Methods and compositions for treatment of peptic ulcers and / or peptic ulcer disease

Abstract

This disclosure provides methods for the treatment of peptic ulcer disease, comprising administering an effective amount of a formulation comprising an HIV protease inhibitor. The HIV protease inhibitor may comprise amprenavir or its prodrug fosamprenavir, darunavir, ritonavir, saquinavir, or a combination thereof. The formulation may also include a bioadhesive polymer, hydrogel, and / or biocompatible gel.

Description

[0001] METHODS AND COMPOSITIONS FOR TREATMENT OF PEPTIC ULCERS AND / OR PEPTIC ULCER DISEASE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 569,875, filed on March 26, 2024, the contents of which are herein incorporated by reference in their entireties.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] N / A

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (65005301134. xml; Size: 1,830 bytes; and Date of Creation: March 25, 2025) is herein incorporated by reference in its entirety.

[0008] FIELD

[0009] This invention relates to the treatment of peptic ulcers and / or peptic ulcer disease. In particular, this invention relates to peptic ulcer treatment via administration of a formulation comprising an HIV protease inhibitor.

[0010] BACKGROUND

[0011] Peptic ulcer disease (PUD) is a common disorder of the digestive system. The estimated lifetime prevalence in Western countries is 5-10% and annual incidence 0.1-0.3%. PUD mainly occurs in the stomach, but can also be present in the proximal duodenum and esophagus. The disorder can lead to life-threatening complications include bleeding, perforation, penetration into adjacent organs and gastrointestinal obstruction. Currently available treatments suffer from reduced efficacy due to antimicrobial resistance, and PUD remains a common cause of hospitalization, with a fatality rate (5-10%) that has remained stable despite a recent decline in disease incidence. Accordingly, a need exists for new and more effective methods of treating PUD.

[0012] SUMMARY

[0013] In one aspect, a method for treating peptic ulcer disease (PUD) is provided. The method comprises administering an effective amount of a formulation comprising an HIV protease inhibitor to a subject having PUD or suspected of having PUD. In some embodiments, the HIV protease inhibitor comprises amprenavir, darunavir, ritonavir, saquinavir, or a combination thereof. In some embodiments, the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir. The formulation may further comprise an alginate, a hydrogel, or a biocompatible gel. The formulation may further comprise chitosan and / or a lectin.

[0014] In another aspect, a use of a formulation including an HIV protease inhibitor for the treatment of peptic ulcer disease (PUD) is provided. In some embodiments, the HIV protease inhibitor comprises amprenavir, darunavir, ritonavir, saquinavir, or a combination thereof. In some embodiments, the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir. The formulation may further comprise an alginate, a hydrogel, or a biocompatible gel. The formulation may further comprise chitosan and / or a lectin.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] N / A

[0017] DETAILED DESCRIPTION

[0018] Methods and compositions for the treatment of peptic ulcer disease (PUD) are disclosed. The methods can include administering an effective amount of a human immunodeficiency7virus (HIV) protease inhibitor (PI). In various aspects, the HIV PI can be in an alginate-complexed formulation, or in a formulation with a bioadhesive polymer, hydrogel, and / or biocompatible gel.

[0019] PUD, a common disorder of the digestive system, is a digestive tract injury that results in a mucosal break greater than 3-5 mm, with a visible depth reaching the submucosa. Mainly occurring in the stomach, but also in the proximal duodenum and esophagus, common etiologies of PUD are Helicobacter pylori infection and use of nonsteroidal anti-inflammatory drugs (NSAIDs). [1] Idiopathic peptic ulcers not due to Helicobacter pylori infection or NSAID use account for approximately 17% of the cases and can be more aggressive, often presenting with bleeding and other ulcer complications and are more difficult to manage. [61, 62] Life-threatening complications include bleeding, perforation, penetration into adjacent organs and gastrointestinal obstruction. [2] The estimated lifetime prevalence in Western countries is 5-10% and annual incidence 0.1 -0.3%. [1,3] Therapeutic strategies include proton pump inhibitors (PPIs), H2 histamine receptor antagonists, antibiotics, and mucosal protectors; however long-term drug therapy is ty pically required and antimicrobial resistance has reduced the efficacy of treatment. [1,4] While the incidence has declined since prior decades, [3,5] widespread use of NSAIDs and selective serotonin reuptake inhibitors (SSRIs), aging populations, increasing societal stress, and poor lifestyle habits pose challenges to treatment, and the case fatality rate remains stable at 5-10 %. [6] PUD remains a common cause of hospitalization [7] and a major source of preventable healthcare expenditures worldwide. [8] The annual direct medical costs of peptic ulcer in the US are estimated at $3. l-4.28bil (1997 and 2001 figures), [9,10] with per patient expenditures estimated at $163-866, or $1883-25,444 for complicated PUD.

[0011]

[0020] Pepsin, a protease produced by chief cells in the gastric mucosa, plays a critical role in the digestion of dietary proteins but can contribute to aerodigestive tract pathophysiology7. It has long been established that both acid and pepsin are required for chronic ulceration in both stomach and duodenum. In 1942. Schiffrin demonstrated that acid alone had little effect on excised cat jejunum, but addition of pepsin resulted in ulceration within 12 hours.

[0012] He and others demonstrated that pepsin and acid clearly caused more severe ulceration of cat stomach, duodenum and ileum than acid alone.

[0013] In a rat model employing gastric perfusion of acid and pepsin. Alphin et al (1977) observed that the degree of gastric ulceration paralleled pepsin proteolytic activity

[0014] and that a pepsin inhibitor (sodium lignosulfinate) protected the gastric mucosa even at the very low pH of 1.3.

[0014] Achord (1981) demonstrated greater basal and stimulated levels of pepsin and acid secretion in patients with acute duodenal ulcer relative to controls or post-healing.

[0015] Other authors have demonstrated that mean normal acid levels are not typically exceeded in gastric peptic ulcers [16,17] implicating other agents such as pepsin or failure of mucosal repair as the primary reason behind the evolution of gastric ulcer. The mucolytic activity7of gastric pepsin promotes its direct interaction with gastric epithelia.

[0018] Pepsin elicits proinflammatory and carcinogenic effects in a variety7of aerodigestive tract epithelia, mediated in part through its receptor-mediated endocytosis which occurs ubiquitously across a variety of cell types (laryngeal, esophageal, pharyngeal, middle ear).

[0019] Similar mechanisms are suspected to contribute to pepsin-induced injury during PUD.

[0021] Interest in pepsin inhibitors for treatment of PUD dates to the 1940s. [20-22] Sulfated polysaccharides were identified as pepsin inhibitors in the 1950s.

[0023] Amylopectin sulphate (SN-263) met success in preclinical surgical or cortisol- induced models of gastric ulceration, [24-26] however the compounds alone were ultimately deemed inadequate for treatment of PUD in clinical trial. [27-29] Pepstatin, a peptidic aspartic protease inhibitor (PI) with 25,000 times the pepsin inhibitory activity of sulfated polysaccharides,

[0030] was described by Umezawa et al (1970).

[0031] Pepstatin prevented gastric ulceration experimentally induced in rats by surgical (pyloric ligation), thermal (23°C for 22h) and indomethacin. [31-34] Despite these promising results, pepstatin was ineffective in clinical studies. [35,36] Its failure was attributed to poor water solubility7and pharmacokinetics related to its peptidic nature with >90% of the oral dose excreted in feces.

[0031] A decade later, the discovery of a viral-encoded aspartic protease essential to the maturation of human immunodeficiency virus (HIV) revitalized interest in drugs to target aspartic Pls.

[0037] Massive efforts to develop novel drugs with reduced peptidic nature and improved pharmacodynamic features yielded the first FDA- approved HIV Pls in 1995.

[0038]

[0022] There are currently ten commercially available HIV Pls.

[0039] Four HIV Pls (amprenavir, ritonavir, saquinavir and darunavir) were previously identified that bind and inhibit pepsin with IC50 in the low micromolar range.

[0040] Darunavir and amprenavir were selected for in vivo study based on in vitro data, cost and tolerability: darunavir exhibited the lowest IC50 for pepsin; amprenavir and darunavir have good safety and tolerability with minimal side effects (diarrhea, nausea and vomiting);

[0039] and a prodrug of amprenavir (fos amprenavir, FOS) existed with improved bioavailability and favorable tolerability. In a mouse model of laryngopharyngeal reflux (LPR) incorporating laryngeal scratch wound and pepsin instillation, oral FOS at the human-equivalent dose for HIV prevented pepsin-mediated laryngeal damage.

[0040] Local delivery by inhalation was effective at l / 20th the dose. These data demonstrated that FOS binds and inhibits pepsin and provided proof of concept that a pepsin inhibitor prevents pepsin-induced aerodigestive tract injury.

[0023] PCT / US2021 / 027758, Aerosolized formulations of HIV protease inhibitors for the treatment of airway reflux, describes the use of HIV PI including FOS as pepsin inhibitors. Subsequent work corroborated the safety of inhaled FOS in mice

[0041] and further substantiated amprenavir protection against pepsin-mediated injury in cell culture models of LPR and gastroesophageal reflux disease (GERD) at serum concentrations achievable provided the manufacturer-recommended dose of FOS for HIV: amprenavir protected against pepsin- induced laryngeal and esophageal damage including cell dissociation, E-cadherin cleavage, and MMP induction. [42,43] Such forms of injury are common to PUD and are risk factors for its progression to gastric cancer. [44,45]

[0024] The primary site of FOS metabolism is the intestine, where alkaline phosphatase (ALP) converts FOS to the active PI amprenavir near the cell membrane permitting its cellular uptake. [46,47] As indicated by the efficacy of both an oral formulation and a reduced dose of inhaled FOS in the LPR mouse model.

[0040] FOS appears to inhibit pepsin both systemically and topically. Mechanisms for local conversion are supported by prior literature. Phosphate ester prodrugs such as FOS are metabolized by ALP in serum

[0048] and may be converted by ALP produced in aerodigestive tract mucosa in response to injury and inflammation as observed in PUD. [49-51]

[0025] Drug formulations that prolong retention at such sites of injury could improve local drug conversion and topical activity and reduce dosing. Alginate, a marine polysaccharide, is one of the most extensively used bioadhesive polymers for drug delivery. It is non-toxic, biocompatible, non-immunogenic. biodegradable, readily available and cost-effective. [52.53] The U.S. FDA recognizes alginate as “Generally Referred As Safe” (GRAS) for alimentary use.

[0054] Its pH-dependent gelation and chemical versatility has made alginate one of the most extensively explored mucoadhesive biomaterials. At excipient concentrations, alginate has been employed to improve gastro-retention and thereby efficacy of therapeutic formulations for gastric and duodenal ulcers. [55-58] At higher concentrations, alginate inhibits pepsin and demonstrates weakly gastroprotective properties. [59,60]

[0026] HIV Pls such as FOS and their alginate-complexed formulations represent a compelling new approach to treatment of PUD by targeting pepsin activity implicated in its pathogenesis. Preclinical studies have demonstrated the role of pepsin in inflammatory’ and carcinogenic changes associated with PUD and the potential efficacy of HIV Pls such as FOS in preventing aerodigestive tract damage associated with PUD and its complications. Future work is warranted in vitro, in vivo and in clinical trial to examine the potential of such formulations for this disease which continues to pose life-threatening risks to patients provided current therapeutic strategies.

[0027] Methods

[0028] Methods for treating peptic ulcer disease (PUD) or a subject suspected of having PUD are disclosed herein. In one aspect. PUD is a digestive tract injury that results in a mucosal break greater than 3-5 mm, with a visible depth reaching the submucosa. In the same or alternative aspects, PUD is a digestive tract injury' that results in a mucosal break greater than 3-5 mm, with a visible depth reaching the submucosa, in the stomach, proximal duodenum, or both. It should be understood that PUD is a condition that is distinct and different from reflux conditions, including but not limited to airway reflux, supraoesophageal reflux, extraoesophageal reflux, gastropharyngeal reflux, laryngopharyngeal reflux, esophagopharyngeal reflux, and gastroesophageal reflux disease.

[0029] In various aspects, the methods for treating PUD in a subject, or in a subject suspected of having PUD, include subjects that do not have a reflux condition, such as airway reflux, supraoesophageal reflux, extraoesophageal reflux, gastropharyngeal reflux, laryngopharyngeal reflux, esophagopharyngeal reflux, and gastroesophageal reflux disease.

[0030] The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. In various aspects, the subject may have digestive tract injury in the stomach and / or proximal duodenum. In the same or alternative aspects, the subject may have a Helicobacter pylori infection, or had an Helicobacter pylori infection; and / or may have a history of prior use of nonsteroidal antiinflammatory drugs (NSAIDs).

[0031] In various aspects, the methods can include administering a formulation comprising an HIV protease inhibitor (PI).

[0032] 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-l-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-y l)methy 1] 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-l-yl)butanamide), saquinavir (IUPAC: (2S)-N-[(2S,3R)-4-[(3S,4aS,8aS)-3-(tert- butylcarbamoyl)-3,4,4a,5,6,7,8,8a-octahydro-lH-isoquinolin-2-yl]-3-hydroxy-l-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-lH-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-l-phenylbutan-2-yl]carbamate), indinavir ((2S)-l-[(2S,4R)-4-benzyl-2-hydroxy-5-[[(lS.2R)-2-hydroxy-2,3-dihydro-lH- inden-l-yl]amino]-5-oxopentyl]-N-tert-butyl-4-(pyridin-3-ylmethyl)piperazine-2- carboxamide), atazanavir (IUPAC: methyl N-[(2S)-l-[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-l-oxobutan-2-yl]carbamate), tipranavir (IUPAC: N-[3-[(lR)-l-[(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-l,3-thiazol-4- yl)methyl]carbamoyl]amino]-4-morpholin-4-ylbutanoyl]amino]-l,6-diphenylhexan-2- yl] carbamate). The HIV protease inhibitor used with the present methods should be capable of binding to and inhibiting the enzymatic activity of pepsin. Thus, in some aspects, the HIV protease inhibitor is amprenavir, darunavir, ritonavir, or saquinavir. In some aspects, the HIV protease inhibitor is amprenavir (IUPAC: [(3S)-oxolan-3-yl] N-[(2S,3R)-4-[(4- aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-l-phenylbutan-2-yl]carbamate) or its prodrug fosamprenavir (IUPAC: [(3S)-oxolan-3-yl] N-[(2S,3R)-4-[(4- aminophenyl)sulfbnyl-(2-methylpropyl)amino]-l-phenyl-3-phosphonooxybutan-2- yl] carbamate). HIV protease inhibitors are known in the art and commercially available.

[0033] 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 aspects, the HIV protease inhibitors for use in the compositions and methods described herein have an IC50 in the micromolar range (pm). In some preferred aspects, the HIV protease inhibitors for use in the compositions and methods described herein have an IC50 in the nanomolar (nm) range.

[0034] The formulations and / or HIV protease inhibitor can be administered by any route. In a preferred aspect, the HIV protease inhibitor is administered orally. In certain aspects, the HIV protease inhibitor in the formulation is administered in an FDA-approved dosing amount and schedule. For example, in some aspects, the HIV protease inhibitor is administered twice daily at about 0.7-1.4g (i.e., a dosage that is FDA-approved for the treatment of HIV and thus safe). In various other aspects, the HIV protease inhibitor in the formulation can be administered based on any specific dosing schedule and amount.

[0035] In various aspects, the formulations when administered orally can allow for absorption at the site of injury, e.g., the stomach and / or proximal duodenum, for at least a fraction of a total dose of the HIV PI, without significantly impeding systemic delivery' via the intestine. In certain aspects, the formulations can increase muco-adhesion and prolong drug delivery' in the stomach and / or proximal duodenum. In various aspects, the residence time in the stomach and / or proximal duodenum is increased by at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 12 hours, at least 18 hours, or at least 24 hours, as compared to an HIV PI administered in the absence of a bioadhesive polymer, hydrogel, or biocompatible gel. In various aspects, the formulations can include any suitable bioadhesive polymer, hydrogel, and / or biocompatible gel. For instance, in certain aspects, the formulations can include alginic acid or a salt thereof. In various aspects, the alginic acid or salt thereof can include sodium alginate, calcium alginate, or both. In a preferred aspect, the formulations can include sodium alginate.

[0036] Alginate is an extensively used bioadhesive polymers for drug delivery7as it is nontoxic, biocompatible, non-immunogenic, biodegradable, mucoadhesive, readily available and cost-effective (See. e.g., PCT / IB2023 / 000485, Sustained-release oral fosamprenavir formulation for treatment of reflux). 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.

[0037] In various aspects, the alginate or sodium alginate can be present in an amount of about 0.01 % w / w to about 10% w / w, about 0. 1 % w / w to about 5% w / w. about 0.2 % w / w to about 3 % w / w, or about 0.5 % w / w to about 3 % w / w, about 1.5 % w / w, or about 2 % w / w. In various aspects, the alginate or sodium alginate is a medium viscosity grade alginate. In various aspects, the alginate or sodium alginate exhibits a viscosity of about 0.001 Pa s, about 0.01 Pa s. about 0.025 Pa s, about 0.05 Pa s, about 0.1 Pa s, about 0.2 Pa s, about 0.3 Pa s. about 0.4 Pa s, about 0.5 Pa s, about 0.6 Pa s, about 0.7 Pa s or about 0.8 Pa s, or a viscosity within a range bounded by any of the foregoing. In an exemplary aspect, the alginate or sodium alginate exhibits a viscosity of about 0.51 Pa s. In one aspect, the alginate or sodium alginate is from Mactocystis pyrifera. In various aspects, the alginate or sodium alginate can exhibit a higher viscosity, e.g., > about 2.93 Pa s. Alginate polymers are available with varying molecular weights (MWs). The formulations may comprise alginate of any suitable MW. In various aspects, the alginate has a mid-range MW of about 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, or 400 kDa, or a MW within a range bounded by any of the foregoing. In an exemplary aspect, the alginate has a MW of about 240 kDa.

[0038] In various aspects, the formulations can include a biocompatible synthetic peptide gel. In one example aspect, the peptide gel can comprise a repeating sequence of amino acids, Arginine (R), Alanine (A), Aspartic Acid (D) and Alanine (A) which can form the RADARADARADARADA (SEQ ID NO: 1) (RADA16) peptide, known as PuraStat®. As used herein, the terms “treat’; “treating” or “treatment” describes the management and care of a subject for the purpose of combating PUD or symptoms associated with PUD. Treating includes the administration of protease inhibitor or formulations of the present disclosure to prevent the onset of the symptoms or complications, to alleviate the symptoms or complications, or to eliminate the disease, condition, or disorder, including PUD and / or symptoms associated with PUD. In preferred aspects, the methods and compositions of the present disclosure reduce mucosal damage and inflammation in the stomach and / or proximal duodenum of the subject. Treatment also includes reducing one or more symptoms of PUD including but not limited to stomach pain, e.g., burning stomach pain, feeling of fullness, e.g., bloating or belching, intolerance to fatty foods, heartbum, nausea, vomiting, including vomiting blood, dark blood in stools, trouble breathing, faint feelings, unexplained weight loss, and appetite changes.

[0039] The terms “effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological or clinical results. In various aspects, that result can be reducing, alleviating, inhibiting or preventing one or more symptoms of a disease or condition, e.g., PUD, reducing, inhibiting or preventing stomach pain, e.g.. burning stomach pain, feeling of fullness, e g., bloating or belching, intolerance to fatty foods, heartbum, nausea, vomiting, including vomiting blood, dark blood in stools, trouble breathing, faint feelings, unexplained weight loss, and appetite changes, or any other desired alteration of a biological system. In some aspects, the effective amount is an amount suitable to provide the desired effect, e.g., reduce one or more of stomach pain, e.g., burning stomach pain, feeling of fullness, e.g., bloating or belching, intolerance to fatty foods, heartbum, nausea, vomiting, including vomiting blood, dark blood in stools, trouble breathing, faint feelings, unexplained weight loss, and appetite changes. The response to a treatment of PUD or a subject suspected of having PUD may be assessed using any standard clinical method including, without limitation, suitable medical imaging techniques, pain, diet, and / or well-being assessments, and stool sample inspection. In the same or alternative aspects, the response to a treatment of PUD or subject suspected of having PUD may be assessed by evaluating a tissue sample taken from the stomach or proximal duodenum of the subject.

[0040] Formulations

[0041] The present disclosure also relates to compositions and / or formulations 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 HIV protease inhibitors and alginate are discussed above.

[0042] The compositions and / or formulations disclosed herein 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.

[0043] The compositions and / or formulations disclosed herein 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).

[0044] The composition may further include a suspending agent, a preservative, a sweetener, a flavoring, water, or 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. In one example aspect, a formulation may include an HIV protease inhibitor, an alginate (or other hydrogel or biocompatible gel), and optionally one or more suspending agents (e.g., Hypromellose and / or Xanthan gum), one or more preservatives (e.g.. Methylparaben, Propylparaben, and / or Sodium Benzoate), one or more sweeteners (Sucralose and / or acesulfame), one or more flavoring agents, and / or water. In various aspects, the HIV protease inhibitor can be present in an amount of amount of about 600 mg / 10 mL (after reconstitution) to about 2000 mg / 10 mL (after reconstitution), about 800 mg / 10 mL (after reconstitution) to about 1800 mg / 10 mL (after reconstitution), about 1000 mg / 10 mL (after reconstitution) to about 1600 mg / 10 mL (after reconstitution), about 1200 mg / 10 mL (after reconstitution) to about 1500 mg / 10 mL (after reconstitution), or about 1400 mg / 10 mL (after reconstitution); the alginate (e.g.. sodium alginate) can be present in an amount of about 5-60 mg / 10 mL (after reconstitution), about 10-50 mg / 10 mL (after reconstitution), about 15-40 mg / 10 mL (after reconstitution), about 20-30 mg / 10 mL (after reconstitution), or about 24.5 mg / 10 mL (after reconstitution). In various aspects, the alginate can be present in a higher amount of about 100 mg / 10 mL (after reconstitution), about 200 mg / 10 mL (after reconstitution), about 300 mg / 10 mL (after reconstitution), about 400 mg / 10 mL (after reconstitution), about 500 mg / 10 mL (after reconstitution), about 600 mg / 10 mL (after reconstitution), about 700 mg / 10 mL (after reconstitution), about 800 mg / 10 mL (after reconstitution), about 900 mg / 10 mL (after reconstitution), or about 1000 mg / 10 mL (after reconstitution). The optional suspending agents, preservatives, sweetener, flavoring agents, and water can be present in suitable amounts to reconstitute the HIV protease inhibitor and alginate in the amounts mentioned herein.

[0045] In various aspects, the formulations may comprise a microparticle loaded with an effective amount of the HIV PI. In various aspects, the HIV PI (e.g., amprenavir or its prodrug fosamprenavir) is present in an aqueous or non-aqueous solution or suspension, such solution or suspension encapsulated in the microparticle. Suitable microparticle formulations are known in the art and include, without limitation, microspheres or beads. In various aspects, the average microparticle size in the formulation may be about 0.1 pm, about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm. about 9 pm, about 10 pm. about 15 pm, about 20 pm, about 25 pm, about 50 pm. about 75 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 500 pm, about 550 pm, about 600 pm, about 650 pm, about 700 pm, about 750 pm, about 800 pm, about 850 pm, about 900 pm, about 950 pm, about 1000 pm. about 2500 pm, or about 5000 pm, or may be within a range bounded by any of the foregoing. In various aspects, the microparticle may further comprise the alginate and one or more additional polymers and / or proteins. The one or more additional polymers and / or proteins may promote mucoadhesion of the microparticle and / or promote floating of the microparticle. In various aspects, the one or more additional polymers and / or proteins may be conjugated to the alginate and / or may be used as a microparticle coating. Suitable polymers and / or proteins for microparticle formulations are known in the art (see, e.g.. [63-68]) and include, without limitation, cellulose and derivatives thereof (e.g., hydroxypropyl methylcellulose (HMPC), ethylcellulose), a lectin (e.g., Concanavalin A), poly(lactic-co-gly colic acid) (PLGA), and chitosan. In one exemplary' aspect, the microparticle (e.g., microsphere) comprises the HIV PI, alginate, chitosan, and a lectin. The microparticle may be prepared using any suitable method known in the art (see, e.g.. [63-68]). In one exemplary’ aspect, the microparticle is prepared in a solution comprising the HIV PI, alginate, chitosan, and lectin. In another exemplary aspect, the microparticle is prepared in a first solution comprising the HIV PI and alginate, then coated in a coating solution comprising chitosan and / or a lectin. The microparticle may be further processed prior to coating (e.g., collected, washed, and / or dried). The alginate may be present in the first solution at any suitable concentration, including without limitation, a concentration of about 0.01 % w / v to about 10% w / v, about 0.1 % w / v to about 5% w / v, about 0.2 % w / v to about 3 % w / v, or about 0.5 % w / v to about 3 % w / v. In an exemplary' aspect, the alginate is present in the first solution at a concentration of about 2% w / v. The chitosan may be present in the first solution or the coating solution at any suitable concentration, including, without limitation, at a concentration of about 0.1 % w / v, about 0.25% w / v, about 0.5% w / v, about 0.75% w / v, about 1.0 % w / v, or about 1.25% w / v, or at a concentration within arange bounded by any of the foregoing. In an exemplary' aspect, the chitosan is present in the first solution or the coating solution at a concentration of about 0.5% w / v. The lectin may be present in the first solution or the coating solution at any suitable concentration, including, without limitation, at a concentration of about 0.01 % w / v, about 0.05 % w / v, about 0.075% w / v, about 0.1% w / v, about 0.125% w / v, about 0.150 % w / v, or about 1% w / v, or at a concentration within a range bounded by any of the foregoing. In an exemplary aspect, the lectin is present in the first solution or the coating solution at a concentration of about 0. 1% w / v. In an exemplary aspect, the microparticle is prepared in a solution comprising 2% w / v of alginate, about 0.25 % w / v to about 1 % w / v of chitosan, and about 0. 1 % w / v of the lectin.

[0046] The compositions and / or formulations 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 present invention has been described in terms of one or more preferred embodiments, or aspects, 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.

[0047] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology7and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter.

[0048] 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.

[0049] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a therapeutic” or “a lectin” should be interpreted to mean "one or more therapeutics” and “one or more lectins,” respectively, unless the context clearly dictates otherwise. As used herein, the term “plurality” means “two or more.”

[0050] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone. C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or "B" or “A and B .”

[0051] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%. it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise. The references cited herein are explicitly incorporated by reference in their entireties, unless explicitly indicated otherwise. No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country’. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein.

[0052] The invention will be more fully understood upon consideration of the following nonlimiting examples.

[0053] EXEMPLARY EMBODIMENTS

[0054] Embodiment 1. A method for treating peptic ulcer disease (PUD), the method comprising: administering an effective amount of a formulation comprising an HIV protease inhibitor to a subject having PUD or suspected of having PUD.

[0055] Embodiment 2. The method of embodiment 1, wherein the HIV protease inhibitor comprises amprenavir, darunavir, ritonavir, saquinavir, or any combination thereof.

[0056] Embodiment s. The method of embodiment 1. wherein the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir.

[0057] Embodiment 4. The method of any one of embodiments 1-3, wherein the formulation further comprises an alginate, such as sodium alginate, calcium alginate, or both, a hydrogel, and / or a biocompatible gel.

[0058] Embodiment s. The method of any one of embodiments 1-4, wherein the formulation further comprises a suspending agent, a preservative, a sweetener, a flavoring agent, water, or a combination thereof.

[0059] Embodiment 6. The method of any one of embodiments 1-5, wherein the formulation is orally administered.

[0060] Embodiment 7. The method of any one of embodiments 1-6, wherein the formulation is in liquid form.

[0061] Embodiment 8. The method of any one of embodiments 1-7, wherein the formulation is administered at a dosage of about 0.7 grams (g) to about 1.4 g of the HIV protease inhibitor.

[0062] Embodiment 9. The method of embodiment 8, wherein the formulation is administered twice daily at a dosage of about 0.7 g to about 1.4 g of the HIV protease inhibitor per administration.

[0063] Embodiment 10. The method of any one of embodiments 1-9, wherein the subject has a digestive tract injury in the stomach.

[0064] Embodiment 1 1. The method of any one of embodiments 1-10, wherein the subject has a digestive tract injury in the proximal duodenum.

[0065] Embodiment 12. The method any one of embodiments 1-11, wherein the subject has a Helicobacter pylori infection, or had an Helicobacter pylori infection. Embodiment 13. The method of any one of embodiments 1-12, wherein the subject has a history of prior use of nonsteroidal anti-inflammatory drugs (NSAIDs).

[0066] Embodiment 14. Use of a formulation comprising an HIV protease inhibitor for the treatment of peptic ulcer disease (PUD).

[0067] Embodiment 15. The use of embodiment 14, wherein the HIV protease inhibitor is amprenavir, darunavir, ritonavir, saquinavir, or any combination thereof.

[0068] Embodiment 16. The use of embodiment 15, wherein the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir.

[0069] Embodiment 17. The use of any one of embodiments 14-16, wherein the formulation further comprises an alginate, such as sodium alginate, calcium alginate, or both, a hydrogel, and / or a biocompatible gel.

[0070] Embodiment 18. The use of any one of embodiments 14-17, wherein the formulation further comprises a suspending agent, a preservative, a sweetener, a flavoring agent, water, or a combination thereof.

[0071] Embodiment 19. The method of any one of embodiments 1-13 or the use of any one of embodiments 14-18. wherein the formulation further comprises chitosan and / or a lectin.

[0072] Embodiment 20. The method or use of embodiment 19, wherein the HIV protease inhibitor, the alginate, and the chitosan and / or the lectin are formulated as a microparticle.

[0073] EXAMPLES

[0074] Example 1; Jn vitro gastric cell culture studies to examine efficacy of various HTV Protease Inhibitors (Pls) for prevention of peptic injury [Prophetic Example]

[0075] Aim 1. Investigation of HIV protease inhibitor rescue of gastric epithelial cell viability in vitro Pepsin is a primary injurious agent during Peptic Ulcer Disease (PUD) which is suspected to contribute to gastric ulceration via promotion of proinflammatory cytokine signaling, mucolytic activity, and breakdown of intercellular adhesion junctions. To demonstrate the efficacy of HIV Pls in treating PUD, HIV Pls harboring high (amprenavir, darunavir, ritonavir, saquinavir) and low- (lopinavir, nelfinavir, indinavir) affinity and inhibitory activity towards pepsin (as previously determined40) will be evaluated in a previously established cell culture model of PUD69: AGS, human gastric adenocarcinoma cell line (American Type Culture Collection. Manassas VA). Experiments will be conducted at pH 4, representative of intragastric pH of patients taking proton pump inhibitors. Cytoprotective activity of HIV protease inhibitors for PUD will be examined per prior study.69 A treatment response cun e will first be performed to identify conditions at which pepsin pH 4 elicits reduction in AGS cell viability. Prior work demonstrates cytotoxicity of epithelial cells to acidified pepsin (e.g. hypopharyngeal cells to O.lmg / ml at pH4 for 3Omin).70’71Briefly, AGS cells maintained per manufacturer specifications will be treated with buffered saline (sham) or O. lmg / ml porcine pepsin at pH 4 for 5, 15, 30, and 60min at 37°C / 5% CO2 in n=8. After three washes in buffered saline. MTT-based In Vitro Toxicology Assay (Sigma Aldrich, St Louis MO) will be conducted. Briefly, cells will be returned to growth medium containing MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) reagent and incubated for 4 hours at 37°C / 5% CO2 prior to addition of MTT Solubilization Solution. Absorbance at a wavelength of 570 nm will be measured spectrophotometrically and mean absorbance compared across groups.

[0076] AGS cells will be co-treated with buffered saline (sham) or pepsin treatment conditions sufficient to reduce cell viability, and a dose range (peak serum concentration and 10-fold greater) of HIV protease inhibitors or dimethyl sulfoxide (diluent control). MTT assay will be conducted as above.

[0077] Anticipated results and data interpretation'. In MTT assay, high absorbance values indicate increased cell viability, as metabolically active cells convert the dye into a purple formazan product. Pepsin treatment conditions that are cytotoxic will reduce mean absorbance relative to sham treatment group; rescue will be indicated by higher mean absorbance relative to pepsin-treated groups. It is expected that treatment with HIV protease inhibitors will increase cell viability, rescuing the cytotoxic effects of pepsin.

[0078] Aim 2. Investigation of HIV protease inhibitor rescue of peptic epithelial inflammatory cytokine signaling in vitro

[0079] Il-ip, IL-6. IL-8, IL- 12. IL- 18, and TNF-a are primary inflammatory mediators of PUD.72,73Many of these cytokines are responsive to pepsin in aerodigestive epithelial cells.71,74A time and dose response cun e will be performed to identify' optimal inflammatory cytokine transcriptional response conditions. Briefly, AGS cells will be treated in biological triplicate with buffed saline (sham) or 0. Img / ml porcine pepsin at pH4 for 5, 30, or 60min at 37°C / 5% CO2 before return to normal growth media for 4, 8. or 24 hours at 37°C / 5% CO2. RNA will be extracted, reverse transcribed, and IL1B, IL6. IL8, IL12, IL18. TNFA. and HPRT1 (housekeeping) will be measured via real-time quantitative PCR in quadruplicate reactions using Taqman gene expression assays (ThermoFisher Scientific, Waltham MA). Threshold cycle (Ct) values <36 will be used for analysis, and gene expression normalized to housekeeping gene HPRT1. Mean HPRT-normalized gene expressions (relative quantitation. RQ) will be compared across groups to identify the pepsin treatment and rest conditions eliciting greatest elevation in most cytokines.

[0080] Experiments will be repeated, treating AGS cells with buffed saline (sham) or empirically determined optimal pepsin treatment, and HIV protease inhibitors exhibiting cytoprotection in Aim 1 or dimethyl sulfoxide (diluent control).

[0081] Anticipated results and data interpretation: Acidified pepsin will promote inflammatory cytokine transcription as indicated by increased mean RQ relative to sham group; rescue will be indicated by reduction of mean RQ relative to pepsin treatment group. It is expected that treatment with HIV protease inhibitors vvi 11 decrease RQ, rescuing the pro- inflammatory effects of pepsin.

[0082] Aim 3. Investigation of HIV protease inhibitor rescue of peptic epithelial disruption in vitro SubAim 1 : Evaluation of HIV protease inhibitor rescue of gastric epithelial monolayer barrier disruption

[0083] We previously established an epithelial cell culture model of ulcerative aerodigestive tract lesions caused by acidified pepsin and its rescue by amprenavir.42,43-75Per the previously established model, AGS cells will be treated with buffered saline (sham) or O. lmg / ml porcine pepsin at pH 4, and HIV protease inhibitors exhibiting cytoprotection in Aim 1 or dimethyl sulfoxide (diluent control) for 1 hour at 37°C / 5% CO2. After two washes in buffered saline, images will be obtained on an inverted microscope (Nikon, Tokyo, Japan and Metamorph Inc., Nashville, TN). The percent cell-free area of a single image from each well (n = 3) will be quantified using PHANTAST plug-in for FIJI (National Institutes of Health, Bethesda, MD) and means compared across groups.

[0084] Anticipated results and data interpretation.' Acidified pepsin will incur significant increase in percent cell-free area relative to sham group; rescue will be indicated by reduction in percent cell-free area relative to pepsin-treated group. It is expected that treatment with HIV protease inhibitors will reduce cell-free area, rescuing the lesion formation induced by pepsin.

[0085] SubAim 2; Evaluation of HIV protease inhibitor rescue of E-cadherin cleavage and MMP dvsregulation

[0086] E-cadherin is the primary protein constituent of adherens junctions. It plays a crucial role in ensuring tissue integrity by immobilizing cells within the epithelium, facilitating contact inhibition of growth, and maintaining proper functioning of tight junctions, the primary permeability barrier of the epithelium. E-cadherin also regulates cell migration and proliferation through interaction with catenins and growth factor signaling pathways. E- cadherin cleavage fragments are present in LPR and GERD specimens, absent in reflux-free controls, suggesting its RIP plays a role in the pathophysiology of acidified pepsin-attributed disease.76,77E-cadherin and MMP dysregulation are similarly implicated in PUD.78'82

[0087] We previously demonstrated that acidified pepsin causes cleavage of E-cadherin in aerodigestive tract epithelia and was rescued by amprenavir.424' Downstream dysregulation of extracellular matrix-degrading matrix metalloproteinases (MMPs) was also rescued by amprenavir.

[0088] To examine rescue of E-cadherin cleavage per our established model, AGS cells will be treated with buffered saline (sham) or O. lmg / ml porcine pepsin at pH4. and HIV protease inhibitors exhibiting cytoprotection in Aim 1 or dimethyl sulfoxide (diluent control) for 30min at 37°C / 5% CO2. Cells will be lysed on the treatment plate in cold radio-immunoprecipitation assay (RIP A) lysis buffer (1%NP4O, 0.5% sodium deoxy cholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 50 mM Tris-Cl, pH 7.4) containing protease inhibitor. Proteins will be separated (4%-20% TGX, Bio Rad Laboratories, Hercules, CA) and transferred to polyvinylidene difluoride. Membranes will be blocked (5% milk and 0.1% Tween-20 in phosphate buffered saline, PBS) and probed with E-cadherin C-terminal (4A2C7, ThermoFisher Scientific) and actin (CP01, Sigma-Aldrich), and horseradish peroxidase (HRP)-conjugated secondary antibodies (Agilent Technologies. Santa Clara, CA). Densitometry will be performed using FIJI; signal corresponding to full-length E-cadherin (120kDa) and its fragments (38 and 33 kDa C-terminal E-cad / CTFl and E-cad / CTF2 fragments) in each lane will be normalized to actin signal from the same lane and mean of three lanes representing biological triplicate compared across groups.

[0089] To examine rescue of MMP dysregulation per our established model, AGS cells will be treated as above but for shorter duration of 15 minutes, rinsed twice in HBSS, and allowed to rest in normal grow th media for 24 hours. RNA will be extracted and qPCR will be performed as in Aim 2 using Taqman gene expression assays for MMP-1, 2, 3, 7,9,14.

[0090] Anticipated results and data interpretation'. Acidified pepsin will result in E-cadherin cleavage as indicated by depletion of full-length 120kDa band and increase in 33 and 38kDa fragment mean actin-normalized band intensities relative to sham group; rescue will be indicated by increase in mean actin-normalized band intensities of full-length 120kDa E- cadherin relative to pepsin-treated group. Pepsin pH 4 will increase MMP RQs 24 h postexposure relative to sham group; rescue will be indicated by reduction of MMP RQs relative to pepsin-treated group. It is expected that treatment with HIV protease inhibitors will reduce E-cadherin cleavage, rescuing the pro-cleavage effects of pepsin.

[0091] Statistical Analysis: Student's t-test will be used to compare groups. For qPCR, RQs will be calculated via 2'AACtmethod. Log transformation will be applied to cell dissociation data if the data is skewed. The genes with p-values < 0.05 and fold change > 1.5 will be included in the evaluation. With 3 treated wells of cells per group, we will have at least 80% power to detect a difference of 3 times SD at the significance level of 0.05. In all Aims, ap-value < 0.05 will be considered as statistically significant.

[0092] Example 2; Gastric Model to Measure Local Delivery of HIV Protease Inhibitors [Prophetic Example]

[0093] Novel formulations of amprenavir, in microspheres coated with chitosan and lectin, (and other active forms of HIV Pls with support from in vitro studies) will be evaluated in an ex vivo gastric model (Aelius Biotech, UK) to assess mucoadhesion and local drug delivery. This ex vivo porcine gastric system provides a clinically relevant, standardized environment to predict drug pharmacokinetics and metabolism in the gastric lumen.

[0094] Ex vivo testing of gastric drug transit and delivery

[0095] These experiments will demonstrate the ability of amprenavir (and other active forms of HIV Pls) to adhere to the gastric mucus layer in an ex vivo porcine stomach. Permeation of the protease inhibitors can be assessed in different formulations. Briefly, fresh porcine stomach will be collected from a local abattoir and placed in cold PBS on ice. Prior to testing, the stomach will be washed internally with deionised water. Sections of a fixed area section of the stomach (e.g. 5x5cm will be cut and placed on a flat plate. Test formulations prepared with 5mL of synthetic gastric juice will then be pipetted gently on to the tissue within a plastic barrier to retain the formulation. Tissue will then be left on a rocker for 30 minutes. After 30 minutes, gastric juice will be washed off and collected for analysis. A further 5mL gastric juice will be added and incubated for 10 minutes, before being tipped off and collected for analysis. After 60 minutes, the final fraction will be collected. All samples will be assessed for amprenavir content to assess levels of adhered and non-adhered material. Amprenavir will be measured in all samples via high-performance liquid chromatography (HPLC).

[0096] Trans-epithelial drug absorption

[0097] One limitation of the whole-stomach approach is the possibility of absorption of amprenavir into the gastric submucosa. To evaluate for this possibility, we will use a transw ell system in which ex vivo porcine gastric specimens are suspended between two chambers within media optimized to sustain cellular activity. To create this model, sections of porcine stomach will be collected fresh from the abattoir and placed in buffer on ice. Gastric mucosal sections will be placed in the transwell system and exposed to different formulations of amprenavir apically. By measuring amprenavir in both the apical and basolateral chambers, the model provides insight into drug absorption into systemic circulation. Specifically, only metabolized drug undergoes transepithelial flux. Apical and basolateral samples will be collected over a 2- hour time course and amprenavir measured by HPLC.

[0098] Anticipated results and data interpretation'. We anticipate that novel formulations of amprenavir in microspheres coated with chitosan and lectin will enhance amprenavir adhesion to the gastric lining, resulting in improved local drug delivery. This will result in greater transepithelial flux of active drug.

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Claims

CLAIMSWhat is claimed:

1. A method for treating peptic ulcer disease (PUD), the method comprising: administering an effective amount of a formulation comprising an HIV protease inhibitor to a subject having PUD or suspected of having PUD.

2. The method of claim 1. wherein the HIV protease inhibitor comprises amprenavir, darunavir, ritonavir, saquinavir, or any combination thereof.

3. The method of claim 1 , wherein the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir.

4. The method of claim 1 , wherein the formulation further comprises an alginate, such as sodium alginate, calcium alginate, or both, a hydrogel, and / or a biocompatible gel.

5. The method of claim 1, wherein the formulation further comprises a suspending agent, a preservative, a sweetener, a flavoring agent, water, or a combination thereof.

6. The method of claim I, wherein the formulation is orally administered.

7. The method of claim 1, wherein the formulation is in liquid form.

8. The method of claim 1, wherein the formulation is administered at a dosage of about 0.7 grams (g) to about 1.4 g of the HIV protease inhibitor.

9. The method of claim 8. wherein the formulation is administered twice daily at a dosage of about 0.7 g to about 1.4 g of the HIV protease inhibitor per administration.

10. The method of claim 1, wherein the subject has a digestive tract injury' in the stomach.

11. The method of claim 1, wherein the subject has a digestive tract injury in the proximal duodenum.

12. The method claim 1, wherein the subject has a Helicobacter pylori infection, or had an Helicobacter pylori infection.

13. The method of claim 1, wherein the subject has a history' of prior use of nonsteroidal anti-inflammatory drugs (NSAIDs).

14. The method of claim 4, wherein the formulation further comprises chitosan and / or a lectin.

15. Use of a formulation comprising an HIV protease inhibitor for the treatment of peptic ulcer disease (PUD).

16. The use of claim 14, wherein the HIV protease inhibitor is amprenavir, darunavir, ritonavir, saquinavir, or any combination thereof.

17. The use of claim 15, wherein the HIV protease inhibitor is amprenavir or its prodrug fosamprenavir.

18. The use of claim 14, wherein the formulation further comprises an alginate, such as sodium alginate, calcium alginate, or both, a hydrogel, and / or a biocompatible gel.

19. The use of claim 14, wherein the formulation further comprises a suspending agent, a preservative, a sweetener, a flavoring agent, water, or a combination thereof.

20. The use of claim 18, wherein the formulation further comprises chitosan and / or a lectin.

21. The method of claim 14 or the use of claim 20, wherein the HIV protease inhibitor, the alginate, and the chitosan and / or lectin are formulated as a microparticle.