Patent Application
20240041764
N/A
A Sequence Listing accompanies this application and is submitted as an XML file of the sequence listing named “650053_00979_Sequence_Listing” which is 2,375 bytes in size and was created on Jul. 25, 2023. The sequence listing is electronically submitted via Patent Center with the application and is incorporated herein by reference in its entirety.
Laryngopharyngeal reflux (LPR), the backflow of gastric contents into the laryngopharynx, is an important health problem. LPR affects children and adults equally, and the clinical spectrum of this disease is extensive.
More than 20% of the US population suffers from laryngopharyngeal reflux. While dietary/lifestyle modifications and alginates provide benefit to some, there is no gold standard medical therapy. Increasing evidence suggests that pepsin is partly, if not wholly, responsible for damage and inflammation caused by laryngopharyngeal reflux. A treatment specifically targeting pepsin would be amenable to local, inhaled delivery and could prove effective for endoscopic signs and symptoms associated with nonacid reflux.
Laryngopharyngeal reflux (LPR), the backflow of gastric contents into the laryngopharynx, is an important health problem. LPR affects both children and adults, and the clinical spectrum is extensive. Unlike patients with gastroesophageal reflux (GER) which is limited to the esophagus, many LPR patients do not experience acid indigestion but present with symptoms due to chronic laryngeal irritation, such as chronic cough, throat-clearing, post-nasal drip, dysphonia, globus, dysphagia, and dyspnea. Significant evidence supports the contribution of chronic LPR to serious and life-threatening illness including airway stenosis, reactive airway disease, and laryngeal cancer. LPR is estimated to affect more than 20% of the United States population and contribute to 10% visits to otolaryngologists. The economic burden of LPR is over $52 billion per year, which is 5.6-fold greater than that of GER; 52% of the burden is attributed to proton pump inhibitors (PPIs).
While PPI therapy is a mainstay in the treatment of GER disease (GERD), its efficacy for LPR is poor. In clinical practice, it was believed that patients with reflux laryngitis require higher doses and longer trials of PPIs than those with typical GERD given the assumption that the upper airway is more sensitive to acid reflux than the esophagus. However, placebo-controlled trials have failed to demonstrate therapeutic benefit of PPIs. While Reichel et al. and Lam et al. reported symptom improvement in randomized, double-blind, placebo-controlled trials, Vaezi argued that improvement was for heartburn rather than throat symptoms. Where laryngeal symptom improvement has been reported it was found proportionally higher in GERD patients than in those without GERD. Given the paucity of data supporting acid-suppression therapy for extraesophageal symptoms, the American Gastroenterological Association guidelines for GERD recommend against its use for acute treatment of patients with potential extraesophageal reflux (EER) syndromes (laryngitis, chronic cough) absent typical GERD symptoms. Despite such advice, treatment for LPR frequently continues to involve empiric therapy with PPIs.
While the acidity of reflux alone can damage the upper airways, combined multichannel intraluminal impedance-pH (MII-pH) monitoring has demonstrated that many episodes of LPR are nonacidic, and that weakly and nonacidic reflux is associated with persistent symptoms in acid-suppressed patients. These symptoms are alleviated by anti-reflux surgery and may be ameliorated by less invasive strategies that limit reflux occurrence or neutralize reflux constituents beyond acid (e.g. dietary and lifestyle modification and over-the-counter alginate products). Thus, one or more nonacid components of gastric refluxate must have a role in laryngeal damage. There is increasing evidence that pepsin, which is present in all refluxate, is partly, if not wholly, responsible for damage and inflammation caused by LPR.
Pepsin is a proteolytic enzyme which is synthesized and secreted as the zymogen pepsinogen by chief cells in the gastric fundus and subsequently cleaved upon introduction to the acidic stomach lumen to produce pepsin. Pepsin is maximally active at pH2 and retains activity up to pH6.5. While stable at pH8, pepsin is irreversibly inactivated at higher pH. The stomach and esophagus have intrinsic defenses against pepsin (mucus, peristalsis, and bicarbonate secretion), however laryngeal tissues do not. Pepsin is thought to play a key role in mucosal damage and inflammation during nonacidic reflux. At neutral pH, pepsin is taken up by laryngeal and hypopharyngeal cells by receptor-mediated endocytosis and retained in intracellular vesicles of low pH where it is presumed to be reactivated. The consequence is chronic inflammation, which in turn, gives rise to symptoms. Endocytosed nonacidic pepsin induces a proinflammatory cytokine gene expression profile in hypopharyngeal cells similar to that which contributes to disease severity during GERD. Inhibition of the proteolytic activity of pepsin abrogates this damage and inflammation.
With compelling evidence of nonacid proximal reflux of pepsin and its association with laryngeal and pharyngeal symptoms and endoscopic findings, the significant cost and risk of prolonged PPI therapy which continues to date despite its inefficacy in the absence of a gold standard medical therapy, and the limitations of alternative non-surgical treatment options such as the short-lived activity of over-the-counter products intended to provide temporary relief and the burden of adherence to dietary and lifestyle modifications, a new medical treatment which specifically targets pepsin would be of great benefit.
We and others have discussed the promise of inhibitors of peptic activity and/or receptor antagonists as potential new therapeutics for LPR.
In one aspect, the 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.
In another aspect, the disclosure provides a method of treating reflux in a subject in need thereof, the method comprising oral administering the formulation described herein to a subject to treat the reflux.
In the present application, the inventors disclose a novel means to treat reflux conditions, including GERD, airway reflux such as laryngopharyngeal reflux (LPR). The deleterious changes in the laryngopharynx observed in LPR develop following direct contact of the mucosa with refluxed gastric contents, which consist of acid as well as pepsin, bile, and pancreatic enzymes.
The present application provides oral alginate formulations that provide sustained release in a subject, reducing one or more symptom of reflux conditions. This new approach would be amenable to local treatment of readily accessible airways affected by LPR allowing lower dosing, the advantage of which is self-evident in that targeted delivery would simultaneously increase efficacy and limit systemic side effects.
Herein, therapeutic compounds were screened for pepsin binding and inhibition. Specific HIV protease inhibitors that inhibited pepsin were administered orally and by inhalation in an LPR mouse model to assess their potential for the treatment of LPR. Oral alginate formulations that provide extended release were developed that provides the most effective delivery.
Local esophageal treatment of GERD has been an area of interest for several decades, however drug delivery to the esophagus is challenging due to the exceedingly short esophageal transit time of orally administered drugs (<16s even when supine). Thickening agents and mucoadhesive substances that prolong esophageal retention and contact time of liquid suspensions have been investigated to overcome this limitation with efforts to date predominantly focused on substances that coat the esophagus and provide topical protection against refluxed acid, termed “esophageal bandages” by Potts et al. This research led to numerous patents for mucoadhesive carbonate formulations (reviewed by Batchelor 2005). Fosamprenavir retained in the esophagus would inactivate extracellular, mucosal-bound pepsin deposited during reflux; its absorption by the mucosa, facilitated by prolonged contact time would also inactivate endocytosed, intracellular pepsin. The desired formulation would permit esophageal absorption of a fraction of total dosage without dramatically impeding systemic delivery via the intestine.
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 related pain and inflammation. Among mucoadhesive excipients, alginate has emerged as an optimal additive for prolonged esophageal residence time of liquid and solid pharmaceutical formulations.
Alginate is an extensively used bioadhesive polymers for drug delivery as it is non-toxic, biocompatible, non-immunogenic, biodegradable, mucoadhesive, readily available and cost-effective. The U.S. FDA recognizes alginate as “Generally Referred As Safe” (GRAS), or safe for alimentary use by qualified experts, listed in the Code of Federal Regulations Title 21 parts 182 and 184. Its pH-dependent gelation and chemical versatility, amenable to modifications to tailor its properties, has made alginate one of the most extensively explored mucoadhesive biomaterials, finding application in various modified drug delivery systems such as hydrogels, microparticles, nanoparticles and adhesive tablets and films for buccal drug delivery.
Our research demonstrated the assessment of the in vitro esophageal retention characteristics of several widely utilized pharmaceutical coating materials for solid pharmaceutical formulations. We found sodium alginate (1.5% w/w; medium viscosity grade from Mactocystis pyrifera) provided superior esophageal adhesion which was capable of ‘self-repairing’ (re-adhering at the next contact point if dislodged). Batchelor et al. demonstrated that alginate (at 2% w/v in 1 ml dose, i.e. 0.02 mg) adheres to the esophageal mucosa in vitro model for periods of up to 60 minutes. Using excised porcine esophagus with saliva wash simulated by constant wash of human saliva at 1 ml/min, we found that lower molecular weight alginates (<75 kDa) bearing lower viscosity (<0.02 Pa s), demonstrated significantly lower esophageal retention than others tested, while alginates with high viscosity (>2.93 Pa s) demonstrated greater esophageal retention at 3 min but similar retention as mid-range viscosity alginates at 15 or 30 min. G/M ratio of alginates had no effect on esophageal adhesion. An alginate of mid-range molecular weight (240 kDa), viscosity (0.51 Pa s) and G/M ratio (44/56) exhibited 21.9±9.5% retention at 30 min when provided at 0.02 mg in 1 ml dose (in water).
Sodium alginate of medium viscosity provided at the MDE of 24.5 mg per 10 ml dose b.i.d. in our formulation would therefore be predicted to confer 20% retention of bound drug (and retention of ˜5 mg alginate) in the esophagus for at least 30 minutes.
Higher doses of alginate in alginate-antacid medications (1000 mg per dose) have a long history of use as a monotherapy for mild to moderate GERD and a complimentary therapy for breakthrough symptoms of those taking PPIs. The therapeutic benefit of alginate is primarily attributed to its raft-forming activity. The alginate raft floats over stomach contents thereby displacing the postprandial acid pocket near the gastroesophageal junction, and effectively reducing acidic reflux events. Secondary mechanisms of anti-reflux activity are thought to be conferred by its mucoadhesion, which prevents diffusion of pepsin and acid such to underlying esophageal mucosa and concomitant epithelial barrier dysfunction, and enzymatic inhibition of pepsin. Notably, Chater et al. found that mid-range viscosity alginate (LF120) at just 0.68 mg/ml inhibited pepsin by 28.46±10.68% (at 1.36 mg/ml inhibition was 44.73±10.98). While this suggests that excipient alginate at the MDE (2.45 mg/ml b.i.d.) could have anti-peptic activity, the therapeutic value of alginate at such low concentrations is unlikely given numerous clinical trials and metanalysis reporting the therapeutic benefit of anti-reflux medications containing 1000 mg sodium alginate per 10 ml dose (Gaviscon Advance, Reckitt Benckiser, Slough, UK), relative to viscosity-matched placebo or products intended primarily as antacids which provided only transient symptom relief regardless of presence of alginate at excipient concentration (Gaviscon tablet or liquid antacid, GlaxoSmithKline Consumer Healthcare, Pennsylvania). The lack of therapeutic benefit of the latter is thought to be due to their inability to form coherent rafts.
Alginate-based anti-reflux medications have recently demonstrated therapeutic efficacy for throat symptoms of LPR. As for esophageal symptoms, therapeutic activity is attributed to raft-formation in the stomach, particularly given improbable contact between orally administered alginate and the larynx. McGlashan et al.27 examined the efficacy of alginates for LPR symptoms and endoscopic findings in 49 patients with RSI and RFS-confirmed LPR randomized to receive liquid alginate suspension (n=24; 10 ml, four times daily post meal and at bedtime; Gaviscon Advance) or no treatment (n=25; control): pre-treatment mean (SD) RSI and RFS scores were similar for the treatment group (23.9 (7.0) and 10.4 (3.6)) and controls (24.6 (7.4) and 10.3 (3.3)), however alginate treatment improved LPR symptoms and findings as indicated by significant differences between treatment and control group RSI at 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 treatments for LPR subtypes (acid to alkaline), Lechien et al.26 found that an anti-reflux diet and thrice daily post meal alginate (Gaviscon Advance) or magaldrate (Riopan, Takeda, Zaventem, Belgium) improved voice quality of patients with HEMII-pH testing-confirmed alkaline LPR (n=48) as indicated by reduced dysphonia and roughness scores (GRBAS scale) and improved jitter, shimmer and noise to harmonic ratio (Lechien et al. 2021).
The present invention provides a sustained-release formulation of oral fosamprenavir, using sodium alginate toincrease muco-adhesion and prolong drug delivery in the esophagus, that will improve esophageal symptoms in the 25-50% LPR patients that also have GERD, and thus have superior efficacy over oral fosamprenavir/Lexiva.
In one embodiment, the formulation contains a low excipient level dose of sodium alginate to prolong drug delivery to the esophagus by increasing muco-adhesion. This is expected to benefit pepsin-mediated esophageal inflammation, mucosal damage and associated symptoms. While high doses of alginate are expected to have therapeutic benefit due to raft formation, the present lower dose formulations are expected to increase muco-adhesion to prolong esophageal retention.
In-vitro tests for esophageal retention will include a texture analyzer for muco-adhesion.
Studies using combined multichannel intraluminal impedance with pH (MII-pH) monitoring have shown that many episodes of LPR are nonacidic, and that weakly and nonacidic reflux is associated with persistent symptoms in acid-suppressed patients (39-42). Pepsin, the chief digestive enzyme in the stomach, has been increasingly implicated as contributing to the damage and inflammation associated with LPR (17-23). Importantly, while the stomach and esophagus have internal defense mechanisms against pepsin, such as mucus, peristalsis, and bicarbonate secretion, laryngeal tissues do not (26). In the airways, which have a neutral pH (below 8), pepsin is enzymatically inactive but stable. However, when pepsin is taken up by laryngeal and hypopharyngeal cells via receptor-mediated endocytosis, it is retained in intracellular vesicles of low pH where it is presumed to be reactivated and cause damage (20, 32, 33, 49, 52). While many episodes of LPR are weakly acidic or nonacidic, pepsin is present in all refluxate (24), and is frequently detected in airway tissue and secretions from patients with LPR. For example, the inventors have demonstrated that endocytosed nonacidic pepsin induces expression of proinflammatory cytokine genes in hypopharyngeal cells. This response is similar to the response that occurs in reflux esophagitis, which contributes to disease severity in GERD patients (21, 31). Importantly, inhibition of pepsin's proteolytic activity (i.e., using pepstatin, curcumin, ecabet sodium, anthocyanin, or pre-incubation at pH 8.0 before decreasing 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.
The present inventors believe that LPR is more dependent on pepsin-mediated damage than on acid-mediated damage, and that drugs that specifically target pepsin should be effective for patients with nonacid reflux. These drugs could finally provide a treatment option for patients who are refractory to proton pump inhibitors (PPI). Pepsin can be inhibited by two mechanisms: (1) via irreversible inactivation, which prevents it from becoming reactivated inside intracellular compartments of lower pH, and (2) via a receptor antagonist, which prevents pepsin uptake by receptor-mediated endocytosis. While the pepsin inhibitor pepstatin is already commercially available, it has poor water-soluble characteristics and pharmacokinetic properties. Thus, new pepsin inhibitor compounds with greater bioavailability are needed.
In the present 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 abilities (see Example 1). Several HIV protease inhibitors have already been approved by the U.S. Food and Drug Administration (FDA) for the treatment of HIV, making these drugs ideal candidates to test the efficacy of pepsin inhibition for 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 drugs might be repurposed to treat LPR. Of the ten commercially available HIV protease inhibitors, the inventors determined that four (i.e., amprenavir, darunavir, ritonavir, and saquinavir) have the ability to bind to and inhibit pepsin activity in vitro (
The present invention provides methods of treating reflux in a subject in need thereof, preferably airway reflux. The methods involve administering a therapeutically effective amount of an formulation comprising an HIV protease inhibitor and alginate to a subject to treat the reflux. As used herein, the term “airway reflux” refers to inflammation of the upper and lower airways caused by reflux of gastric contents. The term airway reflux is used interchangeably with the alternative terms “supraoesophageal reflux” and “extraoesophageal reflux.” These broad terms encompass several related reflux conditions, which include gastropharyngeal reflux (GPR; the backflow of gastric contents up to the esophagus), laryngopharyngeal reflux (LPR; the backflow of gastric contents beyond the esophagus into the laryngopharynx), and esophagopharyngeal reflux (EPR; a similar condition to LPR that is characterized by esophageal abnormalities). Reflux also includes gastroesophageal reflux disease (GERD) which refers to irritation of the esophagus caused by reflux of stomach's contents back up into the esophagus. The reflux treated herein is preferably GERD patients that are refractory to protein pump inhibitor (PPI) therapy.
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 proteolytic cleavage of protein precursors that are necessary for the production of infectious viral particles. Suitable HIV protease inhibitors include those that have been approved by the 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]carbamoyl]amino]butanoyl]amino]-1,6-diphenylhexan-2-yl]carbamate), lopinavir (IUPAC: (2S)-N-[(2S,4S,5S)-5-[[2-(2,6-dimethylphenoxy)acetyl]amino]-4-hydroxy-1,6-diphenylhexan-2-yl]-3-methyl-2-(2-oxo-1,3-diazinan-1-yl)butanamide), saquinavir (IUPAC: (2S)-N-[(2S,3R)-4-[(3S,4aS,8aS)-3-(tert-butylcarbamoyl)-3,4,4a,5,6,7,8,8a-octahydro-1H-isoquinolin-2-yl]-3-hydroxy-1-phenylbutan-2-yl]-2-(quinoline-2-carbonylamino)butanediamide), nelfinavir (IUPAC: (3S,4aS,8aS)-N-tert-butyl-2-[(2R,3R)-2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-phenylsulfanylbutyl]-3,4,4a,5,6,7,8,8a-octahydro-1H-isoquinoline-3-carboxamide), darunavir (IUPAC: [(3aS,4R,6aR)-2,3,3a,4,5,6a-hexahydrofuro[2,3-b]furan-4-yl]N-[(2S,3R)-4-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-1-phenylbutan-2-yl]carbamate), indinavir ((2S)-1-[(2S,4R)-4-benzyl-2-hydroxy-5-[[(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]amino]-5-oxopentyl]-N-tert-butyl-4-(pyridin-3-ylmethyl)piperazine-2-carboxamide), atazanavir (IUPAC: methyl N-[(2S)-1-[2-[(2S,3S)-2-hydroxy-3-[[(2S)-2-(methoxycarbonylamino)-3,3-dimethylbutanoyl]amino]-4-phenylbutyl]-2-[(4-pyridin-2-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 N-[(2R,5R)-5-[[(2S)-2-[[methyl-[(2-propan-2-yl-1,3-thiazol-4-yl)methyl]carbamoyl]amino]-4-morpholin-4-ylbutanoyl]amino]-1,6-diphenylhexan-2-yl]carbamate). The HIV protease inhibitor used with the present invention should be capable of binding to and inhibiting the enzymatic activity of pepsin. Thus, in some embodiments, the HIV protease inhibitor is amprenavir, darunavir, ritonavir, or saquinavir, which were shown to bind to and inhibit pepsin in Example 1. 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 commercially available.
Fosamprenavir is a prodrug of amprenavir that is marketed by ViiV Healthcare as a calcium salt under the trade names Lexiva (U.S.) and Telzir (Europe). The body must metabolize fosamprenavir to form amprenavir, which is the active form of the drug. Thus, administering amprenavir as a prodrug prolongs the duration of time that it is available in the body, acting like a slow release formulation. Further, fosamprenavir has shown excellent pharmacokinetics in mice and because it is already FDA approved, fosamprenavir could be fast-tracked into a pilot clinical trial. In some embodiments, the HIV protease inhibitors for use in the compositions and methods described herein have an IC50 in the micromolar range (μm). In some preferred embodiments, the HIV protease inhibitors for use in the compositions and methods described herein have an IC50 in the nanomolar (nm) range.
In the present methods, the HIV protease inhibitor may be administered using any route that is effective for the treatment of 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 preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration. Administration can be continuous or intermittent.
In some embodiments, the HIV protease inhibitor is administered orally for the treatment of the reflux. For example, in some embodiments, the HIV protease inhibitor is administered twice daily at about 0.7-1.4 g (i.e., a dosage that is FDA-approved for the treatment of HIV and thus safe).
The methods of the present invention are used to treat to reflux in a subject in need thereof. In some embodiments, the reflux may be airway reflux. In other embodiments, the reflux may be GERD, preferably GERD in a subject that is refractory to proton pump inhibition. As used herein, the term “subject in need thereof” or “patient” refers to any human or animal suffering from reflux. In some embodiments, the subject has an airway reflux. In some embodiments, the airway reflux condition selected from laryngopharyngeal reflux (LPR), gastropharyngeal reflux (GPR), and esophagopharyngeal reflux (EPR). In some embodiments, the subject is a subject with reflux episodes caused by weakly acidic or nonacidic reflux. In another embodiment, the subject is a subject refractory to proton pump inhibitor (PPI) therapy.
As used herein, the terms “treat”, “treating” or “treatment” describes the management and care of a subject for the purpose of combating a disease, condition, or disorder. Treating includes the administration of protease inhibitor or composition of present invention to prevent the onset of the symptoms or complications, to alleviate the symptoms or complications, or to eliminate the disease, condition, or disorder. In preferred embodiments, the methods and compositions of the present reduce mucosal damage and inflammation in the airway of the subject. Treatment also includes reducing one or more symptoms of airway reflux, suitably LPR, GPR or ERP, for example, reduction of chronic cough, throat clearing, postnasal drip, hoarseness or dysphonia, globus sensation, dysphagia, dyspnea, or combinations thereof. Treatment also includes reducing chronic laryngeal irritation and inflammation. Treatment in one embodiment also includes reducing one or more symptom of GERD that is refractory to PPI, for example, reducing one or more of the following symptoms: a burning sensation in your chest (heartburn), usually after eating, which might be worse at night, chest pain, difficulty swallowing, regurgitation of food or sour liquid, sensation of a lump in your throat, among others.
The terms “effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological or clinical results. That result can be reducing, alleviating, inhibiting or preventing one or more symptoms of a disease or condition, reducing, inhibiting or preventing laryngeal irritation, reducing or inhibiting laryngeal irritation or mucosal damage, or reducing, alleviating, inhibiting or preventing one or more symptoms of airway reflux, or any other desired alteration of a biological system. In some embodiments, the effective amount is an amount suitable to provide the desired effect, e.g., reduce mucosal damage and inflammation in the airway. The response to a treatment of airway reflux may be assessed using any standard clinical method including, without limitation, visual inspection of the larynx (e.g., fiberoptic laryngeal exam), a reflux symptom index (RSI), reflux finding score (RFS) (e.g., physician reported score based on visual inspection of the larynx), combined esophageal multichannel intraluminal impedance and pH monitoring (MII-pH), reflux symptom score (RSS), reflux sign assessment (RSA), or pepsin activity within the saliva. Alternatively, the response to a treatment of airway reflux may be assessed using by evaluating the inflammation in a tissue sample taken from the airway of the subject, for example, by hematoxylin and eosin (H&E) staining or by detection of the presence of neutrophil infiltrate, keratinization, and necrosis. Another suitable method is measure pepsin activity pre and post 12-week treatment. While it is not expected that the HIV inhibitor will prevent reflux or affect pepsin protein levels, it will inactivate the pepsin enzyme, therefore measuring pepsin activity in saliva post-treatment would confirm that the treatment is inactivating pepsin in the airway. This is currently a research tool to assess efficacy in vivo.
Patients with reflux episodes caused by weakly acidic or nonacidic reflux are largely refractory to proton pump inhibitor (PPI) therapy, which suppresses acid production but does not affect pepsin activity. The methods of the present invention will be of particular benefit to this group of refractory patients, who are in desperate need of an alternative to PPIs. As used herein, the phrase “refectory to treatment” refers to a condition that does not respond to treatment. For example, a patient's reflux may be deemed refractory to PPI therapy if a three-month long, twice-daily treatment with a PPI fails to improve the condition substantially. The response to a treatment of reflux may be assessed using any standard means known in the art including, without limitation, a reflux symptom index (RSI), reflux finding score (RFS), combined esophageal multichannel intraluminal impedance and pH monitoring (MII-pH), reflux symptom score (RSS), or reflux sign assessment (RSA). See the Examples section for a more detailed description of these measures. For example, an effective treatment would decrease the RSI and/or the RFS to normative values, e.g., RSI≤13, RFS≤7 or a combination thereof.
The present invention also provides compositions comprising an oral formulation of an HIV protease inhibitor and an alginate and a pharmaceutically acceptable carrier. Commercially available HIV protease inhibitors are commonly formulated as tablets or oral suspensions for systemic drug delivery. For example, in some embodiments, the composition is formulated for oral administration.
The compositions of the present invention may include any pharmaceutically acceptable carrier that allows for oral delivery. “Pharmaceutically acceptable carriers” are known in the art and include, but are not limited to, for example, suitable diluents, preservatives, solubilizers, emulsifiers, liposomes, nanoparticles, and adjuvants. Pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include isotonic solutions, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
The compositions of the present invention may further include additional components to influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of the HIV protease inhibitor. Suitable components include, without limitation, 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 acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances, and tonicity modifiers (e.g., lactose, mannitol). Additionally, the compositions may be formulated for controlled or sustained release of the HIV protease inhibitor, for example, via formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
The composition of the present invention may further include a suspending agent, a preservative, a sweetener, a flavoring, water, and a combination thereof. Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, Cremophore™ 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, coloring and sweetening agents as appropriate. Preparations for oral administration may also be suitably formulated to give controlled release of the compound, as is well known.
Table 1 provides a proposed formula. It is expected that the API amount will be about 20-30% of the total weight of the dry product formulation.
The compositions may be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome.
The HIV protease inhibitor included in the compositions of the present invention may be any HIV protease inhibitor that is suitable for the treatment of airway reflux, as discussed above. In some embodiments, the HIV protease inhibitor included in the composition is amprenavir, darunavir, ritonavir, saquinavir, or a derivative thereof. In preferred embodiments, the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir. In another embodiment, the HIV protease inhibitor is darunavir.
The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.
It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as “comprising” certain elements are also contemplated as “consisting essentially of” and “consisting of” those elements. The term “consisting essentially of” and “consisting of” should be interpreted in line with the MPEP and relevant Federal Circuit interpretation. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. “Consisting of” is a closed term that excludes any element, step or ingredient not specified in the claim. For example, with regard to sequences “consisting of” refers to the sequence listed in the SEQ ID NO. and does refer to larger sequences that may contain the SEQ ID as a portion thereof.
The references cited herein are explicitly incorporated by reference in their entireties.
The invention will be more fully understood upon consideration of the following non-limiting examples.
Reference is made to Johnston, N., Samuels, T. L., Goetz, C. J, Arnold, L. A., Smith, B. C., Seabloom, D., Wuertz, B., Ondrey, F., Wiedmann, T. S., Vuksanovic, N., Silvaggi, N. R., MacKinnon, A. C., Miller, J., Bock, J. and Blumin, J H. (2022), Oral and Inhaled Fosamprenavir Reverses Pepsin-Induced Damage in a Laryngopharyngeal Reflux Mouse Model. The Laryngoscope. and is incorporated by reference.
To examine whether HIV protease inhibitors bound and inhibited pepsin, we developed assays based on fluorescence polarization which measures size-dependent molecular rotation thereby permitting detection of degradation, association and dissociation events80. A competitive binding assay was designed employing pepstatin, an inhibitor of sub-nanomolar affinity81. Pepstatin-Alexa647 was synthesized by dissolving 1 mg pepstatin A (Sigma-Aldrich) in a 50:50 mixture of dimethylformamide (DMF) and dimethylsulfoxide (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, after which 1 mg Alexa Fluor 647 Cadaverine, Disodium Salt (ThermoFisher Scientific) was added. After 2 hours, the solvents were evaporated under high vacuum (35° C.) and residue partially dissolved in 10% methanol and transferred onto a C18 cartridge (Waters Corporation, Milford, MA). Increasing percentages of methanol were used for the elution. Pepstatin-Alexa647 eluted at 45% methanol. An enzymatic inhibition assay was designed using casein substrate.82 Bovine alpha 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 combined at 2.5 ug/mg label to protein ratio in 0.1M sodium bicarbonate for 15 minutes and labeled casein was separated from unbound label in a Sephadex G-25 (Sigma-Aldrich) column comprised of 90×5 mm packed beads in a glass Pasteur pipette, eluted with dPBS pH7.4 (ThermoFisher Scientific). The fast-moving band (casein-bound fluorophore) was collected in ˜0.4 ml volume. Concentration of resultant probe (casein-Alexa647 in PBS-azide) was estimated via spectrophotometry using Beer's law (Implen Nanophotometer, Implen, Inc. Westlake Village, CA).
Assays were optimized using ranges 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 20 μl volumes in 384-well black optical plates (Nunc, Roskilde, DK) and read on a BioTek Cytation 5 (BioTek Instruments, Winooski, VT) with far red FP filter cube (excitation/emission 620/680 nm). Unlabeled pepstatin dose-response curves were used to ensure that the assays were responsive to pepsin inhibition. Conditions yielding maximal dynamic assay range were used to assess HIV protease inhibitors: 100 nM probe, 0.03 U/ul porcine pepsin A, 37.5% DMSO for competitive binding assay, and 200 nM probe, 0.01 U/μL pepsin, 5% DMSO for peptic activity assay. The HIV protease inhibitors (amprenavir, ritonavir, lopinavir, saquinavir mesylate, nelfinavir mesylate hydrate, darunavir ethanolate, indinavir sulfate salt hydrate; all Sigma-Aldrich) were dissolved in DMSO and tested under optimized assay conditions over three logs concentration. Assays were performed twice with triplicate reactions read for five minutes and mean mP plotted against probe concentration (binding assay) or read at <2 minutes intervals over 30 minutes with mean mP of plotted over time (activity assay). Half maximal inhibitory concentration (IC50) of inhibitors were calculated from kinetic traces analyzed using an online tool (https://icekat.herokuapp.com/icekat)83. mP was normalized to blank (absent inhibitor) to derive percent bound or activity.
Saturated solutions of HIV protease inhibitors (amprenavir, ritonavir and darunavir ethanolate) were prepared in DMSO and centrifuged for 10 minutes at 31,000 rcf Supernatants were added to pepsin (200 mg/ml in water) at 1.6% (v/v) f.c. Due to poor solubility, a solvent for saquinavir mesylate was selected from the CryoSol screen (Molecular Dimensions, Holland, OH). CryoSol mixture SM2 (consisting of 37.5% v/v dioxane, 25% v/v DMSO, 12.5% v/v ethylene glycol, 12.5% v/v 1,2-propanediol, and 12.5% v/v glycerol) was selected as it provided both high solubility and protein compatible conditions for the co-crystallization mixture. Supernatant of saturated saquinivar solution in SM2 was combined with pepsin at 5% f.c. (v/v). Crystallization conditions were optimized by screening 200 mg/ml pepsin in the Salt RX screen (Hampton Research, Viejo, CA). Small bipyramid-shaped crystals formed in 3.5M ammonium chloride and 0.1 M sodium acetate trihydrate pH 4.6 after one week at room temperature served as microseed stock for co-crystallization with amprenavir, ritonavir and darunavir ethanolate per previously described methods84. Diffraction quality crystals (triangular bi-pyramids, approximately 200×100×100 μm) formed after 2-7 days from hanging drops of 2 ul pepsin (180-210 mg/ml) and 1 ul microseed solution serially diluted 10-100× above 3-4M ammonium chloride and 0.1M sodium acetate trihydrate pH4.6. Crystals were cryoprotected by 30% glucose, 5M ammonium chloride and 0.1 M sodium acetate trihydrate pH4.6 and plunged in liquid nitrogen. Co-crystallization with saquinavir was performed in 0.1M acetic acid rather than sodium acetate trihydrate as this permitted large crystal formation without a microseed; crystals were cryoprotected by 30% w/v glucose, 5M ammonium chloride and 0.1 M sodium acetate trihydrate pH 4.6 and plunged in liquid nitrogen.
Diffraction datasets were collected at Life Sciences Collaborative Access Team (LS-CAT) beamlines at the Advanced Photon Source (APS), Argonne National Laboratory, equipped with MAR 300 CCD or Dectris Eiger 9M detectors and data were indexed, integrated and scaled using MOSFLM85 or HKL200086.
Specifically, for pepsin:amprenavir, a 1.9 Å diffraction data set was collected at LS-CAT beamline 21-ID-F with a MAR 300 CCD detector using a 50×50 μm beam at a wavelength of 0.97872 Å. A total of 262 frames were collected from φ=0 to 130.5° with an oscillation range of 0.5° and detector distance of 250 mm. Exposure time was 0.5 seconds. Diffraction data were indexed, integrated and scaled using MOSFLM.
For pepsin:ritonavir, a 2.1 diffraction data set was collected at LS-CAT beamline 21-ID-D with Dectris Eiger 9M detector using a 50×50 μm beam at 1.12721 Å. 900 frames were collected from φ=0 to 180°, while oscillating at a rate of 1°/sec and slicing of 5 images/°. Crystal-to-detector distance was 160 mm. Diffraction data were indexed, integrated and scaled using MOSFLM.
For pepsin:darunavir, a 1.9 Å diffraction data set was collected at LS-CAT beamline 21-ID-G with MAR 300 CCD detector and 50×50 μm beam at 0.97856 Å. 900 frames were collected from φ=0 to 180° with an oscillation range of 0.2° and detector distance of 260 mm. Exposure time was 0.3 seconds. Diffraction data were indexed, integrated and scaled using HKL2000.
For pepsin:saquinavir, a 1.9 Å diffraction data set was collected at LS-CAT beamline 21-ID-F with MAR 300 CCD detector using a 50×50 μm beam at 0.97872 Å. 400 frames were collected from φ=20 to 100° with an oscillation range of 0.2° and detector distance of 200 mm. Exposure time was 0.5 seconds. Diffraction data were indexed, integrated and scaled using MOSFLM.
Initial phases were obtained by molecular replacement in PHASER87. Unliganded porcine pepsin (PDB ID 4PEP) with B factors reset to 20.00 Å and solvent molecules removed was the search model. Model refinement was performed using phenix.refine (PHENIX87-89) and COOT90,91. Geometric restraints for compounds were obtained from CCP4 monomer library92. Models were validated using MolProbity93 as implemented in the PHENIX suite. Models of ritonavir and saquinavir were additionally optimized using PDB-REDO server 94 prior to deposition. Electron density maps were generated via POVSCRIPT and POV-Ray and schematic representation by MarvinSketch,(http://www.ChemAxon.com) and Adobe Illustrator CC 2020.
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 anticipated to suffice for verification of reproducibility in each experiment without excessive use of animal life. The three mice were randomly allocated to treatment groups. No data were excluded from analysis.
Six-week-old female Jackson A/J mice (Jackson Laboratory, Bar Harbor, ME) were fed D-62 powdered Wattenberg diet, 2 g/mouse/day95 and allowed to acclimate for one week upon arrival prior to experiments. In accord with previously established methods for modeling aerodigestive tract damage attributed to GERD and LPR,1,95-99 mechanical injury applied during the first two weeks of a four-week treatment course was used to predispose the laryngeal mucosa to chemical injury by pepsin/acid applied throughout the four weeks. When performed in this manner, mechanical injury increases mucosal susceptibility to subsequent chemical injury while leaving little detectable injury at the conclusion of a four-week treatment course.95 Mechanical injury was performed on all animals (including control) once weekly during the first two weeks of treatment as described (see experimental schema,
In a preliminary experiment to validate the LPR mouse model (i.e. laryngeal damage by pepsin at neutral and acidic pH), 20 μl saline (solvent control) or 0.3 mg/ml pepsin at pH7.0 or 4.0 were provided to mice (n=3) by laryngeal instillation at 24, 48, and 72 hours after mechanical injury during weeks 1 and 2 (
To test the protective effect of HIV protease inhibitors on pepsin-mediated damage in vivo, inhibitors were delivered by aerosol or gavage concurrently with wounding (days 2, 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 sacrificed day 26. Mice were anesthetized with isoflurane (3% in 2.5LPM, 3-5 minutes prior to procedures) as opposed to Avertin due to frequency. Lexiva and Prezista (hereafter referred to by generic: fosamprenavir and darunavir, respectively) were used for gavage, and respective pure drugs for aerosol (fosamprenavir from Anant Pharmaceuticals, Ambernath, Maharashtra India and darunavir from Ambeed, Arlington Heights, IL). Gavage dose was equivalent to that prescribed to HIV patients (20 mg/kg/day fosamprenavir; 8.6 mg/kg/day darunavir). Aerosol was generated as described100. Briefly, a 10 ml suspension of drug in ethanol was placed in the baffle, such that the concentration would remain constant at the equilibrium solubility. Droplets of ethanol containing dissolved drug were generated by an ultrasonic atomizer (nominal frequency 1.7 MHz) and entrained by air at a flow rate of 0.5 LPM with a custom-built glass baffle (UMN Department of Chemistry 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 emanating dry aerosol particles of pure drug were then directed into the exposure chamber. The mass deposited on filters was measured gravimetrically and 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 mass of drug (Minh) for each mouse was defined as Minh=[Aerosol]*RMV*t, where Aerosol is the aerosol concentration of drug, RMV is the respiratory minute volume of the mice (0.025 L/min), and t is the aerosol exposure time. Aerosol concentration was 0.09 mg/L fosamprenavir or 1.2 mg/L darunavir, therefore given the respiratory minute volume of mice (0.025 L/min), the inhaled mass was 0.93 mg/kg/day fosamprenavir or 12 mg/kg/day darunavir. Actual mass deposited was not determined but anticipated to be 10% of inhaled mass (the deposition fraction of 1 m aerosol particles in mice).
Tissues were collected, fixed in paraformaldehyde, embedded in paraffin and 4 um sections stained with hematoxylin and eosin (H&E) via automated stainer. Slides were reviewed by a board-certified pathologist (JM) blinded to treatment groups.
Four of the seven assayed HIV protease inhibitors bound and inhibited pepsin at low micromolar concentrations (
To aid interpretation of the in vitro binding and inhibition data, commercially available porcine pepsin (EC 3.4.23.1) was used for co-crystallization experiments to obtain structural data. Crystallization of human pepsin collected from volunteers failed presumably due to sample heterogeneity. Porcine pepsin shares 86% sequence identity with the human enzyme (PDB ID 1PSN)101 and its structure is nearly identical (root mean square deviation (RMSD) for all Cα atoms=0.50 Å). Minor differences in tertiary structure are localized to a loop of residues (277-282) which is not part of the binding cleft. Residues lining the active site cleft are highly conserved: of 17 making direct contact with inhibitors herein, just two differed (T12 and V291). Thus, porcine was deemed an acceptable substitute for human pepsin for assessing structural biology.
Porcine pepsin was co-crystalized amprenavir, darunavir, ritonavir, and saquinavir (Table 2 and
I/σ(I)
a
aValues in parentheses apply to the high-resolution shell indicated in the resolution row
bMaximum-likelihood based estimates of coordinate error
Pepsin-mediated laryngeal epithelial damage was observed at pH 4 and 7 in the mouse in vivo model which employed pepsin with or without acid exposure following mechanical injury of the larynx (
Fosamprenavir gavage equivalent to the dose used to treat HIV in humans prevented pepsin-mediated laryngeal damage, defined as reactive epithelia, increased intraepithelial inflammatory cells, and apoptosis (
For the past two decades, the treatment of LPR has focused on suppressing gastric acid production. With the introduction of MII-pH technology, it is now understood that LPR is commonly nonacidic and that nonacid proximal events are associated with laryngeal endoscopic signs and symptoms39-46,48-50,102. These findings sparked investigations into the nonacidic components of gastric refluxate.
Although bile induces mucosal damage at weakly and non-acid pH experimentally, it has been argued that “there is no evidence that the same mechanism occurs in the human larynx”57. The clinical relevance of experimental findings has been called into question. Unconjugated bile acids, which cause damage at neutral-high pH such as that of the laryngopharynx, are rarely found in gastric refluxate.56,69 Further, concentrations of bile salts/acids found to damage the larynx and hypopharynx experimentally are 1000-fold greater than those reported in the airways of patients with LPR, GERD and asthma, or lung disease (0.3-50 mM96,103,104 versus 0.8-32 uM105-109) and result in morphologic changes inconsistent with those of LPR patients such as cell membrane ‘blebbing’ 110.
Pepsin is present in all refluxate55. Moreover, it is frequently detected in airway tissue and secretions from LPR patients but absent in MII-pH-confirmed reflux-free subjects, and thus may be predictive of reflux-attributed symptoms and disease20,39,46,50,55,59,65,67,68,111,112 Pepsin at 1 mg/ml in the stomach is diluted by saliva as it is refluxed proximally. A range of concentrations have been reported in airways: 2.5 μg/ml in saliva, 61.5 μg/ml in nasal secretions113,114 and 360 μg/ml in middle ear fluid115. To model chronic LPR within a limited experimental timeframe, 300 μg/ml was employed herein1,77,116,117. Pepsin-mediated damage and inflammatory changes reported in vitro and in vivo, including the histologic changes herein, are consistent with those observed in LPR patients62-64,66,70,118-122. Compelling evidence from multiple groups highlights a major role for pepsin, independent of gastric acid, in reflux-attributed laryngeal symptoms and findings refractory to PPI therapy.
While pepstatin is a potent pepsin inhibitor, its poor water-solubility and pharmacokinetic properties make it a suboptimal therapeutic candidate. Structural data herein indicated that inhibitor binding to the active cleft of pepsin is predominantly stabilized by van der Waals contacts, making rational design of inhibitors difficult. Testing existing inhibitors of other aspartic proteases was therefore deemed the most efficacious route for identification of a pepsin-targeting therapeutic.
There are currently ten commercially available HIV protease inhibitors.123 Seven were amenable to testing in our in vitro binding and inhibition assays and four (amprenavir, ritonavir, saquinavir and darunavir) bound and inhibited pepsin with IC50 in the low micromolar range, validating our hypothesis that existing therapeutic protease inhibitors may exhibit anti-peptic activity. Two drugs were selected for in vivo study based on anti-peptic activity from in vitro assays, cost and reported side effects. While saquinavir exhibits known side effects and interactions (QT prolongation, heart block, high blood lipids and liver problems) and has high cost, amprenavir, ritonavir, and darunavir have minimal side effects (diarrhea, nausea and vomiting).123 Darunavir is more costly than amprenavir and ritonavir, but had the lowest IC50 for pepsin. Darunavir, with the lowest IC50, and fosamprenavir, a prodrug of amprenavir with improved bioavailability and favorable tolerability were therefore selected for assessment in vivo. Given that proximal reflux is inconsistent in surgical models of GER124, we employed a model involving mechanical wounding and pepsin/acid instillation which reliably replicates epithelial alterations similar to that observed in patients with LPR1,63,70,118,119,125. Using this model, the human-equivalent dose of fosamprenavir, but not darunavir, prevented pepsin-mediated laryngeal damage. When administered locally by inhalation, treatment with either compound preserved normal laryngeal histology despite pepsin exposure.
The study herein was intended to investigate whether a pepsin inhibitor may prevent laryngeal damage caused by pepsin exposure in vivo. As with any experimental observation, caution should be exercised when translating in vivo findings from a limited number of animals to the clinical situation. Potential differences between mouse and human respiratory pathobiology should be kept in mind while evaluating the clinical implications of these data. Established methods for in vivo modeling of aerodigestive tract damage attributed to GERD and LPR1, 96-99 were utilized herein and demonstrated mucosal damage consistent with the clinical presentation of LPR supporting their use for assessing drug prevention of LPR-attributed injury: at the four-week conclusion of treatment, no mucosal damage was detectable given mechanical injury and neutral solvent, whereas multi-layered, reactive epithelia with apoptosis was observed in the pepsin and acid treatment groups. The mouse epiglottis occupies a transitional zone from stratified squamous epithelium of the vocal fold to ciliated pseudostratified columnar epithelium at the supraglottis and infraglottis. To avoid misinterpreting squamous epithelium of the vocal folds as signs of injury, representative images were collected rostral to vocal folds, exclusively from tissue with visible thyroid to serve as a guide. Additional features of reactive epithelia (darkened nuclei, variable nuclear diameter, and increased nuclear to cytoplasmic ratio, intraepithelial inflammatory cells, and apoptosis) in pepsin-treated groups, absent in control pH7.0 and those receiving fosamprenavir or darunavir, confirmed epithelial reactivity due to pepsin and the efficacy of HIV protease for prevention of pepsin-mediated injury. While these data are qualitative and would be bolstered by less subjective quantitative measures, the evidence herein provides initial proof-of-concept that a pepsin-targeting therapeutic may reduce mucosal damage akin to that observed in LPR patients and supports more in-depth investigation. Research is ongoing in our laboratory to examine fosamprenavir protection against pepsin-mediated changes in laryngeal cell viability and inflammatory and carcinogenic gene and protein expressions. Further research is also warranted to determine whether laryngeal protection by fosamprenavir aerosol in vivo was due to systemic activity or local conversion to amprenavir. The intestine is the primary site of fosamprenavir metabolism. Conversion of fosamprenavir to amprenavir by alkaline phosphatase (ALP), which is required for its transepithelial flux and subsequent metabolism by cytochrome P450 enzymes, has been shown to occur via intestinal ALP at or near the surface of Caco-2 cells.128,129 It is possible, however, that inhaled fosamprenavir is converted to amprenavir in the airways by serum ALP, just as similar phosphate ester prodrugs are converted by sera collected from healthy subjects. Inhaled fosamprenavir may also be converted by salivary ALP or that expressed by respiratory mucosa and immune cells recruited to tissue injury. Given that ALP is elevated during inflammation132-134 and carcinogenesis including that of the larynx to which LPR contributes,10,74,135-137 ALP may be elevated in LPR-damaged airways thereby increasing fosamprenavir conversion at the desired site of activity. Drug formulations that prolong retention in the aerodigestive tract could further improve local drug conversion and topical activity. Research is ongoing in our laboratory to examine the efficiency of fosamprenavir conversion by laryngeal epithelium, saliva and sera and a dose-response study is underway in the in vivo mouse model to compare the relative efficacies of inhaled fosamprenavir and amprenavir against pepsin-mediated damage.
While additional experimental data will aid our understanding of laryngeal protection by fosamprenavir, LPR symptom improvement will be the ultimate determinant of a successful medical therapy. A randomized placebo-controlled trial therefore represents the best test of a therapeutic compound. Such a trial of fosamprenavir is feasible given that an oral formulation is FDA-approved and an a priori responder definition of clinically meaningful symptom improvement has been established per FDA guidelines.138 Intriguingly, pilot epidemiological data (unpublished) support the therapeutic potential of HIV protease inhibitors for LPR and warrant follow-up: among 2,062 adult HIV patients prescribed an HIV protease inhibitor (Froedtert Memorial Lutheran Hospital, Milwaukee, WI, July 2014-2016; Medical College of Wisconsin Institutional Review Board, 13874) just 0.2% had documented LPR whereas the incidence in the general population is 10-34%.139,140 These data lend preliminary support for clinical investigation of fosamprenavir as a novel therapeutic approach for LPR.
Compelling evidence highlights a major role for pepsin (independent of gastric acid) in reflux-attributed laryngeal symptoms and endoscopic findings refractory to PPI therapy. Fosamprenavir and darunavir, FDA-approved retroviral therapies for HIV/AIDS, bind and inhibit pepsin, abrogating pepsin-mediated laryngeal inflammation and mucosal damage in an LPR mouse model. These drugs target a foreign virus so are ideal to repurpose, allowing a clinical trial to assess efficacy for a much-needed medical treatment for patients faster than could be achieved with novel compounds. Reformulation for local inhaled delivery could further improve outcomes and limit side effects.
Structural data are available in the Worldwide Protein Databank (accession codes 6XCY, 6XCT, 6XCZ, 6XD2; http://www.wwpdb.org/).
SEQ ID NO:1 Synthetic peptide substrate for pepsin Lys-Pro-Ala-Glu-Phe-PNP-Arg-Leu (PNP=paranitrophenylalanine)
The following disclosure can be described in accordance with the following numbered clauses.
This application claims priority to U.S. Application No. 63/392,929, filed on Jul. 28, 2022, the entire contents of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63392929 | Jul 2022 | US |
