Sustained release oral fosamprenavir formulations for treatment of reflux

Abstract

The present invention provides methods of treating respiratory tract regurgitation using HIV protease inhibitors capable of binding to pepsase and inhibiting enzymatic activity thereof. Also provided are compositions comprising the HIV protease inhibitor sustained release formulations for oral administration.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 392,929, filed on July 28, 2022, the entire contents of which are incorporated herein by reference.

[0003] A note about federally funded research

[0004] none

[0005] Sequence Listing

[0006] A sequence listing is attached to this application and is submitted as an XML file named "650053_00979_Sequence_Listing", 2,375 bytes in size, and created on July 25, 2023. The sequence listing is submitted electronically with this application through Patent Center, and its entire contents are incorporated herein by reference. Technical Field

[0007] Laryngopharyngeal reflux (LPR), the reflux of gastric contents into the laryngopharynx, is a significant health problem that affects both children and adults and has a wide range of clinical manifestations.

[0008] More than 20% of the US population suffers from laryngopharyngeal reflux. While some benefit from dietary / lifestyle changes and alginate, there is no gold standard medical therapy. There is growing evidence that the damage and inflammation caused by laryngopharyngeal reflux is due in part, if not entirely, to pepsin. Treatments that specifically target pepsin are amenable to local inhalation delivery and have been shown to be potentially effective for endoscopic signs and symptoms associated with non-acid reflux.

[0009] Laryngopharyngeal reflux (LPR), the reflux of gastric contents into the laryngopharynx, is a significant health problem. Both children and adults are affected by LPR, and the clinical presentation of the disease is wide-ranging. Unlike patients with gastroesophageal reflux (GER), which is confined to the esophagus, many patients with LPR do not have symptoms of acid dyspepsia but instead have symptoms caused by chronic laryngeal irritation, such as chronic cough, throat clearing, postnasal drip, dysphonia, globus, dysphagia, and dyspnea. Substantial evidence suggests that chronic LPR can lead to serious and life-threatening conditions, including airway stenosis, reactive airway disease, and laryngeal cancer. It is estimated that LPR affects more than 20% of the U.S. population and accounts for 10% of otolaryngology visits. The economic burden of LPR exceeds $52 billion per year, 5.6 times that of GER; 52% of this burden is attributable to proton pump inhibitors (PPIs).

[0010] Although PPI therapy is the mainstay of treatment for GER disease (GERD), it has poor efficacy for LPR. In clinical practice, it is believed that patients with reflux laryngitis require a higher dose and longer PPI trial than patients with typical GERD, given that the upper airway is more sensitive than the esophagus to gastric acid reflux. However, placebo-controlled trials have failed to demonstrate a therapeutic benefit of PPIs. Although Reichel et al and Lam et al reported symptom improvement in randomized, double-blind, placebo-controlled trials, Vaezi suggested that the improvement was in heartburn rather than laryngeal symptoms. A higher proportion of laryngeal symptoms have been reported in patients with GERD than in those without GERD. Due to the lack of data supporting acid suppression for extraesophageal symptoms, the American College of Gastroenterology's GERD guidelines recommend against its use for the acute treatment of patients with underlying extraesophageal reflux (EER) syndrome (laryngitis, chronic cough) who do not have typical GERD symptoms. Despite this recommendation, LPR is often treated empirically with PPIs.

[0011] Although the acidity of reflux itself can damage the upper airway, esophageal multichannel intraluminal impedance combined with pH (MII-pH) monitoring has shown that many LPR episodes are nonacidic, and both weakly acidic and nonacidic reflux are associated with persistent symptoms in acid-suppressed patients. These symptoms can be relieved by antireflux surgery and can be improved by noninvasive strategies that limit reflux occurrence or neutralize reflux components other than acid, such as dietary and lifestyle changes and over-the-counter alginate products. Therefore, one or more of the nonacidic components of gastric reflux must cause damage to the larynx. There is growing evidence that the damage and inflammation caused by LPR are due in part, if not entirely, to pepsin, which is present in all refluxate.

[0012] Pepsin is a proteolytic enzyme that is synthesized and secreted by fundic chief cells as the proenzyme pepsinogen, which is subsequently cleaved upon entry into the acidic gastric lumen to produce pepsin. Pepsin is maximally active at pH 2 and remains active until pH 6.5. Although stable at pH 8, pepsin is irreversibly inactivated at higher pH. The stomach and esophagus have intrinsic defenses against pepsin (mucus, peristalsis, and bicarbonate secretion), but laryngeal tissues do not. Pepsin is thought to play a key role in mucosal injury and inflammation during nonacidic reflux. At neutral pH, laryngeal and hypopharyngeal cells take up pepsin via receptor-mediated endocytosis and are retained in low-pH intracellular vesicles where it is presumed to be reactivated. The result is chronic inflammation, which in turn induces symptoms. Internalized nonacidic pepsin induces a proinflammatory cytokine gene expression profile in hypopharyngeal cells that is similar to that contributing to disease severity during GERD. Inhibition of the proteolytic activity of pepsin abrogates this injury and inflammation.

[0013] Given the strong evidence for proximal reflux of nonacidic pepsin and its association with laryngeal and pharyngeal symptoms and endoscopic findings, and the substantial costs and risks of long-term PPI therapy (despite the ineffectiveness of such therapy in the absence of gold standard medical treatments), and the limitations of other nonsurgical treatment options (such as the transient activity of over-the-counter products designed to provide temporary relief and the burden of adhering to dietary and lifestyle changes), new medical treatments / managements that specifically target pepsin would be of great benefit.

[0014] We and others have discussed the promise of peptic activity inhibitors and / or receptor antagonists as potential new treatments for LPR. Summary of the invention

[0015] In one aspect, the present disclosure provides an oral sustained release formulation for treating reflux, comprising: an effective amount of an HIV protease inhibitor; sodium alginate and a pharmaceutically acceptable carrier.

[0016] In another aspect, the present disclosure provides a method of treating reflux in a subject in need thereof, the method comprising orally administering to the subject a formulation described herein to treat reflux. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagrams of assays used to screen for compounds that inhibit pepsin are shown. Assay 1 (top) is a binding assay that measures the extent to which compounds compete with fluorescently labeled pepstatin for pepsin binding sites. Assay 2 (bottom) is a digestion activity assay using fluorescently labeled casein as an enzymatic substrate.

[0018] Figure 2 The percentage of pepsin inhibition produced by the library of pharmacologically active compounds screened using the binding assay is shown.

[0019] Figure 3A-3B The co-crystal structure of amprenavir bound to pepsin is shown, where Figure 3A The 2|Fo|-|Fc| electron density map (green grid) showing amprenavir (purple carbons) bound to the active site of pepsin (yellow carbons) is shown. Figure 3B A schematic diagram of the active site bound to amprenavir is shown, with potential hydrogen bonding interactions indicated by green dashed lines.

[0020] Figure 3C and 3D The crystal structure of the active site of pepsin bound to darunavir and the enzyme-inhibitor interaction are shown.

[0021] Figures 4A-4D .4 depicts the ability of oral Lexiva to prevent pepsin-mediated airway epithelial damage in vivo. Representative animals from the different treatment regimens are shown in the panels: pH 7 ( Figure 4A )、Lexiva( Figure 4B )、0.3mg / ml pepsin (pH 7) ( Figure 4C ) and 0.3 mg / ml pepsin (pH 7) + Lexiva ( Figure 4D ). 20 times magnification. ( Figure 4A , 4B and 4D) Normal-appearing respiratory epithelium, composed of a single layer of ciliated columnar epithelium with basally polarized nuclei and cilia on the apical surface. ( Figure 4C ) Reactive multilayered epithelium with increased nuclear:cytoplasmic (N:C) ratio and loss of cilia.

[0022] Figure 5A and 5B 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.

[0023] Figure 6 Treatment regimen for in vivo studies in mice.

[0024] Fig. 7A and 7B The binding curves of pepsin and HIV protease inhibitors are shown ( Fig. 7A ) and activity curves ( Figure 7B ).

[0025] Figures 8A-8D Structural data for pepsin and HIV protease inhibitors are shown. Figures 8A-8D The left panel shows the active site of porcine pepsin bound to an HIV protease inhibitor. The 2Fo-Fc electron density map contoured at 1.0σ is shown as a magenta grid, and the 2Fo-Fc simulated annealing composite omit map contoured at 1.0σ is shown as a green grid. Figures 8A-8D The right panel depicts a schematic diagram of the active site bound to an HIV protease inhibitor, showing potential hydrogen bonding interactions as indicated by green dashed lines. The electron density map was generated using POVSCRIPT and POV-Ray, and the schematic was created using MarvinSketch and Adobe Illustrator.

[0026] Figures 9A-9H Figure 4 shows the damage to the laryngeal epithelium caused by pepsin and acid in vivo. Representative samples of the treatment group. Figures 9A-9D ) and 200 times ( Figures 9E-9H) magnification of the vocal cords, representing the larynx: pH 7( Fig. 9A and 9E ), pH 4( Fig. 9B and 9F )、0.3mg / ml pepsin (pH 7) ( Fig. 9C and 9G ) and 0.3 mg / ml pepsin (pH 4) ( Fig.9D and 9H ). Fig. 9A and 9E ) Normal airway columnar epithelium (arrow) is approximately one cell thick, with basal polarization of the nucleus and apical surface cilia. ( Fig. 9B and 9F ) Reactive epithelium characterized by thickening (thick arrow) and focal squamous epithelium (long arrow) with loss of cilia. In other areas, relative thickening of the mucosa, moderate increase in the nuclear-cytoplasmic (N:C) ratio, and irregular condensation of chromatin are seen. (c,g) Respiratory epithelium is thickened with pseudostratification of epithelial cells. Multiple foci show keratinization (arrows). In several areas of this treatment group, there is a marked increase in the N:C ratio, loss of nuclear polarization, and reduction of apical cilia. (d,h) Respiratory epithelium is necrotic (arrows) and replaced by inflammatory discharge. Rapid, acute inflammatory infiltration extends to the submucosal area. Scale bar Figures 9A-9D =100 μm; Figures 9E-9H =50μm.

[0027] Fig. 10A and 10B Fosamprenavir gavage and aerosol and darunavir aerosol prevent pepsin-mediated laryngeal injury in vivo. Representative samples at 400x magnification. The laryngeal epithelium in the solvent control group was characterized by a single layer of respiratory epithelium without reactive changes. In mice treated with pepsin-pH 7, the laryngeal epithelium showed reactive epithelial changes and apoptotic debris. Normal histology in mice that received fosamprenavir gavage or aerosol (saline solvent treatment) or fosamprenavir gavage or aerosol (pepsin-pH 7 treatment) showed that fosamprenavir gavage and aerosol prevented pepsin-mediated laryngeal injury. Darunavir gavage caused mild reactivity (rare intraepithelial lymphocytes) in the saline-treated group; pepsin-pH 7-treated darunavir gavage groups had similar findings. Darunavir aerosol was slightly protective against pepsin-mediated injury. Epithelial damage was still present (slight increase in intraepithelial inflammatory cells and reactive epithelial cells), but no apoptosis was observed. Scale bar = 200 μm.

[0028] Fig.11 Certificate of analysis for 1 kg.

[0029] Fig.12 Certificate of analysis for 250 g. DETAILED DESCRIPTION

[0030] In this application, the inventors disclose a novel method for treating reflux disorders, including GERD, respiratory reflux, such as laryngopharyngeal reflux (LPR). The deleterious changes in the laryngopharynx observed in LPR occur after the mucosa comes into direct contact with the refluxed gastric contents, which are composed of acid as well as pepsin, bile, and pancreatic enzymes.

[0031] The present application provides an oral alginate formulation that can be sustained-released in a subject, thereby alleviating the symptoms of one or more reflux disorders. This new approach is suitable for local treatment of the respiratory tract that is affected by LPR and has high accessibility, thereby allowing lower doses, which is an advantage because targeted delivery can simultaneously improve efficacy and limit systemic side effects.

[0032] Here we screened therapeutic compounds for pepsin binding and inhibition. In the LPR mouse model, specific HIV protease inhibitors that inhibit pepsin were given orally and by inhalation to evaluate their potential for treating LPR. An extended release oral alginate formulation was developed to provide the most effective delivery.

[0033] Topical esophageal treatment of GERD has been an area of ​​great interest, however, drug delivery to the esophagus is difficult due to the extremely short transit time of orally administered drugs in the esophagus (less than 16 seconds even when lying flat). To overcome this limitation, thickeners and mucoadhesive substances that can prolong the retention and contact time of liquid suspensions in the esophagus have been studied. To date, the main focus has been on substances that can coat the esophagus and provide local protection against refluxed acid, which Potts et al. called "esophageal bandages." This research has led to the patenting of many mucoadhesive carbonate preparations (Batchelor, 2005, for review). Fosamprenavir retained in the esophagus will inactivate extracellular mucosa-bound pepsin deposited during reflux; due to the prolonged contact time, the mucosa will promote its absorption, which will also inactivate endocytosed intracellular pepsin. The desired formulation will allow the esophagus to absorb a portion of the total dose without significantly hindering systemic delivery through the intestine.

[0034] Viscous and mucoadhesive formulations for esophageal retention have been investigated for local delivery of drugs for diagnosis of Barrett's esophagus, treatment of esophageal cancer and candidiasis, and treatment of GERD and associated pain and inflammation. Among mucoadhesive excipients, alginate has emerged as the best additive for prolonging esophageal retention of liquid and solid drug formulations.

[0035] Alginate is widely used as a bioadhesive polymer for drug delivery because of its non-toxicity, biocompatibility, non-immunogenicity, biodegradability, mucoadhesiveness, easy access and low cost. The U.S. FDA has recognized alginate as a "Generally Referred As Safe" (GRAS) substance, which means that it is safe to eat after expert certification and is listed in Parts 182 and 184 of Title 21 of the Code of Federal Regulations. Alginate has pH-dependent gelation and chemical versatility, and its properties can be adjusted by modification. Therefore, it has become one of the most widely studied mucoadhesive biomaterials and can be used in various improved drug delivery systems (such as hydrogels, microparticles, nanoparticles, and adhesive tablets and films for buccal drug delivery).

[0036] Our study evaluated the in vitro esophageal retention properties of several drug-coating materials widely used in solid drug formulations. We found that sodium alginate (1.5% w / w; medium viscosity grade, derived from giant algae (Mactocystis pyrifera)) has superior esophageal adhesion ability and is able to "self-repair" (re-adhere to the next contact point if detached). Batchelor et al demonstrated that alginate (2% w / v in a 1 ml dose, i.e., 0.02 mg) can adhere to the esophageal mucosa for up to 60 minutes in an in vitro model. Using excised porcine esophagus and saliva flushing (constant flushing at a rate of 1 ml / min to simulate human saliva), we found that lower molecular weight alginate (<75 kDa) with lower viscosity (<0.02 Pa s) had significantly lower esophageal retention than the other tested substances, while high viscosity alginate (>2.93 Pa s) had greater esophageal retention at 3 minutes, but similar retention to medium viscosity alginate at 15 or 30 minutes. The G / M ratio of alginate had no effect on esophageal adhesion. Alginate of medium molecular weight (240 kDa), viscosity (0.51 Pa s) and G / M ratio (44 / 56) presented at 0.02 mg in 1 ml dose (in water) showed 21.9 ± 9.5% retention in 30 minutes.

[0037] Therefore, the medium viscosity sodium alginate provided in our formulation at a dose of 24.5 mg / 10 ml twice daily in MDE is expected to produce 20% retention of bound drug (and retention of approximately 5 mg alginate) in the esophagus for at least 30 minutes.

[0038] Therapeutic Benefits of LPR

[0039] The higher doses of alginate (1000 mg / dose) in alginate antacids have long been used as monotherapy for mild to moderate GERD and as a supplemental therapy for breakthrough symptoms in patients taking PPIs. The therapeutic benefits of alginate are primarily attributed to its raft-forming activity. Alginate rafts float on gastric contents, thereby replacing postprandial acid pockets near the gastroesophageal junction, effectively reducing acid reflux events. The secondary mechanism of antireflux activity is thought to be conferred by its mucoadhesive properties, which prevents the diffusion of pepsin and acid to the underlying esophageal mucosa and the accompanying epithelial barrier dysfunction, as well as enzymatic inhibition of pepsin. Notably, Chater et al. found that only 0.68 mg / ml of medium viscosity alginate (LF120) could inhibit pepsin by 28.46±10.68% (44.73±10.98 inhibition at 1.36 mg / ml). Although this suggests that the excipient alginate in MDE (2.45 mg / ml bid) may have anti-digestive activity, such low concentrations of alginate are unlikely to have therapeutic value, as many clinical trials and meta-analyses have reported therapeutic benefits of anti-reflux medications containing sodium alginate at doses of 1000 mg / 10 ml (Gaviscon Advance, Reckitt Benckiser, Slough, UK) relative to viscosity-matched placebo or products used primarily as antacids, which provided only transient symptom relief regardless of the presence or absence of alginate in the excipient concentration (Gaviscon tablets or liquid antacids, GlaxoSmithKline Consumer Healthcare, Pennsylvania). The lack of therapeutic effect of the latter is thought to be due to their inability to form a coherent raft.

[0040] Recently, alginate-based antireflux agents have been shown to be effective for the treatment of throat symptoms in LPR. As for esophageal symptoms, their therapeutic activity has been attributed to the formation of rafts in the stomach, especially considering that oral administration of alginate would result in little contact with the throat. McGlashan et al. 27The efficacy of alginate on LPR symptoms and endoscopic findings was investigated in 49 patients with RSI and LPR with confirmed RFS who were randomized to receive a liquid alginate suspension (n = 24; 10 ml four times daily after meals and before bed; Gaviscon Advance) or no treatment (n = 25; control): The mean (SD) RSI and RFS scores before treatment were similar in the treatment group (23.9 (7.0) and 10.4 (3.6)) and the control group (24.6 (7.4) and 10.3 (3.3)), but alginate treatment improved LPR symptoms and outcomes, as shown by significant differences in RSI at 2 months (11.2 (7.0) vs. 16.8 (6.4), P = 0.005) and at 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) between the treatment and control groups. Similarly, in a study of personalized treatment for LPR subtypes (acidic to alkaline), Lechien et al. 26 An antireflux diet and three daily doses of alginate (Gaviscon Advance) or magnesium aluminate (Riopan, Takeda, Zaventem, Belgium) after meals were found to improve voice quality in patients (n = 48) with alkaline LPR confirmed by HEMII-pH testing, as demonstrated by reduced dysphonia and roughness scores (GRBAS scale), and improved jitter, flicker, and noise to harmonic ratio (Lechien et al., 2021).

[0041] The present invention provides an oral fosamprenavir sustained-release formulation, using sodium alginate to increase mucosal adhesion in the esophagus and prolong drug delivery, which will improve esophageal symptoms in 25-50% of LPR patients who also suffer from GERD, and thus has a superior therapeutic effect than oral fosamprenavir / Lexiva.

[0042] In one embodiment, the formulation contains sodium alginate at a low excipient level dose to prolong drug delivery to the esophagus by increasing mucoadhesion. This is expected to be beneficial for pepsin-mediated esophageal inflammation, mucosal damage, and related symptoms. While high doses of alginate are expected to have therapeutic benefits due to raft formation, the current low dose formulation is expected to increase mucoadhesion to prolong esophageal retention time.

[0043] In vitro testing will include a texture analyzer for mucoadhesion.

[0044] Studies using esophageal multichannel intraluminal impedance combined with pH (MII-pH) monitoring have shown that many LPR episodes are nonacidic and that both weakly acidic and nonacidic reflux are associated with persistent symptoms in acid-suppressed patients (39-42). Pepsin is the major digestive enzyme in the stomach and is increasingly thought to be responsible for LPR-related damage and inflammation (17-23). ​​Importantly, while the stomach and esophagus have intrinsic defense mechanisms against pepsin, such as mucus, peristalsis, and bicarbonate secretion, laryngeal tissues do not (26). In the airways, where pH is neutral (less than 8), pepsin enzymatic activity is low but stable. However, when pepsin is taken up by laryngeal and hypopharyngeal cells via receptor-mediated endocytosis, it is retained in low-pH intracellular vesicles where it is presumed to reactivate and cause damage (20, 32, 33, 49, 52). Although many LPR episodes are weakly acidic or non-acidic, pepsin is present in all refluxates (24) and is frequently detected in the respiratory tissues and secretions of patients with LPR. For example, the inventors have demonstrated that endocytosed non-acidic pepsin induces the expression of pro-inflammatory cytokine genes in hypopharyngeal cells. This response is similar to that occurring in reflux esophagitis, which increases the severity of disease in patients with GERD (21, 31). Importantly, inhibition of the proteolytic activity of pepsin (i.e., using pepstatin, curcumin, ecabet sodium, anthocyanidins, or preincubation at pH 8.0 followed by 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.

[0045] The inventors believe that LPR is more dependent on pepsin-mediated damage than acid-mediated damage, and believe 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 refractory to proton pump inhibitors (PPIs). Pepsin can be inhibited by two mechanisms: (1) through irreversible inactivation, which prevents its reactivation in intracellular compartments with lower pH; (2) through receptor antagonists, which prevents receptor-mediated endocytosis of pepsin. Although the pepsin inhibitor pepstatin is commercially available, its water solubility and pharmacokinetic properties are poor. Therefore, new pepsin inhibitor compounds with higher bioavailability are needed.

[0046] In the present application, the inventors screened for therapeutic compounds that have the ability to bind to pepsin and inhibit its enzymatic activity, and identified specific HIV protease inhibitors with these abilities (see Example 1). The U.S. Food and Drug Administration (FDA) has approved several HIV protease inhibitors for the treatment of HIV, making these drugs ideal candidates for testing the therapeutic effects of pepsin inhibition on LPR. Using epidemiological data, the inventors demonstrated that the incidence of airway reflux in patients taking HIV protease inhibitors (0.2%) was significantly lower than that in the general population (10-34.4%), which supports the idea that these HIV drugs can be repurposed to treat LPR. Among ten commercially available HIV protease inhibitors, the inventors determined that four of them (i.e., amprenavir, darunavir, ritonavir, and saquinavir) have the ability to bind to pepsin in vitro and inhibit its enzymatic activity ( Fig. 7A and 7B To test these drug candidates in vivo, the inventors established a novel LPR mouse model ( Figure 1 ). These mice will be used to test the ability of HIV protease inhibitors to improve pepsin-mediated laryngeal mucosal damage and inflammation. Mice receive HIV protease inhibitors by oral gavage and nebulized delivery to compare the results of systemic and local delivery, respectively. Based on the results of these animal studies, the inventors will test the efficacy of promising HIV protease inhibitors in a 12-week randomized, double-blind, placebo-controlled clinical trial ( Figure 5A and 5B ).

[0047] method:

[0048] The present invention provides a method for treating reflux, preferably respiratory reflux, in a subject in need thereof. The method comprises administering to a subject a therapeutically effective amount of a preparation comprising an HIV protease inhibitor and an alginate to treat reflux. As used herein, the term "respiratory reflux" refers to inflammation of the upper and lower respiratory tracts caused by reflux of gastric contents. The term respiratory reflux is interchangeable with the alternative terms "supraoesophageal reflux" and "extraesophageal reflux". These broad terms cover several related reflux conditions, including gastropharyngeal reflux (GPR; reflux of gastric contents to the esophagus), laryngopharyngeal reflux (LPR; reflux of gastric contents from the esophagus to 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 esophageal irritation caused by reflux of gastric contents back into the esophagus. The reflux treated herein is preferably refractory to GERD patients with protein pump inhibitor (PPI) therapy.

[0049] As used herein, the term "HIV protease inhibitor" refers to any antiviral drug that inhibits one or more HIV proteases. HIV protease inhibitors prevent viral replication by selectively binding to HIV proteases and blocking the proteolytic cleavage of protein precursors necessary 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, including 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,3S,5S)-3-hydroxy-5-[[(2S)-3-methyl-2-[[methyl-[(2-propan-2-yl-1,3-thiazol-4-yl)methyl]amino [(2S,4S,5S)-5-[[2-(2,6-dimethylphenoxy)acetyl]amino]-4-hydroxy-1,6-diphenylhexan-2-yl]-3-methyl-2-(2-oxo-1,3-diazin-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-isoquinoline quinoline-2-carbonylamino] succinamide), nelfinavir (IUPAC: (3S,4aS,8aS)-N-tert-butyl-2-[(2R,3R)-2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-phenylthiobutyl]-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]hydrazine]-3,3-dimethyl-1-oxobutan-2-yl]carbamate), tipranavir (IUPAC: N-[3-[(1R)-1-[(2R)-4-hydroxy-6-oxo-2-(2-phenylethyl)-2-propyl-

[00136] The HIV protease inhibitor used in the present invention should be able to bind to pepsin and inhibit its enzymatic activity. Therefore, in some embodiments, the HIV protease inhibitor is amprenavir, darunavir, ritonavir or saquinavir, which are shown in Example 1 to be able to bind to pepsin 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-phosphooxybutyl-2-yl]carbamate). HIV protease inhibitors are known in the art and are commercially available.

[0050] Fosamprenavir is a prodrug of amprenavir and is sold by ViiV Healthcare as a calcium salt under the trade names Lexiva (US) and Telzir (Europe). The human body must metabolize fosamprenavir to form its active form, amprenavir. Therefore, administering amprenavir as a prodrug can extend its duration in the body, acting like a slow-release formulation. In addition, fosamprenavir exhibits excellent pharmacokinetics in mice, and because it is already FDA-approved, fosamprenavir can quickly enter pilot clinical trials. In some embodiments, the HIV protease inhibitors used in the compositions and methods described herein have an IC in the micromolar range (μm). 50 In some preferred embodiments, HIV protease inhibitors used in the compositions and methods described herein have IC values ​​in the nanomolar (nM) range. 50 .

[0051] In the present method, the HIV protease inhibitor can be administered using any route that is effective in treating reflux, preferably respiratory reflux, and is preferably provided in a formulation for oral administration. As used herein, the terms "administering" and "administering" 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 / administration can be continuous or intermittent.

[0052] In some embodiments, HIV protease inhibitors are administered orally to treat reflux. For example, in some embodiments, HIV protease inhibitors are administered twice daily at about 0.7-1.4 g (i.e., doses approved by the FDA for the treatment of HIV and therefore safe).

[0053] The methods of the present invention are used to treat reflux in a subject in need thereof. In some embodiments, reflux may be airway reflux. In other embodiments, reflux may be GERD, preferably GERD in a subject refractory to proton pump inhibitors. As used herein, the term "subject in need thereof" or "patient" refers to any person or animal suffering from reflux. In some embodiments, the subject suffers from airway reflux. In some embodiments, the airway reflux condition is selected from laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), and esophageal pharyngeal reflux (EPR). In some embodiments, the subject is a subject with reflux episodes caused by weakly acidic or non-acidic reflux. In another embodiment, the subject is a proton pump inhibitor (PPI) refractory subject.

[0054] As used herein, the terms "treatment", "treating" or "treatment" describe the management and care of a patient for the purpose of combating a disease, disorder or condition. Treatment / treatment includes administering a protease inhibitor or composition of the present invention to prevent the onset of symptoms or complications, alleviate symptoms or complications, or eliminate a disease, disorder or condition. In a preferred embodiment, the methods and compositions of the present invention reduce mucosal damage and inflammation in the respiratory tract of a subject. Treatment / treatment also includes alleviating one or more symptoms of airway reflux, appropriately LPR, GPR or ERP, such as alleviating chronic cough, throat clearing, postnasal drip, hoarseness or dysphonia, globus sensation, dysphagia, dyspnea or a combination thereof. Treatment / treatment also includes alleviating chronic laryngeal irritation and inflammation. In one embodiment, treatment / treatment also includes alleviating one or more symptoms of PPI-refractory GERD, for example, alleviating one or more of the following symptoms: a burning sensation in the chest (heartburn) that usually occurs after eating and may be exacerbated at night, chest pain, dysphagia, regurgitation of food or acidic liquids, a feeling of a lump in the throat, etc.

[0055] 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 can be to reduce, alleviate, inhibit or prevent one or more symptoms of a disease or condition, reduce, inhibit or prevent laryngeal irritation, reduce or inhibit laryngeal irritation or mucosal damage, or reduce, alleviate, inhibit or prevent one or more symptoms of airway reflux, or any other desired change in a biological system. In some embodiments, an effective amount is an amount suitable for providing the desired effect, such as reducing the amount of mucosal damage and inflammation in the respiratory tract. The response to airway reflux treatment can be assessed using any standard clinical method, including but not limited to laryngeal visual inspection (e.g., fiberoptic laryngeal inspection), reflux symptom index scale (RSI), reflux flow sign scoring scale (RFS) (e.g., doctor-reported score based on laryngeal visual inspection), combined esophageal multi-channel intraluminal impedance combined with pH (MII-pH), reflux symptom score (RSS), reflux flow sign assessment (RSA) or pepsin catalytic activity in saliva. Alternatively, the response to treatment of airway reflux can be assessed by assessing inflammation in tissue samples collected from the respiratory tract of the subject (e.g., 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 catalytic activity before and after 12 weeks of treatment. Although HIV inhibitors are not expected to prevent reflux or affect pepsin protein levels, they inactivate pepsin, so measuring pepsin catalytic activity in saliva after treatment will confirm that the treatment is inactivating pepsin in the respiratory tract. This is currently a research tool for assessing in vivo efficacy.

[0056] Most patients with reflux attacks caused by weakly acidic or non-acidic reflux are refractory to proton pump inhibitor (PPI) therapy, which inhibits acid production but does not affect pepsin catalytic activity. The method of the present invention will be particularly beneficial to this refractory patient group, who are in urgent need of alternatives to PPI. As used herein, the phrase "(treatment / treatment) refractory" refers to a condition that is unresponsive to treatment / treatment. For example, if a three-month, twice-daily PPI therapy fails to significantly improve the condition, the patient's reflux can be considered refractory to PPI therapy. The response to reflux therapy can be assessed using any standard method known in the art, including but not limited to the Reflux Symptom Index Scale (RSI), Reflux Sign Rating Scale (RFS), Combined Esophageal Multi-Channel Lumen Impedance Combined pH (MII-pH), Reflux Symptom Score (RSS) or Reflux Sign Assessment (RSA). For a more detailed description of these measures, please refer to the Examples section. For example, effective treatment will reduce RSI and / or RFS to standard values, such as RSI≤13, RFS≤7, or a combination thereof.

[0057] Composition:

[0058] The present invention also provides compositions, it comprises HIV protease inhibitor and alginate and the oral preparation of pharmaceutically acceptable carrier. Commercially available HIV protease inhibitor is usually formulated into tablet or oral suspension for systemic administration. For example, in some embodiments, the composition is configured for oral administration.

[0059] The compositions of the present invention may include any pharmaceutically acceptable carrier that allows oral delivery. "Pharmaceutically acceptable carrier" is known in the art and includes, but is not limited to, for example, suitable diluents, preservatives, solubilizers, emulsifiers, liposomes, nanoparticles, and adjuvants. Pharmaceutically acceptable carriers can 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, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffered media.

[0060] The composition of the present invention further includes additional components that affect the physical state, solubility, stability, in vivo release rate and in vivo clearance rate of HIV protease inhibitors. Suitable components include but are not limited to buffers (e.g. Tris-HCl, acetate, phosphate), additives (such as albumin or gelatin) to prevent surface absorption, detergents (e.g. Tween20, Tween 80, Pluronic F68, bile acid salts), solubilizers (e.g. glycerol, polyethylene glycol), antioxidants (e.g. ascorbic acid, sodium bisulfite), preservatives (e.g. thimerosal, benzyl alcohol, parabens), fillers and tonicity regulators (e.g. lactose, mannitol). In addition, compositions can be formulated for controlled release or sustained release HIV protease inhibitors, e.g., formulated in lipophilic reservoirs (e.g., fatty acids, waxes, oils).

[0061] The composition of the present invention may further include suspending agents, preservatives, sweeteners, flavoring agents, water and combinations thereof. Liquid preparations for oral administration may be in the form of, for example, elixirs, solutions, syrups or suspensions, or may be prepared as dry products for reconstitution with water or other suitable carriers prior to use. Pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous carriers (e.g., almond oil, oily esters of ethanol, cremophore) may be used by conventional means. TM or fractionated vegetable oils); and preservatives (e.g. methyl or propyl parabens, or sorbic acid) to prepare such liquid preparations. The preparations may also contain buffer salts, preservatives, flavoring agents, coloring agents and sweeteners as appropriate. It is well known that preparations for oral administration may also be appropriately formulated as controlled release compounds.

[0062] A suggested formulation is provided in Table 1. It is expected that the amount of API will be approximately 20-30% of the total weight of the dry product formulation. Fig.11 and 12 is an analytical proof of the composition described herein.

[0063] Table 1: Suggested recipes

[0064]

[0065]

[0066] The composition can be prepared into a unit dosage form for administration to a subject. The amount and timing of administration are determined by the treating physician to achieve the desired effect.

[0067] The HIV protease inhibitor included in the composition of the present invention can be any HIV protease inhibitor suitable for treating airway reflux, as described 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.

[0068] The present invention has been described in terms of one or more preferred embodiments, but it should be understood that many equivalents, alternatives, variations and modifications besides those explicitly described are possible and within the scope of the present invention.

[0069] It will be apparent to those skilled in the art that, in addition to what has been described, many additional modifications can be made without departing from the inventive concept. In interpreting the present disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variants of the term "comprising" should be interpreted as referencing elements, components or steps in a non-exclusive manner, so that the referenced elements, components or steps can be combined with other elements, components or steps that are not explicitly referenced. The embodiments referred to as "comprising" certain elements are also considered to be "essentially composed of (these elements)" and "consisting of (these elements)". The terms "essentially composed of..." and "consisting of..." should be interpreted in accordance with the interpretation of the PEP and the relevant Federal Circuit. The transitional term "essentially composed of..." limits the scope of the claim to the specified substances or steps and "those that do not essentially affect the basic and new characteristics" of the claimed invention. "Consisting of..." is a closed term that does not include any elements, steps or ingredients not specified in the claim. For example, "consisting of..." with respect to a sequence refers to the sequence listed in SEQ ID NO., and does refer to a larger sequence that may contain SEQ ID as part of it.

[0070] References cited herein are hereby incorporated by reference in their entirety.

[0071] A more complete understanding of the present invention can be obtained upon consideration of the following non-limiting examples.

[0072] Example

[0073] Example 1: Oral and inhaled fosamprenavir reverses pepsin-induced damage in a mouse laryngopharyngeal reflux model

[0074] Reference: 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. and Blumin, JH (2022). Oral and Inhaled Fosamprenavir Reverses Pepsin-Induced Damage in a Laryngopharyngeal Reflux Mouse Model. The Laryngoscope., which is incorporated herein by reference.

[0075] method

[0076] Binding and activity assays

[0077] To examine whether HIV protease inhibitors can bind and inhibit pepsin, we developed a fluorescence polarization-based assay that measures size-dependent molecular rotation, allowing detection of degradation, association, and dissociation events. 80 A competitive binding assay was designed using the subnanomolar affinity inhibitor pepstatin 81. Pepstatin-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) DMF. The mixture was stirred for 1 hour, and then 1 mg of Alexa Fluor 647 cadaverine disodium salt (ThermoFisher Scientific) was added. After 2 hours, the solvent was evaporated under high vacuum (35° C.), and the residue was partially dissolved in 10% methanol and transferred to a C18 column (Waters Corporation, Milford, MA). Elution was performed using increasing percentages of methanol. Pepstatin-Alexa647 was eluted with 45% methanol. Enzyme inhibition assays were designed using casein substrate. 82 Bovine α-casein (Sigma-Aldrich, St. Louis, MO) was labeled with Alexa Fluor 647 carboxylate succinimidyl ester (Thermo Fisher Scientific, Waltham, MA) as described in the paper 82 In brief, the two were mixed in 0.1 M sodium bicarbonate at a ratio of 2.5 ug / mg label to protein for 15 minutes, and the labeled casein was then separated from the unbound label by a Sephadex G-25 (Sigma Orrich) column consisting of 90 x 5 mm packed beads in a glass Pasteur pipette and eluted with dPBS (pH 7.4) (Thermo Fisher Scientific). The fast moving band (casein-bound fluorophore) was collected in a volume of approximately 0.4 ml. The concentration of the resulting probe (casein-Alexa 647 in PBS-azide) was estimated spectrophotometrically using Beer's law (Impon Nano Spectrophotometer, Impon, Westlake Village, California, USA).

[0078] The assay was optimized using a concentration range of 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 (HIV protease inhibitor diluent) in 0.1 M HCl (pH 1) with 0.01% v / v Tween-20 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 (excitation / emission 620 / 680 nm). Unlabeled pepstatin dose response curves are used to ensure that the assay is responsive to pepsin inhibition. Conditions that produce the maximum dynamic range of the assay are used to evaluate HIV protease inhibitors: 100nM probe, 0.03U / ul porcine pepsin A, 37.5% DMSO for competitive binding assays, 200nM probe, 0.01U / μL pepsin, 5% DMSO for digestion activity assays. HIV protease inhibitors (amprenavir, ritonavir, lopinavir, saquinavir mesylate, nelfinavir mesylate hydrate, darunavir glycolate, indinavir sulfate hydrate; all products of Sigma Orrich) were dissolved in DMSO and tested for three logarithmic concentrations under optimized assay conditions. The assay was performed twice, three sets of replicate reactions were read every five minutes, and a plot of the average mP versus probe concentration was drawn (binding assay), or read at intervals of <2 minutes within 30 minutes, and a plot of the average mP over time was drawn (activity assay). The kinetic trajectories were analyzed using an online tool (https: / / icekat.herokuapp.com / icekat) to calculate the half-maximal inhibitory concentration (IC 50 ) 83 The mP was normalized to blank (no inhibitor present) to give percent binding or activity.

[0079] crystallization

[0080] Saturated solutions of HIV protease inhibitors (amprenavir, ritonavir and darunavir glycolate) were prepared in DMSO and centrifuged at 31,000rcf for 10 minutes. The supernatant was added to pepsin (200mg / ml in water) with 1.6% (v / v) fc. Due to poor solubility, solvents for saquinavir mesylate were selected from CryoSol screening (Molecular Dimensions, Holland Township, Ohio). 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-propylene glycol and 12.5% ​​v / v glycerol) was selected because it provides high solubility and protein compatibility conditions for the cocrystallization mixture. The supernatant of the saturated saquinivar solution in SM2 was mixed with pepsin at 5% fc (v / v). Crystallization conditions were optimized by screening 200 mg / ml pepsin in a Salt RX screen (Hampton Research, Mission Viejo, CA). After one week at room temperature, small bipyramidal crystals formed in 3.5 M ammonium chloride and 0.1 M sodium acetate trihydrate (pH 4.6) were used as microseed stocks for co-crystallization with amprenavir, ritonavir, and darunavir glycolate as previously described. 84 . A hanging drop of 2ul of pepsin (180-210mg / ml) and 1ul of microseed solution (serially diluted 10-100 times in 3-4M ammonium chloride and 0.1M sodium acetate trihydrate (pH 4.6)) formed diffraction quality crystals (trigonal bipyramids, approximately 200×100×100μm) after 2-7 days. The crystals were cryoprotected with 30% glucose, 5M ammonium chloride and 0.1M sodium acetate trihydrate (pH 4.6) and immersed in liquid nitrogen. Co-crystallization with saquinavir was performed in 0.1M acetic acid rather than sodium acetate trihydrate because this allows large crystals to form in the absence of microseeds; the crystals were cryoprotected with 30% w / v glucose, 5M ammonium chloride and 0.1M sodium acetate trihydrate (pH 4.6) and immersed in liquid nitrogen.

[0081] Diffraction data sets were collected at the Advanced Photon Source (APS) equipped with a MAR 300 CCD or Dectris Eiger 9M detector at the Life Sciences Collaborative Access Team (LS-CAT) beamline at Argonne National Laboratory and using MOSFLM 85 or HKL2000 86 Index, integrate, and scale your data.

[0082] Specifically, for pepsin:amprenavir, a wavelength of A 50 × 50 μm beam was collected at LS-CAT beamline 21-ID-F using a MAR 300 CCD detector. Diffraction data set. A total of 262 frames were collected from 1 to 130.5° with an oscillation range of 0.5° and a detector distance of 250 mm. The exposure time was 0.5 s. The diffraction data were indexed, integrated and scaled using MOSFLM.

[0083] For pepsin: ritonavir, use a wavelength of A 50 × 50 μm beam was collected at LS-CAT beamline 21-ID-D using a Dectris Eiger 9M detector Diffraction data set. 900 frames were acquired from 10° to 180° while oscillating at 1° / s and slicing 5 images / °. The crystal to detector distance was 160 mm. The diffraction data were indexed, integrated and scaled using MOSFLM.

[0084] For pepsin: darunavir, use a wavelength of A 50 × 50 μm beam was collected at LS-CAT beamline 21-ID-G using a MAR 300 CCD detector. Diffraction data set. A total of 900 frames were collected from 10° to 180° with an oscillation range of 0.2° and a detector distance of 260 mm. The exposure time was 0.3 seconds. The diffraction data were indexed, integrated and scaled using HKL2000.

[0085] For pepsin: saquinavir, use a wavelength of A 50 × 50 μm beam was collected at LS-CAT beamline 21-ID-F using a MAR 300 CCD detector. Diffraction data set. A total of 400 frames were collected from 10° to 100° with an oscillation range of 0.2° and a detector distance of 200 mm. The exposure time was 0.5 s. The diffraction data were indexed, integrated and scaled using MOSFLM.

[0086] The initial phase is PHASER 87 The search model was unliganded porcine pepsin (PDB ID 4PEP), and its B-factor was reset to The solvent molecules were removed. Using phenix.refine (PHENIX 87-89 ) and COOT 90,91Model refinement was performed. The geometric constraints of the compounds were from the CCP4 monomer library 92 Using the MolProbity implementation in the PHENIX suite 93 Validate the model. Before deposition, the PDB-REDO server was also used 94 The models of ritonavir and saquinavir were optimized. The electron density map was generated by POVSCRIPT and POV-Ray, and the schematic was drawn by MarvinSketch (http: / / www.ChemAxon.com) and Adobe Illustrator CC 2020.

[0087] In vivo mouse model

[0088] Experiments were approved by the University of Minnesota (UMN) Institutional Animal Care and Use Committee (1712-35415A) and performed at UMN. Three replicate animals per treatment condition were expected to be sufficient to verify the reproducibility of each experiment without excessive use of animal resources. Three mice were randomly assigned to treatment groups. No data were excluded from the analysis.

[0089] Six-week-old female Jackson A / J mice (Jackson Laboratory, Bar Harbor, ME) were fed a D-62 powdered Wattenberg diet at a dose of 2 g / mouse / day. 95 The rats were acclimated for one week before the experiment. 1,95-99 , mechanical injury was applied during the first two weeks of the four-week treatment course, making the laryngeal mucosa susceptible to chemical injury by pepsin / acid applied during the four weeks. When performed in this manner, mechanical injury increased the sensitivity of the mucosa to subsequent chemical injury, leaving little detectable injury at the end of the four-week treatment course. 95 During the first two weeks of treatment, all animals (including the control group) were subjected to weekly mechanical injury as described (see Experimental Protocol, 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-tipped curved (135°) needle was used to pull from distal to proximal to cause lesions in the subglottis, glottis, and supraglottis, resulting in slight abrasions.

[0090] In preliminary experiments, to validate the LPR mouse model (i.e., laryngeal injury caused by pepsin under neutral and acidic pH conditions), mice (n = 3) were given 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 h after mechanical injury at weeks 1 and 2. Figure 6 ); in the 3rd and 4th weeks, non-invasive laryngeal instillation was continued (3 days / week). Before each trauma and laryngeal instillation, mice were anesthetized by intraperitoneal administration of 225-240 mg / kg of avertin (2,2,2-tribromoethanol). At the end of the fourth week, the mice were sacrificed.

[0091] To test the protective effect of HIV protease inhibitors on pepsin-mediated in vivo damage, the inhibitors were delivered by aerosol or gavage at the same time as the trauma (days 2 and 8) and solvent / pepsin instillation (days 3-5, 9-11, 16-18 and 23-25). Aerosol or gavage was provided on days 1-5, 8-12, 15-19 and 22-25, and mice were killed on day 26. Due to frequency, mice were anesthetized with isoflurane (3% at 2.5 LPM, 3-5 minutes before the operation) instead of avertin. Lexiva and Prezista (hereinafter referred to as generic names: fosamprenavir and darunavir) were used for gavage, and the corresponding pure drugs were used for aerosol administration (fosamprenavir from Anant Pharmaceuticals, Ambernath, Maharashtra, India, and darunavir from Ambeed, Arlington Heights, Illinois). The doses administered by tube were equivalent to the prescribed doses for HIV patients (20 mg / kg / day of fosamprenavir; 8.6 mg / kg / day of darunavir). Aerosol generation was as described above. 100 . Briefly, 10 ml of drug suspension in ethanol was placed in the baffle to keep the concentration constant at equilibrium solubility. Ethanol droplets containing dissolved drug were generated by an ultrasonic nebulizer (nominal frequency 1.7 MHz) and entrained with air at a flow rate of 0.5 LPM using a custom glass baffle (UMN Department of Chemistry Glass Shop). The aerosol cloud was then passed through a cylindrical drying column containing activated carbon rings. The ethanol was removed and the emitted pure drug dry aerosol particles were then directly delivered to the exposure chamber. The mass deposited on the filter was measured gravimetrically and the total output rate (mg / min) was determined. The aerosol concentration (mass / volume of air) was calculated by dividing the total output rate by the air flow rate (0.5 LPM). The inhaled drug mass (M) of each mouse was inh ) is defined as M inh= [Aerosol] * RMV * t, where [Aerosol] is the aerosol concentration of the drug, RMV is the respiratory minute volume of the mouse (0.025 L / min), and t is the aerosol exposure time. The aerosol concentration is 0.09 mg / L fosamprenavir or 1.2 mg / L darunavir, so considering the minute volume of the mouse (0.025 L / min), the inhaled amount is 0.93 mg / kg / day fosamprenavir or 12 mg / kg / day darunavir. The actual deposition has not been determined, but it is expected to be 10% of the inhaled mass (deposition fraction of 1 μm aerosol particles in the mouse body).

[0092] Tissues were harvested, fixed with paraformaldehyde, embedded in paraffin, and 4 μm sections were stained with hematoxylin and eosin (H&E) by an automated stainer. Sections were reviewed by a certified pathologist (JM) who was blinded to the treatment groups.

[0093] result

[0094] Binding and activity assays

[0095] Four of the seven HIV protease inhibitors tested were able to bind and inhibit pepsin at low micromolar concentrations ( Fig. 7A and 7B ): amprenavir, darunavir, ritonavir, and saquinavir. The in vitro activity of these four HIV protease inhibitors against pepsin provides basic support for further research.

[0096] Structural data

[0097] To aid in the interpretation of the 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 and human enzyme (PDB ID 1PSN) 101 The sequences are 86% identical and their structures are almost identical (RMS deviation of all Cα atoms). ). The minor differences in the tertiary structure are located in a loop of residues (277-282) that are not part of the binding cleft. The residues in the active site cleft are highly conserved: of the 17 residues that make direct contact with the inhibitors described herein, only two are different (T12 and V291). Therefore, porcine pepsin is considered an acceptable alternative to human pepsin in terms of evaluating the structural biology.

[0098] Porcine pepsin co-crystallized with amprenavir, darunavir, ritonavir, and saquinavir (Table 2 and Figures 8A-8D). All of these are peptidomimetics; the alcohol of the central phenylalaninol residue mimics the tetrahedral intermediate of peptide bond cleavage, bound between the catalytic aspartate residues D32 and D215. The binding directionality (phenylalaninol amino group on the prime side of the binding site) is similar to that of pepstatin. 101 Binding depends on van der Waals contacts between inhibitor side chains and binding site residues; few hydrogen bonds are observed (5-6). For example, in the pepsin-ritonavir complex ( Fig. 8A ), the β-homophenylalanine side chain is bound in the P1 subsite, making van der Waals contacts with F111, F117, and I120. The phenylalaninol side chain is bound in the P1 subsite, making contacts with I213, M289, V291, and I300. The thiazole and isopropylthiazole groups of ritonavir do not have any stabilizing interactions with the active site. Accordingly, the electron density for these groups is poorly defined, while the B-factors (reflecting the precision of atomic positions) for these parts of the molecule are very high. Structure of the pepsin-saquinavir complex ( Figure 8B ), in which the side chain of the phenylalaninol residue interacts with the P1' subsite, but the two ends of the molecule, the quinoline and decahydroisoquinoline moieties, also have poor density and high B factors. Figure 8C ) and darunavir ( Fig.8D ) follow the same pattern. The phenylalaninol residue of both inhibitors occupies the P1' site and interacts with I213, M289, V291 and I300. The isobutyl group mimics a leucine residue and occupies the P1 site, interacting with F111, F117 and I120. In 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 has no polar contacts with the active site. At the other end of the molecule, where the two compounds differ, the tetrahydrofuranyl group of amprenavir forms a hydrogen bond with the phenolic oxygen of Y189. However, the di-tetrahydrofuranyl group of darunavir is unable to make such interactions with the active site and is limited to van der Waals contacts with I73, T74, I128 and Y189. The similarities in the structures and binding modes of amprenavir and darunavir do not explain their differences in IC 50 The difference in .

[0099] Table 2. Crystallographic data collection and model refinement statistics

[0100]

[0101]

[0102]

[0103] a The values ​​in brackets apply to the high-resolution shell indicated in the Resolution row.

[0104] b Coordinate Error Estimation Based on Maximum Likelihood

[0105] In vivo mouse model

[0106] In an in vivo mouse model, when pepsin was applied after mechanical injury to the larynx (with or without acid exposure), pepsin-mediated laryngeal epithelial damage was observed at pH 4 and 7 ( Figures 9A-9H ). The laryngeal epithelium of animals in the pH 7 control group was normal, 1-2 cells thick, ciliated, and without inflammation, keratinization, or necrosis; the results indicate that the control group had no detectable mucosal damage due to mechanical trauma within the first two weeks of treatment or pH 7 solvent. The laryngeal epithelium of the pH 4 group was reactive, thickened (3-4 cells thick), and keratinized, with lost cilia. The laryngeal epithelium from the pepsin-pH 7 group was of moderate thickness (2-3 cells) with signs of keratinization, an increased nuclear-to-cytoplasmic ratio, and loss of polarization. The epithelium of the pepsin-pH 4 group was completely lost due to necrosis and infiltration of inflammatory cells.

[0107] Fosamprenavir gavage at doses equivalent to those used to treat HIV in humans prevented pepsin-mediated laryngeal injury, defined as reactive epithelial cell injury, increased intraepithelial inflammatory cells, and increased apoptosis ( Fig. 10A and 10B ). Oral darunavir caused mild reactions (no reaction in the darunavir aerosol group; Figures 9A-9H ) obscured the ability to detect its effect on pepsin-mediated injury. Fosamprenavir aerosol can prevent pepsin-mediated laryngeal injury ( Figures 9A-9H Darunavir aerosol provided moderate protection against pepsin-mediated injury: although epithelial damage was present (mild increase in intraepithelial inflammatory cells and reactive epithelial cells), no apoptosis was observed, as in mice treated with pepsin-pH 7 and sham inhalation.

[0108] discuss

[0109] Over the past two decades, the treatment of LPR has focused on inhibiting gastric acid secretion. With the introduction of MII-pH technology, it is now understood that LPR is often non-acidic and that non-acidic proximal events are associated with laryngeal endoscopic signs and symptoms. 39-46,48-50,102 These findings have led to research into the non-acidic components of gastric reflux.

[0110] Although bile has been shown to cause mucosal damage in both mildly acidic and non-acidic pH conditions in experiments, it is argued that "there is no evidence that the same mechanism occurs in the human larynx." 57 The clinical significance of the results has been questioned. Unconjugated bile acids can cause damage under neutral-high pH conditions (such as the laryngopharynx) but are rarely found in gastric reflux fluid.56,69 In addition, the concentrations of bile salts / acids found to damage the larynx and hypopharynx were 1000-fold higher (0.3-50 mM) than those reported in the respiratory tract of patients with LPR, GERD, and asthma or lung disease. 96,103,104 Compare 0.8-32uM 105-109 ), and lead to morphological changes inconsistent with those in LPR patients, such as cell membrane "blebbing" 110 .

[0111] Pepsin is present in all reflux 55 In addition, pepsin is frequently detected in respiratory tissues and secretions of LPR patients but is absent in non-reflux subjects confirmed by MII-pH, thus predicting reflux-induced symptoms and illness. 20,39,46,50,55,59,65,67,68,111,112 1 mg / ml of pepsin in the stomach is diluted by saliva when it flows proximally. It is reported that the concentration range in the respiratory tract is: 2.5 μg / ml in saliva and 61.5 μg / ml in nasal secretions. 113,114 , 360μg / ml in middle ear fluid 115 In this paper, 300 μg / ml was used to establish a chronic LPR model within a limited experimental time frame. 1,77,116,117 The reported in vitro and in vivo pepsin-mediated injury and inflammatory changes, including the histological changes described herein, are consistent with those observed in LPR patients. 62-64,66,70,118-122 Strong evidence from multiple groups highlights the important role of pepsin, whereas gastric acid is not involved, in the finding of reflux-induced laryngeal symptoms and failure to respond to PPI therapy.

[0112] Although pepstatin is a potent pepsin inhibitor, its poor water solubility and poor pharmacokinetic properties make it an undesirable therapeutic candidate. The structural data presented here suggest that the binding of the inhibitor to the pepsin-promoting cleft is stabilized primarily by van der Waals contacts, which makes rational inhibitor design difficult. Therefore, testing existing inhibitors of other aspartic proteases is considered the most effective approach to identify therapeutics targeting pepsin.

[0113] There are currently ten HIV protease inhibitors commercially available. 123 Seven of these could be tested by our in vitro binding and inhibition assays, and four of them (amprenavir, ritonavir, saquinavir, and darunavir) bound to and inhibited pepsin, IC 50In the low micromolar range, this validated our hypothesis that existing therapeutic protease inhibitors could exhibit anti-digestive activity. Two drugs were selected for in vivo studies based on in vitro assayed anti-digestive activity, cost, and reported side effects. Saquinavir exhibits known side effects and interactions (QT interval prolongation, heart block, hyperlipidemia, and liver problems) 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 low IC for pepsin. 50 Therefore, choose IC 50 The lowest-bioavailable darunavir and the more bioavailable and well-tolerated amprenavir prodrug fosamprenavir were evaluated in vivo. 124 We used a model involving mechanical trauma and pepsin / acid instillation that reliably reproduces epithelial changes similar to those observed in LPR patients. 1,63,70,118,119,125 Using this model, human-equivalent doses of fosamprenavir, but not darunavir, prevented pepsin-mediated laryngeal injury. When administered topically by inhalation, treatment with either compound preserved normal laryngeal histology despite exposure to pepsin.

[0114] This study was designed to investigate whether pepsin inhibitors can prevent laryngeal damage caused by pepsin exposure in vivo. As with any experimental observation, caution should be exercised when translating in vivo findings from limited numbers of animals to the clinical setting. Potential differences between mouse and human respiratory pathobiology should be kept in mind when assessing the clinical relevance of these data. This paper utilized established in vivo models of GERD and LPR. 1,96-99The methods of aerodigestive injury induced by pepsin and demonstrated that mucosal damage is consistent with the clinical manifestations of LPR, supporting their use to evaluate drugs to prevent LPR-induced damage: at the end of four weeks of treatment, no mucosal damage was measured due to mechanical injury and neutral solvents, while multi-layered reactive epithelial cell apoptosis was observed in the pepsin- and acid-treated groups. The mouse epiglottis is located in the transition zone between the stratified squamous epithelium of the vocal cords and the ciliated pseudostratified columnar epithelium of the supraglottic and subglottic regions. To avoid mistaking the squamous epithelium of the vocal cords for signs of injury, we only collected representative images from the visible thyroid tissue at the anterior end of the vocal cords for reference. The pepsin-treated groups also showed additional features of reactive epithelium (darkening of the nuclei, variable nuclear diameters, increased nuclear-to-cytoplasmic ratio, intraepithelial inflammatory cells, and apoptosis), which were absent in the control pH 7.0 group and the groups treated with fosamprenavir or darunavir, confirming the epithelial reactivity induced by pepsin and the efficacy of HIV protease in preventing pepsin-mediated injury. Although these data are qualitative and could be further validated by less subjective quantitative measures, the evidence presented here provides preliminary proof of concept that treatment targeting pepsin could reduce mucosal damage similar to that observed in patients with LPR and supports further investigation. Studies are ongoing in our laboratory to examine the protective effects of fosamprenavir on pepsin-mediated changes in laryngeal cell viability and expression of inflammatory and oncogenes and proteins. Further studies are needed to determine whether the protective effects of fosamprenavir aerosol on the larynx in vivo are due to systemic activity or local conversion to amprenavir. The intestine is the major site of fosamprenavir metabolism. Conversion of fosamprenavir via alkaline phosphatase (ALP) is required for its transepithelial flux and subsequent metabolism by cytochrome P450 enzymes, and studies have shown that this process is carried out by intestinal ALP located on or near the surface of Caco-2 cells. 128,129 However, inhaled fosamprenavir may be converted to amprenavir in the respiratory tract via serum ALP, just as serum collected from healthy subjects converts similar phosphate prodrugs. Inhaled fosamprenavir may also be converted by salivary ALP or by ALP expressed by immune cells recruited to the respiratory mucosa and tissue damage. 132-134 and carcinogenesis (including LPR-induced laryngeal inflammation), 10,74,135-137 ALP may be elevated in the LPR-injured airways, thereby increasing fosamprenavir conversion to the desired active site. Drug formulations that prolong aerodigestive tract residence time could further improve local drug conversion and local activity. Studies are ongoing in our laboratory to examine the efficiency of fosamprenavir conversion via the laryngeal epithelium, saliva, and serum, and to perform a dose-response study in an in vivo mouse model to compare the relative efficacy of inhaled fosamprenavir and amprenavir against pepsin-mediated injury.

[0115] Although additional experimental data will help us understand the protective effects of fosamprenavir on the larynx, improvement in LPR symptoms will be the ultimate determinant of the success of drug therapy. Therefore, randomized placebo-controlled trials are the best approach to testing therapeutic compounds. Such trials are feasible for fosamprenavir, given that the oral formulation is FDA-approved and an a priori responder definition of clinically meaningful symptom improvement has been established based on FDA guidance. 138 Interestingly, preliminary epidemiologic data (unpublished) support the therapeutic potential of HIV protease inhibitors for LPR and warrant follow-up: among 2,062 adult HIV patients prescribed HIV protease inhibitors (Froedtert Memorial Lutheran Hospital, Milwaukee, WI, July 2014–2016; Medical College of Wisconsin Institutional Review Board, 13874), only 0.2% developed LPR, compared with an incidence of 10–34% in the general population. 139,140 These data provide preliminary support for clinical investigation of fosamprenavir as a novel treatment for LPR.

[0116] in conclusion

[0117] These strong evidences highlight the important role of pepsin, while gastric acid is not involved, in reflux-induced laryngeal symptoms and endoscopic findings in patients refractory to PPI therapy. Fosamprenavir and darunavir, FDA-approved retroviral therapies for HIV / AIDS, bind to and inhibit pepsin and abolish pepsin-mediated laryngeal inflammation and mucosal damage in the LPR mouse model. These drugs are well suited for repurposing because they target foreign viruses and can be evaluated in clinical trials for much-needed medical treatments in patients more rapidly than novel compounds. Reformulation for topical inhalation administration could further improve outcomes and limit side effects.

[0118] Data availability

[0119] Structural data are available at the Worldwide Protein Databank (accession codes 6XCY, 6XCT, 6XCZ, 6XD2; http: / / www.wwpdb.org / ).

[0120] sequence

[0121] SEQ ID NO: 1 Synthetic peptide substrate for pepsin

[0122] Lys-Pro-Ala-Glu-Phe-PNP-Arg-Leu (PNP = p-nitrophenylalanine)

[0123] References

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[0264] 141.Nikki Johnston, PhD;Tina L.Samuels, MS;Christopher J.Goetz, BS;Leggy A.Arnold, PhD;Brian C.Smith, PhD;Donna Seabloom, BS;Beverly Wuertz, BS;Frank Ondrey, MD, PhD;Timothy S.Wiedmann, PhD;Nemanja Vuksanovic, PhD;Nicholas R.Silvaggi, PhD;Alexander C.MacKinnon, MD, PhD;James Miller, MD, MPH;Jonathan Bock, MD;Joel H.Blumin, MD.Oral and Inhaled Fosamprenavir Reverses Pepsin-Induced Damage in a Laryngopharyngeal Reflux Mouse Model. Laryngoscope, 00:1–11, 2022.

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[0268] Other aspects

[0269] The following disclosures may be described according to the following numbered clauses.

[0270] Item 1. An oral sustained-release preparation for treating reflux, comprising: an effective amount of an HIV protease inhibitor; sodium alginate and a pharmaceutically acceptable carrier.

[0271] Item 2. The oral sustained-release formulation according to Item 1, wherein the HIV protease inhibitor and sodium alginate form improves sustained-release for at least 30 minutes.

[0272] Clause 3. The oral formulation according to Clause 1 or 2, wherein the HIV protease inhibitor is amprenavir, darunavir, ritonavir, saquinavir or any combination thereof.

[0273] Clause 4. The oral formulation according to any one of the preceding clauses, wherein the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir.

[0274] Clause 5. The oral formulation according to any one of the preceding clauses, comprising one or more of a suspending agent, a preservative, a sweetener, a flavoring agent, water, and combinations thereof.

[0275] Clause 6. The oral formulation according to any one of the preceding clauses, wherein the formulation is a liquid.

[0276] Clause 7. A method of treating reflux in a subject in need thereof, the method comprising orally administering to the subject a formulation as described in any of Clauses 1-5 to treat reflux.

[0277] Clause 8. The method according to Clause 7, wherein the HIV protease inhibitor is capable of binding to pepsin and inhibiting its enzymatic activity.

[0278] Clause 9. The method according to 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.

[0279] Clause 10. The method according to any one of Clauses 7-9, wherein the subject has an airway reflux disorder selected from laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR) and esophageal reflux (EPR).

[0280] Clause 11. The method according to Clause 10, wherein the subject's condition is a proton pump inhibitor (PPI) refractory condition.

[0281] Clause 12. The method according to any one of clauses 7-10, wherein the method reduces laryngeal mucosal damage and inflammation.

[0282] Clause 13. The method according to any one of Clauses 7-12, wherein the subject suffers from gastroesophageal reflux disease (GERD), preferably GERD refractory to proton pump inhibition.

[0283] Clause 14. Use of a composition according to any one of clauses 1-5 for treating reflux in a subject in need thereof, wherein the subject suffers from a condition selected from the group consisting of laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), esophageal reflux (EPR) or GERD refractory to protein pump inhibition.

Claims

1. An oral sustained-release preparation for treating reflux, comprising: An effective amount of an HIV protease inhibitor; Sodium alginate and a pharmaceutically acceptable carrier.

2. The oral sustained-release formulation according to claim 1, wherein the HIV protease inhibitor and sodium alginate form improves sustained release for at least 30 minutes.

3. The oral formulation according to claim 1, wherein the HIV protease inhibitor is amprenavir, darunavir, ritonavir, saquinavir or any combination thereof.

4. The oral formulation according to claim 1, wherein the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir.

5. The oral formulation according to claim 1, comprising one or more of a suspending agent, a preservative, a sweetener, a flavoring agent, water and a combination thereof. The oral formulation according to claim 1 , wherein the formulation is a liquid.

7. A method of treating reflux in a subject in need thereof, the method comprising orally administering to the subject the formulation of claim 1 to treat reflux.

8. The method of claim 7, wherein the HIV protease inhibitor is capable of binding to pepsin and inhibiting its enzymatic activity.

9. The method of claim 7, wherein the HIV protease inhibitor is administered twice daily at a dose of about 1.4 g or less.

10. The method of claim 7, wherein the subject has an airway reflux disorder selected from laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), and esophageal reflux (EPR).

11. The method of claim 10, wherein the subject's condition is a proton pump inhibitor (PPI) refractory condition.

12. The method of claim 7, wherein the method reduces laryngeal mucosal damage and inflammation.

13. The method of claim 7, wherein the subject suffers from gastroesophageal reflux disease (GERD), preferably GERD refractory to proton pump inhibition.

14. Use of the composition of claim 1 for treating reflux in a subject in need thereof, wherein the subject suffers from a condition selected from the group consisting of laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), esophagopharyngeal reflux (EPR), or GERD refractory to protein pump inhibition.

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