coined "esophageal bandages." This research has led to numerous patents on mucoadhesive carbonate formulations (reviewed by Batchelor 2005). Fosamprenavir, retained in the esophagus, inactivates extracellular, mucosally-bound pepsin deposited during reflux. Mucosal absorption is enhanced by extended contact time, which also inactivates intracellular pepsin. A desirable formulation would allow a portion of the total administered dose to be absorbed in the esophagus without significantly impeding systemic delivery via the intestine.

[0019] Viscous and mucoadhesive formulations that remain in the esophagus have been investigated for the diagnosis of Barrett's esophagus, the treatment of esophageal cancer and candidiasis, and the local delivery of drugs for the treatment of GERD and associated pain and inflammation. Among mucoadhesive excipients, alginate has emerged as the additive of choice for extending the esophageal residence time of liquid and solid pharmaceutical formulations.

[0020] Alginate is widely used as a bioadhesive polymer for drug delivery because it is nontoxic, biocompatible, nonimmunogenic, biodegradable, mucoadhesive, readily available, and cost-effective. The U.S. FDA has recognized alginate as "Generally Regarded as Safe" (GRAS), meaning it is recognized by qualified experts as safe for use in food, and it is listed in Title 21 of the Code of Federal Regulations, Parts 182 and 184. Alginate's pH-dependent gelation and chemical versatility, which allow it to be modified to tailor its properties, have made it one of the most extensively studied mucoadhesive biomaterials and it has been used in a variety of engineered drug delivery systems, including hydrogels, microparticles, nanoparticles, and adhesive tablets and films for oral drug delivery.

[0021] Our study demonstrated the in vitro esophageal retention characteristics of several pharmaceutical coating materials commonly used in solid pharmaceutical formulations. Sodium alginate (1.5% w / w, medium-viscosity grade derived from Mactocystis pyrifera) was found to exhibit excellent esophageal adhesion and to be capable of "self-repair" (i.e., re-adhesion at the next contact point after detachment). Batchelor et al. demonstrated that alginate (2% w / v, i.e., 0.02 mg in a 1 ml dose) adheres to the esophageal mucosa for up to 60 minutes in an in vitro model. Using an excised porcine esophagus and a saliva lavage solution simulating a constant flow of human saliva at 1 ml / min, they found that low-molecular-weight alginates (<75 kDa) with low viscosity (<0.02 Pas) had significantly lower esophageal retention than the other alginates tested. Meanwhile, high-viscosity alginates (>2.93 Pas) exhibited significant esophageal retention at 3 minutes and comparable retention to medium-viscosity alginates at 15 and 30 minutes. The G / M ratio of alginate did not affect esophageal adhesion. Alginate with a medium molecular weight (240 kDa), viscosity (0.51 Pas), and G / M ratio (44 / 56) showed a retention rate of 21.9 ± 9.5% at 30 min when administered at a dose of 0.02 mg in 1 ml of water.

[0022] Thus, medium viscosity sodium alginate provided in our formulation at 24.5 mg MDE per 10 ml twice daily would be predicted to result in 20% retention of bound drug (and ∼5 mg alginate retention) in the esophagus for at least 30 minutes.

[0023] [Treatment effect on LPR] High doses of alginate, such as those found in alginate antacids (1000 mg per dose), have been used for many years as monotherapy for mild to moderate GERD and as adjunctive therapy for episodic symptoms in patients taking PPIs. The therapeutic effect of alginate is primarily due to its raft-forming properties. Alginate rafts float on top of gastric contents, thereby mobilizing the acid pocket near the gastroesophageal junction after a meal and effectively reducing acid reflux. A secondary mechanism of anti-reflux activity is thought to be mucoadhesion. Mucoadhesion prevents pepsin and acid from diffusing into the esophageal submucosa, causing concomitant epithelial barrier dysfunction and enzyme inhibition of pepsin. In particular, Chater et al. found that a medium-viscosity alginate (LF120) inhibited pepsin by 28.46 ± 10.68% at only 0.68 mg / ml (44.73 ± 10.98% at 1.36 mg / ml). This suggests that the excipient alginate in MDE (2.45 mg / ml, twice daily) may have anti-digestive properties. However, the therapeutic value of alginate at such low concentrations is considered low, given numerous clinical trials and meta-analyses reporting the therapeutic efficacy of an anti-reflux medication containing 1000 mg of sodium alginate per 10 ml dose (Gaviscon Advance, Reckitt Benckiser, Slough, UK) compared with a viscosity-matched placebo or a product intended primarily as an antacid (Gaviscon tablets or antacid liquid, GlaxoSmithKline Consumer Healthcare, Pennsylvania), which provides only temporary symptomatic relief regardless of the presence of alginate excipient concentrations. The lack of therapeutic efficacy of the latter is thought to be due to an inability to form cohesive rafts.

[0024] Alginate-based antireflux medications have recently demonstrated therapeutic efficacy for the throat symptoms of LPR. Regarding esophageal symptoms, the therapeutic effect is thought to be due to gastric raft formation, especially considering the low likelihood of contact between orally administered alginate and the larynx. McGlashan et al. 27 investigated the efficacy of alginate on LPR symptoms and endoscopic findings in 49 patients with confirmed RSI and RFS. Patients were randomly assigned to receive alginate suspension (n = 24, 10 ml, 4 times daily after meals and before bedtime, Gaviscon Advance) or no treatment (n = 25, control group). Pretreatment mean (SD) RSI and RFS scores were similar in the treatment (23.9 (7.0) and 10.4 (3.6)) and control (24.6 (7.4) and 10.3 (3.3)) groups, but alginate treatment significantly improved LPR symptoms and findings after 2 months (11.2 (7.0) vs. 16.8 (6.4), P = 0.005) and 6 months (11.2 (8.1) vs. 18.3 (9.4), P = 0.008) and RFS at 6 months (7.1 (2.8) vs. 9.5 (3.4), P = 0.005). Similarly, in a study of personalized treatment for LPR subtypes (acidic to alkaline), Lechien et al. 26 found that an anti-reflux diet and administration of alginate (Gaviscon Advance) or magaldrate (Riopan, Takeda, Zaventem, Belgium) three times daily after meals improved voice quality in patients with alkaline LPR (n = 48) confirmed by HEMII-pH testing. Improvement was indicated by a reduction in dysphonia and voice roughness scores (GRBAS scale), jitter, shimmer, and noise-to-harmonic ratio (Lechien et al. 2021).

[0025] The present invention provides a sustained-release formulation of oral fosamprenavir that uses sodium alginate to enhance mucoadhesion and prolong drug delivery in the esophagus, improving esophageal symptoms in 25-50% of LPR patients who also have GERD, and therefore has superior efficacy to oral fosamprenavir / Lexiva.

[0026] In one embodiment, the formulation contains a low excipient level of sodium alginate to enhance mucoadhesion and thereby prolong drug delivery to the esophagus. This is expected to be beneficial for pepsin-mediated esophageal inflammation, mucosal damage, and related conditions. While high doses of alginate are expected to have a therapeutic effect through raft formation, low-dose formulations of the present invention are expected to enhance mucoadhesion and prolong esophageal retention.

[0027] In vitro tests for esophageal retention include mucoadhesive texture analyzers.

[0028] Studies combining multichannel intraluminal impedance and pH (MII-pH) monitoring have shown that many episodes of LPR are nonacidic, and that both weakly acidic and nonacidic reflux are associated with persistent symptoms in acid-suppressed patients (39-42). Pepsin, the main digestive enzyme in the stomach, is increasingly suspected of contributing to the damage and inflammation associated with LPR (17-23). Importantly, the stomach and esophagus possess internal defenses against pepsin, such as mucus, peristalsis, and bicarbonate secretion, whereas laryngeal tissue lacks them (26). In the airway, where pH is neutral (<8), pepsin is enzymatically inactive but stable. However, once pepsin is internalized by laryngeal and hypopharyngeal cells via receptor-mediated endocytosis, it is retained in low-pH intracellular vesicles, where it is thought 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. For example, we have demonstrated that endocytosed non-acidic pepsin induces proinflammatory cytokine gene expression in hypopharyngeal cells. This response is similar to that occurring in reflux esophagitis, which contributes to disease severity in GERD patients (21, 31). Importantly, inhibition of pepsin's proteolytic activity (i.e., the use of pepstatin, curcumin, ecabet sodium, anthocyanins, or preincubation at pH 8.0 before lowering the pH to 7.0) has been shown to abolish this damage and inflammation (5, 7, 22, 33, 52, 54-56), making pepsin a promising therapeutic target for the treatment of airway reflux.

[0029] We believe that LPR depends more on pepsin-mediated damage than acid-mediated damage, and that drugs that specifically target pepsin should 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 (preventing reactivation in low-pH intracellular compartments) and (2) receptor antagonists (preventing 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.

[0030] In this application, the inventors screened therapeutic compounds for their ability to bind to pepsin and inhibit its enzymatic activity and identified specific HIV protease inhibitors with these capabilities (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 in the treatment of LPR. Using epidemiological data, the inventors 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 for the treatment of LPR. Of the 10 commercially available HIV protease inhibitors, the inventors confirmed that four (amprenavir, darunavir, ritonavir, and saquinavir) have the ability to bind to pepsin and inhibit its activity in vitro (Figures 7A and 7B). To test these drug candidates in vivo, we established a novel mouse model of LPR (Figure 1). These mice will be used to test the ability of HIV protease inhibitors to reduce pepsin-mediated laryngeal mucosal damage and inflammation. The mice will be administered HIV protease inhibitors both orally and by aerosol, and the results of systemic and local administration will be compared. 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 (Figures 5A and 5B).

[0031] [method:] The present invention provides a method for treating reflux, preferably airway reflux, in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a formulation comprising an HIV protease inhibitor and an alginate salt 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 into the esophagus), laryngopharyngeal reflux (LPR; reflux of gastric contents beyond the esophagus into the laryngopharynx), and esophagopharyngeal reflux (EPR; a condition similar to LPR but characterized by esophageal abnormalities). Reflux disease also includes gastroesophageal reflux disease (GERD), in which gastric contents reflux into the esophagus irritates the esophagus. The reflux patients treated here are preferably GERD patients who are unresponsive to protein pump inhibitor (PPI) therapy.

[0032] As used herein, the term "HIV protease inhibitor" refers to an antiviral drug that inhibits one or more HIV proteases. HIV protease inhibitors selectively bind to HIV protease and prevent viral replication by inhibiting the proteolytic cleavage of protein precursors required for the production of infectious viral particles. Suitable HIV protease inhibitors include those 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)propan-2-yl), and ribovir (IUPAC: 1,3-thiazol-5-ylmethyl)propan-2-yl). N-[(2S,3S,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-butylcarbamate) [(3aS,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-ylphenyl)methyl]hydrazinyl]-3,3-dimethyl-1-oxobutan-2-yl]carbamate), tipranavir (IUPAC: N-[3-[(1R)-1-[(2R)-4-hydroxy-6-oxo-2-(2-phenylethyl)-2-propyl-3H-pyran-5-yl]propyl]phenyl]-5-(trifluoromethyl)pyridine-2-sulfonamide), and cobicistat (IUPAC: 1,3-thiazol-5-ylmethyl The HIV protease inhibitors used in the present invention must be capable of binding to pepsin and inhibiting its enzymatic activity. 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.

[0033] Fosamprenavir is a prodrug of amprenavir, sold as a calcium salt by ViiV Healthcare under the trade names Lexiva (USA) and Telzir (Europe). The body metabolizes fosamprenavir to form the active form of the drug, amprenavir. Thus, administering amprenavir as a prodrug increases its availability in the body, acting like a sustained-release formulation. Furthermore, fosamprenavir has demonstrated excellent pharmacokinetics in mice and is already FDA-approved, allowing fosamprenavir to rapidly progress into pilot clinical trials. In some embodiments, the HIV protease inhibitors used in the compositions and methods described herein have IC values in the micromolar range (μm). 50 In some preferred embodiments, the HIV protease inhibitors used in the compositions and methods described herein have IC in the nanomolar range (nm). 50 I have.

[0034] In this method, the HIV protease inhibitor can be administered by any route effective for treating reflux, preferably airway reflux, and preferably provides a formulation for oral administration. As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical formulation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration. Administration can be continuous or intermittent.

[0035] In some embodiments, the HIV protease inhibitor is administered orally to treat reflux. For example, in some embodiments, the HIV protease inhibitor is administered at about 0.7 to 1.4 g twice daily (i.e., a dose that is FDA-approved and therefore safe for the treatment of HIV).

[0036] The methods of the present invention are used to treat reflux in a subject in need of treatment. In some embodiments, the reflux can be airway reflux. In other embodiments, the reflux can be GERD, preferably GERD in a subject that is unresponsive to proton pump inhibitors. 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 that is unresponsive to proton pump inhibitor (PPI) therapy.

[0037] As used herein, the terms "treat," "treating," or "treatment" refer to the management and care of a subject for the purpose of combating a disease, condition, or disorder. Treatment 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 a subject's airway. Treatment also includes reducing one or more symptoms of airway reflux, suitably 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 reducing one or more symptoms of GERD that are unresponsive to PPIs, such as one or more of the following: a burning sensation in the chest (heartburn) that usually occurs after meals and may be worse at night; chest pain; difficulty swallowing; regurgitation of food or sour liquids; or a feeling of a lump in the throat.

[0038] The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological or clinical result. The 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 or suppression 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 adequate to produce a desired effect, such as reducing 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 a visual examination of the larynx), esophageal multichannel intraluminal impedance and pH monitoring (MII-pH), reflux symptom score (RSS), reflux symptom assessment (RSA), or salivary pepsin activity. Alternatively, the response to treatment for airway reflux can be assessed by assessing 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 treatment has inactivated pepsin in the airways. This is currently a research tool for assessing in vivo efficacy.

[0039] Patients with reflux episodes caused by weakly acidic or non-acidic reflux often respond poorly to proton pump inhibitor (PPI) therapy, which suppresses acid production but does not affect pepsin activity. The methods of the present invention are particularly beneficial for this non-responsive patient group, who are in dire need of an alternative to PPIs. As used herein, the phrase "non-responsive to treatment" refers to a condition that does not respond to treatment. For example, a patient's reflux may be considered non-responsive 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), Reflux Finding Score (RFS), Esophageal Multichannel Intraluminal Impedance and pH Monitoring (MII-pH), Reflux Symptom Score (RSS), or Reflux Sign Assessment (RSA). See the Examples section for a detailed description of these measures. For example, an effective treatment is one that reduces the RSI and / or RFS to normal values (eg, RSI≦13, RFS≦7, or a combination thereof).

[0040] [Composition:] The present invention also provides a composition comprising an oral formulation of an HIV protease inhibitor and alginate salt and a pharmaceutically acceptable carrier.Commercially available HIV protease inhibitors are usually formulated as tablets or oral suspensions for systemic drug delivery.For example, in some embodiments, the composition is formulated for oral administration.

[0041] The compositions of the present invention may contain any pharmaceutically acceptable carrier that allows oral 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 include 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).

[0042] 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, detergents (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). Additionally, the compositions may be formulated for controlled or sustained release of the HIV protease inhibitor, for example, by formulation in a lipophilic depot (e.g., fatty acids, waxes, oils).

[0043] The compositions of the present invention may further comprise suspending agents, preservatives, sweeteners, flavoring agents, water, and combinations thereof. Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or may be provided as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats and oils), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophor, etc.). TM The pharmaceutical compositions may be prepared by conventional means using pharmaceutically acceptable additives such as hydroxybenzoates (e.g., methyl or propyl-p-hydroxybenzoates or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring agents, coloring agents, and sweeteners as needed. Preparations for oral administration may also be suitably formulated to give controlled release of the compound, as is well known.

[0044] The proposed formulation is shown in Table 1. The amount of API is expected to be approximately 20-30% of the total weight of the dry product formulation. Figures 11 and 12 are Certificates of Analysis for the compositions described herein.

[0045] [Table 1]

[0046] The compositions may be prepared in unit dosage form for administration to a subject. The amount and timing of administration will be left to the discretion of the treating clinician to achieve the desired result.

[0047] The HIV protease inhibitor contained in the composition of the present invention can be any HIV protease inhibitor suitable for treating airway reflux disease, as described above. In some embodiments, the HIV protease inhibitor contained 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.

[0048] While the invention has been described in terms of one or more preferred embodiments, it should be understood that many equivalents, alternatives, variations, and modifications, aside from those expressly described, are possible and within the scope of the invention.

[0049] It will be apparent to those skilled in the art that many additional modifications beyond those already described are possible without departing from the inventive concept. In interpreting this disclosure, all terms should be interpreted as broadly as possible within the context. Variations of the term "comprising" should be interpreted as a non-exclusive reference to an element, component, or step, such that the referenced element, component, or step can be combined with other elements, components, or steps not expressly referenced. Embodiments referred to as "comprising" certain elements are also considered to "essentially comprise" and "consist of" those elements. The terms "essentially comprise" and "consist" should be interpreted consistent with the interpretations of the MPEP and the relevant Federal Circuit Court. The transitional phrase "essentially comprise" limits the scope of a claim to certain materials or steps that "do not materially affect the basic and novel characteristics" of the claimed invention. "Consisting of" is a limiting term that excludes elements, steps, or ingredients not specifically recited in the claim. For example, "consisting of" with respect to a sequence refers to the sequence recited in SEQ ID NO. and to a larger sequence that may include the SEQ ID NO.

[0050] All references cited herein are expressly incorporated by reference in their entirety.

[0051] The present invention will be more fully understood in light of the following non-limiting examples. [Example]

[0052] Example 1: Oral and inhaled fosamprenavir reverses pepsin-induced damage in a mouse model of laryngopharyngeal reflux. Johnston, N., Samuels, TL, Goetz, CJ, Arnold, LA, Smith, BC, Seabloom, D., Wuertz, B., Ondrey, F., Wiedmann, TS, Vuksanovic, N., Silvaggi, NR, MacKinnon, AC, Miller, J., Bock, J., Blumin, JH (2022), "Oral and inhaled fosamprenavir reverses pepsin-induced injury in a mouse model of laryngopharyngeal reflux." The Laryngoscope. Incorporated by reference.

[0053] <method> Binding and Activity Assays To investigate whether HIV protease inhibitors bind and inhibit pepsin, we developed a fluorescence polarization-based assay that measures size-dependent molecular rotation and allows detection of degradation, binding, and dissociation events. 80 A competitive binding assay was designed using the subnanomolar affinity inhibitor pepstatin. 81Pepstatin-Alexa647 was synthesized by dissolving 1 mg of pepstatin A (Sigma-Aldrich) in a 50:50 mixture of dimethylformamide (DMF) and dimethyl sulfoxide (DMS), followed by the addition of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (0.6 mg) and trimethylamine (10 μL) in DMF. The mixture was stirred for 1 h, after which 1 mg of Alexa Fluor 647 cadaverine disodium salt (ThermoFisher Scientific) was added. After 2 h, the solvent was evaporated under high vacuum (35 °C), and the residue was partially dissolved in 10% methanol and transferred to a C18 cartridge (Waters Corporation, Milford, MA). Increasing percentages of methanol were used for elution. Pepstatin-Alexa647 was eluted with 45% methanol. An enzyme inhibition assay was designed using a casein substrate. 82 Bovine α-casein (Sigma-Aldrich, St. Louis, MO) was labeled with Alexa Fluor 647 carboxylic acid, succinimidyl ester (ThermoFisher Scientific, Waltham, MA) as described. 82 Briefly, the two were mixed in 0.1 M sodium bicarbonate at a label-to-protein ratio of 2.5 μg / mg for 15 minutes, and the labeled casein was separated from unbound label on a Sephadex G-25 (Sigma-Aldrich) column packed with 90x5 mm beads using a glass Pasteur pipette and eluted with dPBS pH 7.4 (ThermoFisher Scientific). 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).

[0054] The assay was optimized using 0.3–1000 μM unlabeled pepstatin, 100–500 nM pepstatin-Alexa647 or casein-Alexa647 probe, 0.003–3 U / μl porcine pepsin (Worthington Biochemical Corporation, Lakewood, NJ), and 5–37.5% DMSO in 0.1 M HCl, pH 1, containing 0.01% v / v Tween-20 (HIV protease inhibitor diluent) in a 20 μl volume in a 384-well black optical plate (Nunc, Roskilde, Denmark) and read on a BioTek Cytation 5 (BioTek Instruments, Winooski, VT) equipped with a far-red FP filter cube (excitation / emission 620 / 680 nm). An unlabeled pepstatin dose-response curve was used to confirm that the assay responded to pepsin inhibition. To evaluate HIV protease inhibitors, conditions yielding the largest dynamic assay range were used. For competitive binding assays, the assay conditions were 100 nM probe, 0.03 U / µL porcine pepsin A, and 37.5% DMSO; for pepsin activity assays, the assay conditions were 200 nM probe, 0.01 U / µL pepsin, and 5% DMSO. HIV protease inhibitors (amprenavir, ritonavir, lopinavir, saquinavir mesylate, nelfinavir mesylate hydrate, darunavir ethanolate, and indinavir sulfate hydrate, all from Sigma-Aldrich) were dissolved in DMSO and tested under optimized assay conditions across 3 log concentrations. Assays were performed in duplicate; triplicate reactions were read for 5 min and the mean mP plotted against probe concentration (binding assay), or read at 2 min intervals over 30 min and the mean mP plotted over time (activity assay). The half-maximal inhibitory concentrations (IC) of the inhibitors were determined. 50 ) is an online tool (https: / / icekat.herokuapp.com / icekat) 83 The mP was calculated from kinetic traces analyzed using the .mP was normalized to the blank (no inhibitor) and the percentage of binding or activity was derived.

[0055] (crystallization) Saturated solutions of HIV protease inhibitors (amprenavir, ritonavir, and darunavir ethanolate) were prepared in DMSO and centrifuged at 31,000 rcf for 10 minutes. The supernatant was added to pepsin (200 mg / ml in water) at 1.6% (v / v) FC. Due to its low solubility, the solvent for saquinavir mesylate was selected from a CryoSol screen (Molecular Dimensions, The Netherlands, OH). CryoSol mixture SM2 (composed 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) was selected to provide both high solubility and protein compatibility for the cocrystallization mixture. The supernatant of the saturated saquinavir solution in SM2 was mixed with pepsin at 5% FC (v / v). Crystallization conditions were optimized by screening 200 mg / ml pepsin with a Salt RX screen (Hampton Research, Viejo, CA). After 1 week at room temperature in 3.5 M ammonium chloride and 0.1 M sodium acetate trihydrate, pH 4.6, small bipyramidal crystals formed. This was used as a microseed stock and was then grown as previously described. 84 Co-crystallization with amprenavir, ritonavir, and darunavir ethanolate was performed according to the protocol described in [1]. Diffraction-quality crystals (triangular bipyramidal, approximately 200 × 100 × 100 μm) formed within 2–7 days after the addition of 2 μl of pepsin (180–210 mg / ml) and 1 μl of microseed solution, serially diluted 10–100 times in 3–4 M ammonium chloride and 0.1 M sodium acetate trihydrate, pH 4.6. The crystals were cryoprotected in 30% glucose, 5 M ammonium chloride, and 0.1 M sodium acetate trihydrate, pH 4.6, and immersed in liquid nitrogen. Co-crystallization with saquinavir was performed in 0.1 M acetic acid rather than sodium acetate trihydrate, which allows for the formation of large crystals without microseeds. The crystals were cryoprotected in 30% w / v glucose, 5 M ammonium chloride, and 0.1 M sodium acetate trihydrate, pH 4.6, and immersed in liquid nitrogen.

[0056] Diffraction data sets were collected at the Life Sciences Collaborative Access Team (LS-CAT) beamline at the Advanced Photon Source (APS) at Argonne National Laboratory equipped with MAR300CCD or Dectris Eiger 9M detectors, and data were collected using MOSFLM. 85 or HKL2000 86 Indexed, integrated, and scaled using

[0057] Specifically, for pepsin:amprenavir, a 1.9 Å diffraction dataset was collected on a MAR300 CCD detector at LS-CAT beamline 21-ID-F using a 50 x 50 μm beam with 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. The exposure time was 0.5 seconds. Diffraction data were indexed, integrated, and scaled using MOSFLM.

[0058] For pepsin:ritonavir, a 2.1 Å diffraction dataset was collected on a Dectris Eiger 9M detector at LS-CAT beamline 21-ID-D using a 50 × 50 μm beam with a wavelength of 1.12721 Å. 900 frames were collected from φ = 0 to 180°, oscillating at a rate of 1° / s and slicing five images per degree. The crystal-to-detector distance was 160 mm. Diffraction data were indexed, integrated, and scaled using MOSFLM.

[0059] For pepsin:darunavir, a 1.9 Å diffraction data set was collected at LS-CAT beamline 21-ID-G using a MAR300 CCD detector and a 50 × 50 μm beam of 0.97856 Å. 900 frames were collected from φ = 0 to 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.

[0060] For pepsin:saquinavir, a 1.9 Å diffraction dataset was collected at LS-CAT beamline 21-ID-F using a 50 × 50 μm beam at 0.97872 Å using a MAR300 CCD detector. 400 frames were collected in the range 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.

[0061] The initial phase is PHASER 87 The model was obtained by molecular replacement with . The B-factor was reset to 20.00 Å and the solvent molecules were removed from unliganded porcine pepsin (PDB ID 4PEP) as the search model. The model was refined using phenix.refine (PHENIX 87-89 ) and COOT 90,91 The geometric constraints of the compounds were performed using the CCP4 monomer library. 92 The model was taken from MolProbity, which is implemented in the PHENIX suite. 93 The ritonavir and saquinavir models were validated using the PDB-REDO server prior to deposition. 94 Electron density maps were generated by POVSCRIPT and POV-Ray, and graphical representations were generated by MarvinSketch (http: / / www.ChemAxon.com) and Adobe Illustrator CC2020.

[0062] (in vivo mouse model) Experiments were approved by the University of Minnesota (UMN) Animal Care and Use Committee (1712-35415A) and conducted at UMN. Three animal replicates per treatment condition were expected to be sufficient to verify the reproducibility of each experiment without excessive use of animal life. Three mice were randomly assigned to treatment groups. No data were excluded from analysis.

[0063] Six-week-old female Jackson A / J mice (Jackson Laboratory, Bar Harbor, ME) were fed 2 g of D-62 powdered Wattenberg chow per mouse per day. 95 The mice were allowed to acclimate for 1 week after arrival before the experiment. This is a previously established method to model aerodigestive tract damage caused by GERD and LPR. 1,95-99 According to

[14] , mechanical injury was administered during the first 2 weeks of a 4-week treatment course to sensitize the laryngeal mucosa to chemical injury with pepsin / acid administered throughout the 4 weeks. When performed in this manner, mechanical injury increases the sensitivity of the mucosa to subsequent chemical injury, but little detectable injury remains at the end of the 4-week treatment course. 95 Mechanical injury was performed on all animals (including controls) once a week during the first two weeks of treatment as described (see experimental scheme, Figure 6). 95 Briefly, anesthetized mice were suspended by their upper teeth on an inclined board under a surgical microscope. Under 6x magnification, a blunt, curved (135°) needle was drawn from distal to proximal to create a mild abrasion, injuring the subglottis, glottis, and supraglottis.

[0064] In preliminary experiments to validate the LPR mouse model (larynx injury by pepsin at neutral and acidic pH), mice (n = 3) were administered 20 μl of saline (solvent control) or 0.3 mg / ml pepsin (pH 7.0 or 4.0) by laryngeal instillation at 24, 48, and 72 hours after mechanical injury in weeks 1 and 2 (Figure 6). Laryngeal instillation without wounding (3 days per week) was continued in weeks 3 and 4. Mice were anesthetized with intraperitoneal avertin (2,2,2-tribromoethanol) 225–240 mg / kg prior to wounding and laryngeal instillation. Mice were sacrificed at the end of week 4.

[0065] To test the protective effect of HIV protease inhibitors against pepsin-mediated damage in vivo, we administered the inhibitors by aerosol or orally simultaneously with wounding (days 2 and 8) and vehicle / pepsin injection (days 3–5, 9–11, 16–18, and 23–25). Aerosol or oral administration occurred on days 1–5, 8–12, 15–19, and 22–25, and mice were sacrificed on day 26. Mice were anesthetized with isoflurane (3% in 2.5 LPM, 3–5 minutes before treatment) rather than Avertin due to frequency. Lexiva and Prezista (hereafter referred to as generic names fosamprenavir and darunavir, respectively) were used for oral administration, and the respective pure drugs were used for aerosol administration (fosamprenavir was manufactured by Anant Pharmaceuticals, Ambernath, Maharashtra, India; darunavir was manufactured by Ambeed, Arlington Heights, Illinois). Oral doses were equivalent to those prescribed for HIV patients (fosamprenavir 20 mg / kg / day, darunavir 8.6 mg / kg / day). Aerosols were generated as described. 100 Briefly, 10 ml of a drug suspension in ethanol was placed in a baffle to maintain a constant concentration at equilibrium solubility. Ethanol droplets containing the dissolved drug were generated using an ultrasonic nebulizer (nominal frequency 1.7 MHz) and entrained in air at a flow rate of 0.5 LPM through a custom-made glass baffle (UMN Chemistry Department Glass Shop). The aerosol cloud was 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. The mass deposited on the filter was weighed to determine 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). The inhaled mass of drug (M inh ) is M inhThe RMV was defined as = [aerosol] * RMV * t, where aerosol is the aerosol concentration of the drug, RMV is the respiratory volume per minute of the mouse (0.025 L / min), and t is the aerosol exposure time. The aerosol concentrations were 0.09 mg / L for fosamprenavir and 1.2 mg / L for darunavir. Considering the respiratory volume of the mouse (0.025 L / min), the inhaled mass was 0.93 mg / kg / day for fosamprenavir and 12 mg / kg / day for darunavir. The actual deposited mass was not measured but is expected to be 10% of the inhaled mass (the deposition rate of 1 μm aerosol particles in mice).

[0066] Tissues were collected, fixed in paraformaldehyde, embedded in paraffin, and 4-µm sections were stained with hematoxylin and eosin (H&E) using an automated stainer. Slides were examined by a board-certified pathologist (JM) who was blinded to the treatment groups.

[0067] <result> Binding and Activity Assays Of the seven HIV protease inhibitors assayed, four bound to and inhibited pepsin at low micromolar concentrations (Figures 7A and 7B): amprenavir, darunavir, ritonavir, and saquinavir. The in vitro activity of these four HIV protease inhibitors against pepsin provided the basis for further study.

[0068] (Structural Data) To aid in the interpretation of in vitro binding and inhibition data, commercially available porcine pepsin (EC 3.4.23.1) was used in co-crystallization experiments to obtain structural data. Crystallization of human pepsin collected from volunteers failed, likely due to sample heterogeneity. Porcine pepsin was synthesized from the human enzyme (PDB ID 1PSN). 101It shares 86% sequence identity with human pepsin, and its structure is nearly identical (root mean square deviation (RMSD) for all Cα atoms = 0.50 Å). The only minor differences in the tertiary structure are localized in a loop of residues (277-282) that are not part of the binding groove. Residues inside the active site groove are highly conserved, and of the 17 residues that make direct contact with the inhibitor, only two (T12 and V291) differ. Therefore, porcine pepsin was considered an acceptable substitute for human pepsin for evaluating its structural biology.

[0069] Porcine pepsin was co-crystallized with amprenavir, darunavir, ritonavir, and saquinavir (Table 2 and Figures 8A-8D). All are peptidomimetics. The alcohol of the central phenylalaninol residue, mimicking the tetrahedral intermediate of peptide bond cleavage, is bound between the catalytic aspartic acid residues D32 and D215. The binding directionality of each (the amino group of phenylalaninol on the prime side of the binding site) was the same as that of pepstatin 101. Binding relies on van der Waals contacts between the inhibitor side chain and residues lining the binding site, with a small number (5-6) of hydrogen bonds observed. For example, in the pepsin-ritonavir complex (Figure 8A), 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 contacts with I213, M289, V291, and I300. The thiazole and isopropylthiazole groups of ritonavir do not make stabilizing interactions with the active site. The electron density for these groups is correspondingly poorly defined, and the B factor, which reflects the precision of the atomic positions in these parts of the molecule, is very high. The structure of the pepsin-saquinavir complex (Figure 8B) is similar in that the side chain of the phenylalaninol residue interacts with the P1' subsite, but both ends of the molecule, the quinoline and decahydroisoquinoline moieties, also have low density and high B factors. The structures of amprenavir (Figure 8C) and darunavir (Figure 8D) follow the same pattern. The phenylalaninol residue in both inhibitors occupies the P1' site, interacting with I213, M289, V291, and I300. An 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 forms a hydrogen bond with the backbone amide of T77. The aniline group does not make polar contact with the active site. At the opposite end of the molecule, where the two compounds differ, 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. The structures and binding poses of amprenavir and darunavir are similar, resulting in IC. 50 did not explain the difference.

[0070] [Table 2] JPEG2025526506000004.jpg185160

[0071] (Mouse biological model) In a mouse in vivo model using pepsin with or without acid exposure after mechanical injury of the larynx, pepsin-mediated laryngeal epithelial damage was observed at pH 4 and 7 (Figures 9A-9H). Animals in the pH 7 control group had normal laryngeal epithelium 1-2 cells thick, with cilia present and no inflammation, keratinization, or necrosis. No mucosal damage was detected in the control group during the first 2 weeks of treatment or after mechanical injury with pH 7 solvent. The laryngeal epithelium in the pH 4 group was reactive, thickened (3-4 cells thick), and keratinized with loss of cilia. The laryngeal epithelium in the pepsin pH 7 group was moderately thick (2-3 cells), with evidence of keratinization, an increased nuclear-to-cytoplasmic ratio, and loss of polarity. The pepsin pH 4 group experienced complete epithelial loss due to necrosis and inflammatory cell infiltration.

[0072] Oral administration of fosamprenavir at doses equivalent to those used in human HIV treatment prevented pepsin-mediated laryngeal damage, defined as reactive epithelium, increased intraepithelial inflammatory cells, and apoptosis (Figures 10A and 10B). The mild response induced by oral darunavir (not seen in the darunavir aerosol group, Figures 9A–9H) made it difficult to detect the effect of darunavir on pepsin-mediated damage. Fosamprenavir aerosol prevented pepsin-mediated laryngeal damage (Figures 9A–9H). Darunavir aerosol provided moderate protection against pepsin-mediated damage. Although epithelial damage was present (a mild increase in intraepithelial inflammatory cells and reactive epithelial cells), apoptosis, as seen in mice treated with pepsin-pH7 and sham inhalation, was not observed.

[0073] <Consideration> For the past 20 years, treatment of LPR has focused on suppressing gastric acid production. With the introduction of MII-pH technology, it is now understood that LPR is generally non-acidic and that non-acidic proximal events are associated with signs and symptoms of laryngoscopy. 39-46,48-50,102 These findings stimulated investigation of the non-acidic components of gastric reflux fluid.

[0074] Experimentally, bile can cause mucosal damage at both slightly acidic and non-acidic pH, but "there is no evidence that the same mechanism occurs in the human larynx." 57 It has been argued that the clinical relevance of experimental findings has been questioned. Unconjugated bile acids cause damage in the laryngopharynx and other areas with neutral to high pH, but are rarely found in gastric reflux fluid. 56,69 Furthermore, the concentrations of bile salts / acids found experimentally to be damaging to the larynx and hypopharynx are 1000 times higher than those reported in the airways of patients with LPR, GERD, asthma, or pulmonary disease (0.3–50 mM). 96,103,104 0.8 to 32uM 105~109 ), "blebbing" of the cell membrane 110 These results in morphological changes that are inconsistent with those seen in LPR patients.

[0075] Pepsin is present in all reflux fluids 55Furthermore, pepsin is frequently detected in airway tissues and secretions from patients with LPR but is absent in subjects with no reflux confirmed by MII-pH, potentially predicting reflux-related symptoms and disease. 20,39,46,50,55,59,65,67,68,111,112 Pepsin in the stomach, 1 mg / ml, is diluted by saliva upon proximal reflux. Various concentrations have been reported in the respiratory tract: 2.5 μg / ml in saliva and 61.5 μg / ml in nasal secretions. 113,114 , and 360 μg / ml in middle ear fluid. 115 To model chronic LPR within a limited experimental time frame, 300 μg / ml was used here. 1,77,116,117 Pepsin-mediated injury and inflammatory changes have been reported in vitro and in vivo, including the histological changes reported in this study, and are consistent with those observed in patients with LPR. 62-64,66,70,118-122 Compelling evidence from multiple groups highlights the major role of pepsin, independent of gastric acid, in reflux laryngeal symptoms and findings that are unresponsive to PPI therapy.

[0076] Although pepstatin is a potent pepsin inhibitor, its poor water solubility and poor pharmacokinetic properties make it a suboptimal therapeutic candidate. Our structural data suggest that inhibitors that bind to the active groove of pepsin are primarily stabilized by van der Waals contacts, making rational inhibitor design challenging. Therefore, testing existing inhibitors of other aspartic proteases was deemed the most effective approach to identify therapeutic agents targeting pepsin.

[0077] There are currently 10 HIV protease inhibitors on the market. 123 Seven of these are testable in our in vitro binding inhibition assay, with four (amprenavir, ritonavir, saquinavir, and darunavir) having IC in the low micromolar range. 50We tested our hypothesis that existing therapeutic protease inhibitors may exhibit antipepsin activity by binding to and inhibiting pepsin in vitro. Two drugs were selected for in vivo studies based on their antipepsin activity in in vitro assays, cost, and reported side effects. Saquinavir has known side effects and interactions (QT prolongation, heart block, elevated blood lipids, liver damage) and is costly, whereas amprenavir, ritonavir, and darunavir have minimal side effects (diarrhea, nausea, and vomiting). 123 Darunavir is more expensive than amprenavir and ritonavir, but has a higher IC against pepsin. 50 Therefore, IC 50 Darunavir, which has the lowest GER, and fosamprenavir, a prodrug of amprenavir with improved bioavailability and good tolerability, were selected for in vivo evaluation. 124 Given the inconsistency of proximal reflux in previous surgical models, we employed a model involving mechanical wounding and pepsin / acid injection. This model is consistent with the LPR model. 1,63,70,118,119,125 This reliably reproduces epithelial changes similar to those observed in patients with rheumatoid arthritis. Using this model, human-equivalent doses of fosamprenavir, but not darunavir, prevented pepsin-mediated laryngeal damage. When administered topically by inhalation, treatment with either compound maintained normal laryngeal histology despite exposure to pepsin.

[0078] The present study aimed to investigate whether pepsin inhibitors could prevent laryngeal damage caused by pepsin exposure in vivo. As with any experimental observation, caution is required when extrapolating in vivo findings from a limited number of animals to the clinical situation. Potential differences in respiratory pathobiology between mice and humans must be kept in mind when evaluating the clinical implications of these data. Here, we investigated the role of pepsin inhibitors in preventing laryngeal damage caused by pepsin exposure in vivo. 1,96-99Established methods for in vivo modeling of aerodigestive tract injury caused by LPR were used to demonstrate mucosal damage consistent with clinical findings in LPR, supporting the use of these methods in evaluating pharmacological prevention of LPR-induced injury. At the end of 4 weeks of treatment, no mucosal damage was detected considering mechanical and neutral solvent treatments, but multilayered reactive epithelium with apoptosis was observed in the pepsin- and acid-treated groups. The mouse epiglottis occupies the transitional region from stratified squamous epithelium of the vocal fold to ciliated pseudostratified columnar epithelium in the supraglottis and subglottis. To avoid misinterpreting the squamous epithelium of the vocal fold as a sign of injury, representative images were collected anterior to the vocal fold, only from tissue with a visible thyroid gland as a guide. Additional features of reactive epithelium in the pepsin-treated group (nuclear darkening, changes in nuclear diameter, increased nucleus-to-cytoplasm ratio, intraepithelial inflammatory cells, and apoptosis) were not observed in the pH 7.0 control and fosamprenavir- or darunavir-treated groups, confirming pepsin-induced epithelial reactivity and the efficacy of HIV protease in preventing pepsin-mediated damage. While these data are qualitative and supported by less subjective quantitative measures, the evidence here provides initial proof-of-concept that therapeutics targeting pepsin may attenuate mucosal damage similar to that seen in LPR patients and supports more detailed investigation. Our laboratory is currently conducting studies to examine the protective effects of fosamprenavir on pepsin-mediated changes in laryngeal cell viability and inflammatory and oncogenic gene and protein expression. Further studies are also needed to determine whether in vivo laryngeal protection by fosamprenavir aerosol is due to systemic effects or local conversion to fosamprenavir. Fosamprenavir is primarily metabolized in the intestine. Conversion of fosamprenavir to amprenavir by alkaline phosphatase (ALP), which is required for its epithelial penetration and subsequent metabolism by cytochrome P450 enzymes, has been shown to occur via intestinal ALP at or near the surface of Caco-2 cells. 128,129However, inhaled fosamprenavir may be converted to amprenavir in the airways by serum alkaline phosphatase (ALP), similar to the conversion of similar phosphate prodrugs by serum from healthy subjects. Inhaled fosamprenavir may also be converted by salivary alkaline phosphatase (ALP) or by ALP expressed by immune cells recruited to the respiratory mucosa and tissue injury. ALP is a key regulator of inflammation. 132-134 and increases during carcinogenesis, such as laryngeal carcinogenesis, in which LPR is involved. 10,74,135-137 In airways damaged by LPR, ALP is elevated, potentially increasing the conversion of fosamprenavir at the intended site of action. Drug formulations that extend residence time in the aerodigestive tract may further improve local drug conversion and activity. Our laboratory is currently conducting studies to examine the efficiency of fosamprenavir conversion by laryngeal epithelium, saliva, and serum, and a dose-response study comparing the relative efficacy of inhaled fosamprenavir and fosamprenavir against pepsin-mediated injury is underway in an in vivo mouse model.

[0079] Although additional experimental data will contribute to our understanding of fosamprenavir's laryngeal protection, improvement of LPR symptoms is the ultimate determinant of drug therapy success. Therefore, a randomized, placebo-controlled trial would be the best test of a therapeutic compound. Given that the oral formulation is FDA-approved and a pre-response definition of clinically meaningful symptom improvement has been established according to FDA guidelines, such a trial of fosamprenavir is feasible. 138 Interestingly, pilot epidemiological data (unpublished) support the therapeutic potential of HIV protease inhibitors for LPR, which warrants follow-up. Among 2,062 adult HIV patients prescribed HIV protease inhibitors (Froedtert Memorial Lutheran Hospital, Milwaukee, Wisconsin, July 2014–2016, Medical College of Wisconsin Institutional Review Board, 13874), only 0.2% had documented LPR, compared with a general population incidence of 10–34%. 139,140These data preliminarily support the clinical investigation of fosamprenavir as a new treatment for LPR.

[0080] [Conclusion] Compelling evidence highlights the critical role of pepsin (independent of gastric acid) in reflux-related laryngeal symptoms and endoscopic findings unresponsive to PPI therapy. The FDA-approved HIV / AIDS retroviral therapies, fosamprenavir and darunavir, bind to and inhibit pepsin, suppressing pepsin-mediated laryngeal inflammation and mucosal damage in the LPR mouse model. Because these drugs target a foreign virus, they are ideal for repurposing, allowing clinical trials to assess the efficacy of much-needed treatments for patients more rapidly than can be achieved with novel compounds. Reformulation for local inhaled delivery may further improve outcomes and limit side effects.

[0081] Data availability Structural data are available in the Worldwide Protein Data Bank (accession codes 6XCY, 6XCT, 6XCZ, 6XD2, http: / / www.wwpdb.org / ).

[0082] [array] SEQ ID NO: 1 Synthetic peptide substrate for pepsin Lys-Pro-Ala-Glu-Phe-PNP-Arg-Leu (PNP = paranitrophenylalanine)

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[0084] [Additional Information] The following disclosures can be explained according to the following numbered clauses:

[0085] Clause 1. An oral sustained release formulation for the treatment of reflux disease, comprising an effective amount of an HIV protease inhibitor, sodium alginate, and a pharmaceutically acceptable carrier.

[0086] Clause 2. The oral sustained release formulation of clause 1, wherein the HIV protease inhibitor and the sodium alginate form provide sustained release for at least 30 minutes.

[0087] Clause 3. The oral formulation of clause 1 or 2, wherein said HIV protease inhibitor is amprenavir, darunavir, ritonavir, saquinavir, or any combination thereof.

[0088] Clause 4. The oral formulation of any of the preceding clauses, wherein the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir.

[0089] Clause 5. The oral formulation of any one of the preceding clauses, wherein the formulation comprises one or more of a suspending agent, a preservative, a sweetener, a flavoring agent, water, and combinations thereof.

[0090] Clause 6. An oral formulation according to any one of the preceding clauses, wherein said formulation is a liquid.

[0091] Clause 7. A method of treating reflux disease in a subject in need thereof, comprising orally administering to said subject a formulation according to any one of clauses 1 to 5 for the treatment of reflux disease.

[0092] Clause 8. The method of clause 7, wherein the HIV protease inhibitor is capable of binding to pepsin and inhibiting its enzymatic activity.

[0093] Clause 9. The method of any one of clauses 7-8, wherein the HIV protease inhibitor is administered twice daily at a dose of about 1.4 g or less.

[0094] Clause 10. The method of any one of clauses 7 to 9, wherein the subject suffers from an airway reflux disease selected from laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), and esophagopharyngeal reflux (EPR).

[0095] Clause 11. The method of clause 10, wherein the subject's condition is refractory to treatment with a proton pump inhibitor (PPI).

[0096] Clause 12. The method of any one of clauses 7 to 10, wherein said method reduces damage and inflammation of the laryngeal mucosa.

[0097] Clause 13. The method of any one of clauses 7 to 12, wherein the subject suffers from gastroesophageal reflux disease (GERD), preferably GERD refractory to proton pump inhibition.

[0098] Clause 14. Use of the composition of any one of clauses 1 to 5 for the treatment of reflux in a subject in need thereof, wherein said subject has a condition selected from the group consisting of laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), esophagopharyngeal reflux (EPR), or GERD unresponsive to protein pump inhibition.

Claims

[Claim 1] An effective dose of HIV protease inhibitor, Sodium alginate and a pharmaceutically acceptable carrier, An oral sustained-release formulation for the treatment of reflux disease, including [specifically, a product name].

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