Larazotide peptide formulations

Abstract

This invention partially provides compositions comprising a peptide, which is a laprazole peptide or a laprazole peptide derivative, or a salt thereof, wherein the peptide or salt thereof is contained within a matrix providing controlled-release and sustained-release formulations. This invention envisions these compositions, formulations, and methods for the treatment of diseases and conditions of the small intestine.

Description

Technical Field

[0001] The present invention provides compositions, formulations, and methods for treating small intestinal diseases and conditions such as, but not limited to, fatty liver disease.

[0002] Cross-reference to related applications

[0003] This application claims the benefits of U.S. Provisional Application No. 63 / 009,768, filed April 14, 2020, and U.S. Provisional Application No. 62 / 888,052, filed August 16, 2019, the entire contents of which are incorporated herein by reference. Background Technology

[0004] The intestinal epithelium is the layer of cells that forms the luminal surface of the small and large intestines, which are part of the gastrointestinal tract (GI), and represents the largest interface (over 400m) between the external and internal environments. 2 The intestinal epithelium has two important functions: absorbing nutrients and providing a barrier against harmful environmental substances such as bacteria, viruses, toxins, and food allergens.

[0005] The barrier properties of the intestinal epithelium are regulated by specialized plasma membrane structures called tight junctions. Alterations in tight junctions can lead to disruption of the intestinal barrier function and increased intestinal permeability. An intact intestinal barrier prevents pathogens, antigens, endotoxins, and other pro-inflammatory substances from penetrating the body, while an incomplete intestinal barrier allows them to enter, which can trigger local or systemic inflammation and disease.

[0006] For example, nonalcoholic steatohepatitis (NASH) is a serious liver disease caused by inflammation and fat accumulation within the organ. In the United States, NASH affects as many as approximately 2%–5% of the population. Another 10%–30% of Americans have fat in their livers without inflammation or liver damage; this condition is called NAFLD or “fatty liver.” The underlying cause of NASH is unclear, but it most commonly occurs in middle-aged and overweight or obese individuals. Lipotoxicity and the gut-hepatic axis (and its two components) are major contributing factors to the pathogenesis of NASH. Disruption of the intestinal epithelial lining and barrier integrity leads to increased epithelial permeability and the passage of harmful toxins, and also causes antigenic components to “cross-talk” into the liver via circulation, resulting in inflammation and damage to hepatocytes. Dysbiosis of the microbiota and alterations in intestinal immunity lead to greater translocation of bacteria and bacterial products into the systemic circulation. Consequently, bacteria or bacterial products can reach the liver via the portal vein.

[0007] In the liver, conserved motifs / structures of bacteria and bacterial products (PAMPs) contribute to hepatic steatosis, inflammation, and fibrosis. Chronic liver diseases, including NAFLD / NASH, may be associated with perturbation of the intestinal epithelial lining and disruption of barrier integrity, leading to increased permeability of the normal gut. This "leaky gut" allows harmful toxins and antigenic components to "crosstalk" into the liver via the bloodstream, causing inflammation and damage to hepatocytes and other organs or cells. Complications can lead to liver fibrosis, then cirrhosis, and finally liver cancer. These chronic liver diseases represent major causes of liver transplantation and primary liver cancer.

[0008] Therefore, effective treatments for intestinal barrier dysfunction are needed to treat, improve, and slow the progression of diseases including NAFLD and NASH. Summary of the Invention

[0009] This invention provides, in various aspects and embodiments, pharmaceutical compositions for delivering laprazole peptide or its derivatives or salts to the small or large intestine, including delivery to the small intestine, to treat fatty liver diseases such as NASH. In some embodiments, the compositions provide sustained release of laprazole peptide or its derivatives to the jejunum and ileum of a human subject. In other aspects and embodiments, this invention provides treatment of inflammatory liver diseases and fatty liver diseases with the compositions disclosed herein.

[0010] In various embodiments, laprazole peptides or derivatives are administered in sustained-release, controlled-release, or modulated-release formulations. Sustained-release, controlled-release, or modulated-release formulations improve the dose-response relationship of the active agent. For example, a formulation may deliver and / or functionally release 0.25 to about 5 mg of laprazole peptides or derivatives. In various embodiments, sustained-release, controlled-release, or modulated-release formulations deliver at least about 0.25 mg, or at least about 0.5 mg, or at least about 1 mg, or at least 2 mg of laprazole peptides or derivatives.

[0011] Sustained-release, controlled-release, or modulated-release formulations can functionally release peptides over a period of at least about 2 hours, or at least about 2.5 hours, or at least about 3 hours, or at least about 4 hours, or at least about 5 hours. In some embodiments, the sustained-release or controlled-release composition begins to release the peptide within about 10 to about 30 minutes of exposure to simulated intestinal fluid, and the release of the peptide continues for at least about 180 minutes, or at least about 210 minutes, or at least about 240 minutes, or at least about 280 minutes of exposure to simulated intestinal fluid. For example, release profiles can be created using compositions with different enteric polymer coatings and / or polymer coatings of different thicknesses. In some embodiments, the present invention provides a composition comprising an effective amount of a peptide or salt thereof of a laprazole peptide or a laprazole peptide derivative, said peptide or salt thereof being contained within a biodegradable or erosive polymer matrix, said composition further comprising an enteric coating.

[0012] In some embodiments, the beads also comprise an enteric coating that substantially resists dissolution in simulated gastric fluid. The composition remains substantially intact or may be substantially insoluble in gastric fluid. The stability of the gastric fluid-resistant coating can be pH-dependent. For example, the enteric coating can prevent the peptide from being released in large quantities in simulated gastric fluid with a pH of about 5.5, as well as in simulated intestinal fluid. In some embodiments, the matrix provides sustained release of the peptide in simulated intestinal fluid with a pH of about 6 or higher, such as about 6.5 to about 7.0. Thus, the enteric coating is stable in simulated gastric fluid but unstable in simulated intestinal fluid with a pH above about 6.0. The enteric coating in these embodiments substantially does not release the peptide in the duodenum, but rather delays release until the composition enters the jejunum, then provides sustained release in the jejunum and ileum.

[0013] In some embodiments, the composition is a capsule for oral delivery comprising a cluster of beads containing an effective amount of laprazole peptide or a laprazole peptide derivative or a salt thereof contained within an erosive polymer matrix, the beads further comprising an enteric coating comprising a copolymer of methyl acrylate, methyl methacrylate, and methacrylic acid. The ratio of free carbonyl groups to ester groups in the copolymer may be about 1:10 (e.g., EUDRAGIT F30D). In such embodiments, the enteric coating may be about 20% to about 30% of the total weight of the composition. In some embodiments, the erosive matrix comprises microcrystalline cellulose. In some embodiments, the composition provides the release of less than about 15% of the peptide after about 2 hours in simulated gastric fluid. Furthermore, the composition provides the release of less than about 25% of the peptide after about 2 hours in simulated intestinal fluid having a pH of about 5.5. In various embodiments, the composition releases at least about 40% but no more than about 80% of the peptide after about 2 hours in simulated intestinal fluid having a pH of about 7.0. In various implementations, 100% release from the simulated intestinal fluid with a pH of about 7 is achieved after at least three hours, or in some implementations, after at least about 3.5 or at least about four hours.

[0014] In other aspects, the present invention provides methods for treating small bowel conditions, symptoms, and / or diseases. Such small bowel conditions are generally associated with impaired intestinal barrier function and increased intestinal permeability. For example, impaired intestinal barrier function and increased intestinal permeability may be associated with various inflammatory liver diseases, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and cirrhosis (e.g., alcoholic cirrhosis). In some embodiments, the subject suffers from one or more liver disease-related conditions, such as kidney disease (e.g., chronic kidney disease), viral hepatitis, and diabetes, hypertriglyceridemia, and / or insulin resistance.

[0015] In some embodiments, patients may receive adjunctive therapy, which in some embodiments synergistically works with laprazole peptide treatment. In some embodiments, the present invention relates to a regimen for administering laprazole peptide (or a derivative of laprazole peptide) to a subject to improve glycemic control. In various embodiments, the laprazole peptide regimen enhances the effectiveness of conventional pharmacological interventions such as metformin, basal insulin, GLP-1 receptor agonists (e.g., liraglutide), sodium-glucose cotransporter-2 (SGLT-2) inhibitors, gastric inhibitory peptide (GIP), sulfonylureas, pPAR-γ agonists, obeticholic acid, etc. According to the present invention, the laprazole peptide regimen prevents hyperglycemic complications, including cardiovascular complications and organ damage.

[0016] In another aspect, the present invention provides compositions and methods for treating patients with cancer, as well as methods for enhancing cancer immunotherapy. The compositions described herein can improve cancer immunotherapy by reducing the epithelial permeability of the small intestine and / or large intestine. See PCT / US2019 / 022885, which is incorporated herein by reference in its entirety. Furthermore, cancer can be any cancer that can be treated with immunotherapy, including immune checkpoint inhibitor therapy, including primary cancers, metastatic cancers, and hematologic malignancies. In some embodiments, administration of a pharmaceutical composition comprising laprazole peptide is used to enhance the efficacy of immunotherapy (e.g., immune checkpoint inhibitor therapy), including in subjects who have not shown a response or have only shown a partial response to prior treatment with immune checkpoint inhibitor therapy.

[0017] Other aspects and embodiments of the invention will become apparent from the following detailed description. Attached Figure Description

[0018] Figure 1 The results showed that mice in the Western diet group had significantly higher serum glucan levels (a measure of intestinal integrity) compared to the normal diet group.

[0019] Figure 2 This study design illustrates the administration of laprazole acetate to mice fed a Western diet with impaired intestinal integrity. Eight groups of mice were placed on a Western diet for 16 weeks and administered laprazole acetate, a pioglitazone comparator, or a mediator between weeks 8 and 16. Serum dextran concentrations were measured at baseline and at the end of the study.

[0020] Figure 3 The results showed that oral administration of laprazole peptide to mice fed a Western diet resulted in improved intestinal integrity.

[0021] Figure 4 An exemplary laprazole peptide formulation for sustained release into the early jejunum and ileum is shown.

[0022] Figure 5 The results of the dissolution test for enteric-coated tablets (F23-2 in Example 3) are shown.

[0023] Figures 6A-6F The results of in vitro dissolution tests for various formulations are illustrated: Core particle A, with an S100 enteric coating, showed a 50% weight increase ( Figure 6A Core particle B-1, with S100 enteric coating, 30% weight increase ( Figure 6B Core particle B-2, with S100 enteric coating, 90% weight increase ( Figure 6C Core particles B-3, with F30D enteric coating, 10% weight increase ( Figure 6D Core particle B-4, with F30D enteric coating, 23% weight increase ( Figure 6E ); and core particle B-5, with F30D enteric coating, 50% weight increase ( Figure 6F ).

[0024] Figure 7 An animal model was used to illustrate the in vivo release distribution of laprazole peptide from the delayed and slow-release formulation B-4 in the porcine gastrointestinal (GI) tract. Probe 1 was pointed into the duodenum (8-10 cm from the pylorus); probe 2 was pointed approximately 20 cm from probe 1; probe 3 was pointed approximately 50 cm from probe 1; and probe 4 was pointed into the cecum-ileum junction.

[0025] Figure 8 The in vivo release distribution of a delayed-release laprazole peptide formulation is illustrated. The formulation contains two groups of beads that deliver laprazole peptide acetate to the duodenum and jejunum and is intended for the treatment of celiac disease.

[0026] Figure 9 The in vivo release distribution of delayed and extended release B-4 formulations is shown.

[0027] Figure 10 The results of measuring TER (transepithelial resistance) in an isolated ischemic jejunum model were illustrated by administering various concentrations (0.1 μM, 0.5 μM, or 1 μM) of laprazole peptide in fractional doses every 45 minutes. Detailed Implementation

[0028] This invention provides, in various aspects and embodiments, pharmaceutical compositions for delivering laprazole peptide or its derivatives or salts into the small and / or large intestine for the treatment of fatty liver diseases such as NASH, and for treating cancer by enhancing immunotherapy. In some embodiments, the compositions provide sustained release of laprazole peptide or its derivatives into the jejunum and ileum of a human subject. In other aspects and embodiments, the invention provides treatment of inflammatory liver diseases and fatty liver diseases with the compositions disclosed herein. In other aspects and embodiments, the invention provides treatment of patients with cancer with the compositions disclosed herein, and methods for enhancing immune checkpoint inhibitor therapy.

[0029] Laprazole peptide is a peptide agent that promotes the integrity of tight junctions in the gastrointestinal tract (GI). Laprazole peptide has the amino acid sequence: Gly Gly Val Leu Val Gln Pro Gly (SEQ ID NO:1) and can be formulated for targeted release within the GI moiety. Laprazole peptide has shown benefits in clinical trials, particularly at lower doses (e.g., 0.5 mg), in alleviating celiac disease symptoms. See US 2016 / 0022760, which is incorporated herein by reference. Higher doses (e.g., 1 mg and 2 mg doses) showed diminished activity or no activity at all. It is believed that exopeptidases such as aminopeptidases located within the brush border of the luminal surface can produce laprazole peptide-derived fragments, including fragments lacking the N-terminal glycine residue. For example, fragments GVLVQPG (SEQ ID NO:2) and VLVQPG (SEQ ID NO:3) are inactive as tight junction regulators. Furthermore, when these two fragments are mixed with the full-length laprazole peptide, the activity is completely lost. The localized accumulation of these inactive laprazole peptide fragments (due to laprazole peptide overdose) may in fact compete with and block the peptide's function. This would explain clinical observations that low doses of laprazole peptide exert their optimal effect by avoiding the accumulation of competing inactive fragments. Therefore, in some embodiments, controlled-release, sustained-release, or modulated-release formulations are employed to increase the effectiveness of laprazole peptides or derivatives.

[0030] Microdose administration of the active laprazole peptide can prevent local accumulation of inactive fragments. Figure 10 For example, formulations that release small amounts of laprazole peptide over a prolonged period of time improve the efficacy of the active agent. It is believed that sustained release of laprazole peptide will be more effective than large releases of the active agent around the same location.

[0031] In some embodiments, the active agent is a laprazole peptide. In other embodiments, the active agent is a laprazole peptide derivative, for example, having one or more amino acid modifications such as amino acid substitution, deletion, and / or insertion. For example, the derivative may have 1, 2, 3, or 4 amino acid modifications relative to SEQ ID NO:1, said amino acid modifications being independently selected from amino acid deletion, insertion, and / or substitution. Exemplary laprazole peptide derivatives are described in US 8,785,374, US 8,957,032, and US 9,279,807, which are incorporated herein by reference in their entirety. Further laprazole peptide derivatives are disclosed in PCT / US2019 / 19350, which is also incorporated herein by reference.

[0032] For example, in some embodiments, a laprazole peptide derivative exhibiting resistance to exopeptidases such as aminopeptidases is applied, thereby avoiding the accumulation of large amounts of inactive peptide fragments. Exemplary modifications include amino acid substitutions at the N-terminus and / or C-terminus to reduce exopeptidase digestion, extension of the N-terminus and / or C-terminus to delay exopeptidase digestion of functional peptides, incorporation of D amino acids, and cyclization. Exemplary laprazole peptide derivatives are disclosed in PCT / US2019 / 19350, which is incorporated herein by reference in its entirety.

[0033] In various embodiments, the peptide has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight D-amino acids. In one embodiment, each amino acid (except Gly) of the laprazole peptide derivative is a D-amino acid, and the laprazole peptide derivative is optionally a retro-inverso peptide. The retro-inverso peptide contains a reverse amino acid sequence (e.g., GPQVLVGG), with all non-glycine amino acids present as D-amino acids. The retro-inverso peptide maintains a side-chain topology similar to the original L-amino acid peptide and makes the peptide more resistant to proteolytic degradation. In some embodiments, the N-terminal Gly of the retro-inverso peptide is replaced with Ala, Leu, Ile, Val, or allylglycine. In these or other embodiments, one or both C-terminal Gly residues of the retro-inverso peptide are independently replaced with amino acids selected from Ala, Leu, Ile, Val, or allylglycine.

[0034] In other embodiments, the peptide having the amino acid sequence of SEQ ID NO:1 has one or two D-amino acids at the N-terminus and optionally the C-terminus, with all other amino acids in the L-configuration. For example, the L-Pro of the peptide having the amino acid sequence of SEQ ID NO:1 is replaced with D-Pro, with all other amino acids in the L-configuration. In these embodiments, the N-terminus and / or C-terminus are substituted or extended such that the peptide does not have glycine at the terminal (Gly does not have both D- and L-configurations). In some embodiments, the terminal Gly residue is replaced with D-Ala. The terms "lalazole peptide" or "lalazole peptide treatment" refer to treatment with a laprazole peptide or derivative that promotes tight linkage integrity.

[0035] In some embodiments, the laprazole peptide derivative is d-lprazole peptide, i.e., having a D-amino acid at every position other than Gly. Compared to laprazole peptide, D-lprazole peptide offers advantages in dosage and / or efficacy.

[0036] Laprazole peptides or derivatives may be administered as salts in any suitable form. For example, laprazole peptides or derivatives may be administered as acetates. Salts of laprazole peptides (including acetates and hydrochlorides) are described in US 2013 / 0281384, which is incorporated herein by reference in its entirety. Alternative salts may be used, including any pharmaceutically acceptable salt of the peptide such as those listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. PHStahl and CGWermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are incorporated herein by reference in their entirety.

[0037] In various embodiments, laprazole peptides or derivatives are administered in a sustained-release or controlled-release formulation. Sustained-release, controlled-release, or modulated-release formulations avoid adverse dosing reactions, wherein the sustained-release, controlled-release, or modulated-release formulation does not overwhelm the laprazole peptide receptor at the delivery target site with an inactive peptide. For example, the formulation may deliver and / or functionally release 0.25 to about 5 mg of laprazole peptide or derivative, or about 0.25 to about 4 mg of laprazole peptide or derivative, or about 0.25 to about 3 mg of laprazole peptide or derivative, or about 0.25 to about 2 mg of laprazole peptide or derivative, or about 0.25 to about 1 mg of laprazole peptide or derivative. In various embodiments, sustained-release, controlled-release, or modulated-release formulations deliver at least about 0.25 mg, or at least about 0.5 mg, or at least about 1 mg, or at least 2 mg of laprazole peptide or derivative. For example, the formulation may contain about 1 mg to about 5 mg of laprazole peptide or derivative, or about 1 mg to about 3 mg of laprazole peptide or derivative. In some embodiments, the formulation may contain about 0.25 mg to about 1 mg of laprazole peptide or a derivative, or about 0.5 mg to about 2 mg of laprazole peptide or a derivative. As used herein, the term "about" includes ±10% of the relevant value.

[0038] Sustained-release, controlled-release, or modulated-release formulations can release peptides over a period of at least about 2 hours, or at least about 2.5 hours, or at least about 3 hours, or at least about 4 hours, or at least about 5 hours. In some embodiments, the sustained-release or controlled-release composition (e.g., comprising peptide-containing particles, gels, emulsions, or biodegradable or erosive matrices) begins to release the peptide within about 10 to about 30 minutes of exposure to simulated intestinal fluid, with peptide release continuing for at least about 180 minutes, or at least about 210 minutes, or at least about 240 minutes, or at least about 280 minutes of exposure to simulated intestinal fluid. For example, release profiles can be created using compositions with different enteric polymer coatings and / or polymer coatings of different thicknesses.

[0039] In some embodiments, the present invention provides a composition comprising an effective amount of a peptide, or a salt thereof, of which is a laprazole peptide or a laprazole peptide derivative, said peptide or salt thereof, contained within a biodegradable or erosive polymer matrix. The matrix provides sustained release of the peptide in simulated intestinal fluid for at least about 120 minutes. In some embodiments, the matrix provides sustained release of the peptide in simulated intestinal fluid for at least about 180 minutes, or at least about 210 minutes, or at least about 240 minutes. The simulated intestinal fluid may have a pH of at least about 6.0, or at least about 6.2, or at least about 6.5. In some embodiments, the simulated intestinal fluid has a pH of about 7.

[0040] In some embodiments, the beads also comprise an enteric coating that is substantially resistant to dissolution in simulated gastric juice. The composition remains substantially intact or may be substantially insoluble in gastric juice. The stability of the gastric juice-resistant coating (referred to herein as "enteric coating") can be pH-dependent. A pH-dependent delayed-release coating will be substantially stable in acidic environments (e.g., pH of about 5.5 or lower) and substantially unstable in near-neutral to alkaline environments (e.g., pH greater than about 6.0). For example, an enteric coating may be employed that will substantially disintegrate or dissolve in near-neutral to alkaline environments such as those found in the small intestine (such as the jejunum and ileum). Examples of simulated gastric juice and simulated intestinal juice include, but are not limited to, those disclosed in 2005 Pharmacopeia 23NF / 28USP in Test Solutions and / or other simulated gastric juice and simulated intestinal juice known to those skilled in the art, such as prepared enzyme-free simulated gastric juice and / or intestinal juice.

[0041] For example, an enteric coating can prevent the peptide from being released in large quantities in simulated gastric fluid and simulated intestinal fluid with a pH of about 5.5. In some embodiments, the matrix provides sustained release of the peptide in simulated intestinal fluid with a pH of about 6 or higher, such as about 6.5 to about 7.0. Thus, the enteric coating is stable in simulated gastric fluid but unstable in simulated intestinal fluid with a pH above about 6.0. The enteric coating in these embodiments essentially does not release the peptide in the duodenum, but rather delays release until the composition enters the jejunum, and then provides sustained release in the jejunum and ileum. The composition essentially does not deliver the peptide to the colon.

[0042] Alternatively, the stability of a delayed-release coating can be enzyme-dependent. An enzyme-dependent delayed-release coating will be substantially stable in a fluid lacking the specific enzyme and substantially unstable in a fluid containing that enzyme. The coating will substantially disintegrate or dissolve in a fluid containing the appropriate enzyme. Enzyme-dependent control can be achieved, for example, by using materials that release the active ingredient only upon exposure to enzymes in the intestine, such as galactomannan.

[0043] Various types of enteric coatings are known for the delayed but high-volume delivery of active agents to the gastric tract. In some embodiments, the sustained-release composition comprises an enteric solvent that is substantially stable in acidic environments and substantially unstable in near-neutral to alkaline environments. In one embodiment, the sustained-release coating contains an enteric solvent that is substantially stable in gastric juices. The enteric solvent may be selected from, for example, solutions or dispersions of: methacrylic acid copolymers, cellulose phthalate acetate, hydroxypropyl methyl cellulose phthalate, polyvinyl acetate phthalate, carboxymethyl ethyl cellulose, and... Type polymers (poly(methacrylic acid, methyl methacrylate), hydroxypropyl methyl cellulose acetate succinate, cellulose trimellitate acetate, shellac or other suitable enteric coating polymers). Type polymers include, for example FS 30D, L 30D-55, L 100-55, L 100, L 12.5, L 12.5 P, RL 30 D, RL PO, RL 100, RL 12.5, RS 30 D, RSPO, RS 100, RS 12.5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12.5, and S 12.5 P. In some implementations, the following are used: One or more of the following: FS 30D, L 30D-55, L 100-55, L 100, L 12,5, L 12,5 P, RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40D, NM 30 D, S100, S 12,5, and S 12,5 P. The enteric solvent can be a combination of the aforementioned solutions or dispersions. In some embodiments, the enteric solvent is EUDRAGIT F30D, which comprises a copolymer of methyl acrylate, methyl methacrylate, and methacrylic acid. The copolymer has a free carbonyl group to ester group ratio of approximately 1:10.

[0044] In another embodiment, when in aqueous solution, the sustained-release coating can degrade over time, independent of the pH and / or the presence of enzymes in the solution. Such a coating may comprise a water-insoluble polymer. Therefore, the solubility of the coating in aqueous solution is pH-independent. The term "pH-independent" as used herein means that the water permeability of the polymer and its ability to release the pharmaceutical ingredient are independent of pH and / or depend only very slightly on pH. Such coatings can be used to prepare, for example, sustained-release formulations. Suitable water-insoluble polymers include pharmaceutically acceptable non-toxic polymers that are substantially insoluble in aqueous media (e.g., water) and independent of the pH of the solution. Suitable polymers include, but are not limited to, cellulose ethers, cellulose esters, or cellulose ether-esters, i.e., cellulose derivatives in which some hydroxyl groups on the cellulose backbone are substituted with alkyl groups and some hydroxyl groups are modified with alkyl groups. Examples include ethyl cellulose, acetyl cellulose, nitrocellulose, etc. Other examples of insoluble polymers include, but are not limited to, varnishes, and acrylate polymers and / or methacrylate polymers, polymers or copolymers of acrylates or methacrylates with low quaternary ammonium content, or mixtures thereof. Other examples of insoluble polymers include EUDRAGIT. EUDRAGIT EUDRAGIT Polyvinyl ester, polyvinyl alcohol acetal, polyacrylate, butadiene-styrene copolymer, etc.

[0045] In some embodiments, the composition relates to coated tablets or coated beads or granules having, for example, a delayed-release feature as described in U.S. Patent No. 8,168,594, the entire contents of which are incorporated herein by reference. Exemplary enteric coatings comprise copolymers of acrylates and methacrylates, which in some embodiments are 1:1 copolymers. Other fillers, binders, and plasticizers (including those for sealing or topcoating layers) are described in US 8,168,594, which is incorporated herein by reference.

[0046] For example, the composition may include one or more release layers. The release layer separates the core tablet or particle from the delayed-release coating. The release layer can be applied to the core via a coating or layering process, typically used with coating equipment such as a coating pan, a coating granulator, or a fluidized bed apparatus that uses water and / or organic solvents for the coating process. Alternatively, the release layer can be applied to the core material using powder coating technology. Materials used for the release layer are pharmaceutically acceptable compounds such as sugars, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, hydroxypropyl cellulose, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, etc., used alone or in combination. The release layer may also contain additives such as plasticizers, colorants, pigments, fillers, anti-sticking agents, and antistatic agents, such as magnesium stearate, titanium dioxide, talc, and other additives.

[0047] The enteric coating composition can be dispersed or dissolved in water or a suitable organic solvent and applied to the core particles by methods well known to those skilled in the art. One or more delayed-release coatings can be applied to the coated core particles.

[0048] Enteric coatings or other coatings may contain one or more inert processing aids, including but not limited to talc, silica, magnesium stearate, etc. Enteric coating compositions may also contain pharmaceutically acceptable plasticizers to obtain desired mechanical properties such as flexibility and hardness. Such plasticizers include, but are not limited to, glyceryl acetate, citrate, phthalates, dibutyl sebacate, cetyl alcohol, polyethylene glycol, polysorbate, or other plasticizers.

[0049] For example, in some embodiments, the coated particles or tablets may be further covered with an outer coating layer. The outer coating material is a pharmaceutically acceptable compound such as sugars, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, hydroxypropyl cellulose, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, etc., used alone or in combination. The outer coating material can prevent potential aggregation of particles coated with enteric coatings, protect the coating from breakage during compression, or enhance the tableting process.

[0050] Therefore, in some embodiments, the matrix comprises one or more binders, fillers, or plasticizers. Such components comprise one or more of cellulose or cellulose derivatives, fatty acid salts, or synthetic polymers. For example, the binder, filler, or plasticizer may comprise a synthetic polymer, and the polymer is optionally a copolymer of vinylpyrrolidine and vinyl acetate. Alternatively, the binder, filler, or plasticizer comprises a cellulose derivative, optionally comprising one or more of ethyl cellulose, hydroxypropyl methylcellulose, and carboxymethyl cellulose. In some embodiments, the binder, filler, or plasticizer comprises a fatty acid salt, optionally selected from C8 to C18 fatty acid salts, optionally a stearate (e.g., magnesium stearate). In some embodiments, the enteric coating comprises a plasticizer, optionally triethyl citrate.

[0051] The oral dosage composition may be in the form of capsules comprising granules or beads, or in the form of enteric-coated tablets, or other forms. In some embodiments, the composition comprises a group of beads or granules containing a matrix and an enteric coating, which may be contained within a capsule. For example, in some embodiments, the beads comprise an enteric coating comprising a copolymer of methyl acrylate, methyl methacrylate, and methacrylic acid, and said copolymer may optionally have a free carbonyl to ester ratio of about 1:10. Such an enteric coating may be from about 15% to about 40% by weight of the composition. In some embodiments, the enteric coating is from about 20% to about 30% by weight of the composition, or from about 20% to about 25% by weight of the composition.

[0052] The polymer matrix can be selected to degrade or erode in a substantially pH-independent manner. In other embodiments, the polymer matrix degrades or erodes in a pH-dependent manner. Exemplary polymer matrices comprise polysaccharide matrices, such as matrices comprising one or more of the following: cellulose, chitin, chitosan, alginate, amylose, pectin, callosity, laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, galactomannan, xanthan gum, dextran, welan gum, gellan gum, diutan gum, pullulan, hyaluronic acid, and derivatives thereof. Cellulose derivatives include, for example, alkyl, hydroxyl, and carboxylated derivatives. In some embodiments, the matrix comprises microcrystalline cellulose. In yet other embodiments, the matrix comprises various biodegradable synthetic polymers known in the art.

[0053] In some embodiments, the composition is a capsule for oral delivery comprising a cluster of beads containing 0.25 to 2 mg of laprazole peptide or a laprazole peptide derivative or a salt thereof contained within an erosive polymer matrix. The beads also contain an enteric coating comprising a copolymer of methyl acrylate, methyl methacrylate, and methacrylic acid. The ratio of free carbonyl groups to ester groups in the copolymer can be about 1:10 (e.g., EUDRAGIT F30D). In such embodiments, the enteric coating constitutes about 20% to about 30% of the total weight of the composition. In some embodiments, the erosive matrix comprises microcrystalline cellulose. The composition may also contain a sealing layer or a top coating.

[0054] In some embodiments, the composition provides the release of less than about 15% of peptides after about 2 hours in simulated gastric fluid. Additionally, the composition provides the release of less than about 25% of peptides after about 2 hours in simulated intestinal fluid having a pH of about 5.5. In various embodiments, the composition releases at least about 40% but no more than about 80% of peptides after about 2 hours in simulated intestinal fluid having a pH of about 7. In various embodiments, 100% release in simulated intestinal fluid having a pH of about 7 is achieved after at least three hours, or in some embodiments, after at least about 3.5 or at least about four hours.

[0055] In other aspects, the present invention provides methods for treating small bowel disorders, symptoms, and / or diseases. Such small bowel disorders are generally associated with impaired intestinal barrier function and increased intestinal permeability. For example, impaired intestinal barrier function and increased intestinal permeability may be associated with various inflammatory liver diseases, including nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and cirrhosis (e.g., alcoholic cirrhosis). In some embodiments, the present invention provides methods for preventing and / or treating celiac disease.

[0056] According to certain embodiments, laprazole peptides or derivatives are administered once or more daily to promote the integrity of GI tight junctions. For example, laprazole peptides or derivatives may be administered once daily, about twice daily, or about three or more times daily. In various embodiments, the regimen of laprazole peptides or derivatives is administered for an extended period. In some embodiments, the regimen of laprazole peptides or derivatives is administered for at least about 1 month, at least about 2 months, at least about 4 months, and at least about 8 months. For example, the regimen of laprazole peptides or derivatives is administered for at least about 6 months. In some embodiments, treatment is continuous to delay or prevent disease progression.

[0057] In some implementations, the subject has fatty liver disease, including but not limited to NAFLD, NASH, alcoholic steatohepatitis (ASH), or fatty liver disease caused by: hepatitis, obesity, diabetes, insulin resistance, hypertriglyceridemia, chronic kidney disease, abeta-lipoproteinemia, glycogen storage disease, Weber-Christian disease, Wolman's disease, acute fatty liver of pregnancy, and lipodystrophy. In some implementations, improved intestinal barrier function limits the amount of toxins (such as LPS) entering the circulation and ultimately exacerbating or promoting disease progression. In some implementations, the subject has NASH.

[0058] In some embodiments, the present invention provides treatment for patients suffering from NAFLD. NAFLD represents a range of diseases that occur without alcohol abuse. NAFLD is characterized by the presence of steatosis (fat in the liver) and can represent liver manifestations of metabolic syndrome (including obesity, diabetes, and hypertriglyceridemia). The severity of NAFLD ranges from relatively benign, isolated, primarily macrovesicular steatosis (i.e., non-alcoholic fatty liver or NAFL) to non-alcoholic steatohepatitis (NASH). NASH is characterized by the histological presence of steatosis, cytological ballooning, scattered inflammation, and pericellinal fibrosis. Liver fibrosis caused by NASH can progress to cirrhosis or liver failure and, in some cases, may lead to hepatocellular carcinoma. In some embodiments, the methods of the present invention reduce or improve one or more symptoms of NAFLD or NASH and can improve or maintain liver function. In some embodiments, the methods of the present invention prevent or slow the progression of NAFLD or NASH.

[0059] In some embodiments, the present invention provides a method for treating or preventing inflammatory liver disease in a subject. The method includes administering the compositions disclosed herein to a subject in need. In some embodiments, the subject has fatty liver disease. In some embodiments, the subject has inflammatory liver disease selected from nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty acid liver disease (NAFLD). In yet other embodiments, the subject has alcoholic steatohepatitis (ASH).

[0060] In some embodiments, the present invention provides treatment for patients suffering from hepatitis. In exemplary embodiments, hepatitis may be caused by viruses, alcohol, drugs, etc. In one embodiment, the present invention provides treatment for patients suffering from hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E. In another embodiment, the present invention provides treatment for alcoholic hepatitis. In yet another embodiment, the present invention provides treatment for autoimmune hepatitis. Symptoms of hepatitis include fatigue, flu-like symptoms, dark urine, pale stools, abdominal pain, loss of appetite, unexplained weight loss, and jaundice. Chronic hepatitis is also associated with cirrhosis and hepatocellular carcinoma. In various embodiments, the methods of the present invention reduce, improve, or eliminate one or more symptoms of hepatitis.

[0061] In some implementations, the subject has one or more liver disease-related conditions, such as kidney disease (e.g., chronic kidney disease), viral hepatitis, diabetes, hypertriglyceridemia, and / or insulin resistance.

[0062] In some embodiments, patients may receive adjunctive therapy, which in some embodiments synergistically works with laprazole peptide therapy. In some embodiments, the present invention relates to a regimen for administering laprazole peptide (or a derivative of laprazole peptide) to a subject to improve glycemic control. In various embodiments, the laprazole peptide regimen enhances the effectiveness of conventional pharmacological interventions such as metformin, basal insulin, GLP-1 receptor agonists (e.g., liraglutide), gastric inhibitory peptide (GIP), sulfonylureas, pPAR-γ agonists, obeticholic acid, etc. According to the present invention, the laprazole peptide regimen prevents complications of hyperglycemia, including cardiovascular complications and organ damage. It is not intended to be bound by any single theory that uncontrolled hyperglycemia may lead to or be associated with intestinal barrier dysfunction and the development of fatty liver disease. These dysfunctions reduce the efficacy of conventional pharmacological interventions for hyperglycemia and diabetes and allow microorganisms and toxins (e.g., lipopolysaccharide or LPS) to diffuse from the intestinal lumen into the lamina propria and systemic circulation, which in turn leads to systemic infection or tissue and organ damage. Furthermore, in subjects with intestinal barrier dysfunction caused by hyperglycemia, glucose may leak into the circulation through damaged epithelial tight junctions, which may affect the efficacy of medications used for glycemic control.

[0063] In other embodiments, the compositions described herein are administered to patients exhibiting symptoms of intestinal permeability, including but not limited to patients with celiac disease, inflammatory bowel syndrome, Crohn's disease, chronic kidney disease, and various autoimmune conditions such as diabetes.

[0064] In other aspects, the present invention provides methods for treating subjects with cancer, and methods for enhancing immunotherapy. Methods for treating cancer and / or enhancing immune checkpoint inhibitor therapy by administering laprazole peptide or a derivative thereof to a subject in need are described in International Application No. PCT / US19 / 22885, filed March 19, 2019, the entire contents of which are incorporated herein by reference. Without wishing to be bound by theory, it is believed that maintaining a healthy intestinal mucosa (e.g., by administering laprazole peptide or a derivative thereof) can enhance the efficacy of immunotherapies, including checkpoint inhibitor therapy, lymphocyte stimulation therapy, or T-cell therapy (e.g., CAR-T cell therapy).

[0065] In some embodiments, such methods for treating cancer and / or enhancing immune checkpoint inhibitor therapy include treating subjects who have received checkpoint inhibitor therapy and / or subjects who are scheduled to receive immune checkpoint inhibitor therapy. In some embodiments, the subject has not shown a response or has only shown a partial response to prior treatment with immune checkpoint inhibitor therapy. In some embodiments, the subject has not achieved at least stable disease with prior treatment using immune checkpoint inhibitor therapy. In some embodiments, the prior immune checkpoint inhibitor therapy is PD-1 blockade therapy (e.g., anti-PD-1 or anti-PD-L1).

[0066] In some implementations, one or more immune checkpoint inhibitors are selected from the following inhibitors: programmed death-ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CDI37, CDI60, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell co-stimulatory molecule), KIR, LAIRI, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, and VTCNI. In some implementations, the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1. In some implementations, the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, pembrolizumab, and nivolumab.

[0067] In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody, such as an anti-CTLA-4, anti-PD-1, or anti-PD-L1 and / or PD-L2 agent (e.g., YERVOY, OPDIVO, or KEYTRUDA, or equivalent agents). In various embodiments, these agents may be administered in multiple doses, such as 4 to 12 doses or 4 to 8 doses, which in some embodiments may be administered over a period of 1 to 4 months (e.g., 1 or 2 months in some embodiments).

[0068] In some implementations, immunotherapy includes the administration of agonists of lymphocyte co-stimulatory molecules such as OX40 or OX40L, CD28 or 4-1BB.

[0069] In some embodiments, the compositions comprising or releasing laprazole peptide or derivatives thereof described herein are administered at least once daily. In some embodiments, the compositions are administered at a regimen comprising 1 to 5 times daily, such as 1 to 3 times daily. In some embodiments, the regimen is initiated prior to immunotherapy (e.g., checkpoint inhibitor therapy), for example at least one week prior to the initiation of immunotherapy (e.g., checkpoint inhibitor therapy), or in some embodiments at least 2 weeks, at least 3 weeks, or at least 4 weeks (about 1 month) prior to the initiation of immunotherapy (e.g., checkpoint inhibitor therapy). In these or other embodiments, the regimen continues throughout the entire immunotherapy (e.g., checkpoint inhibitor therapy) and optionally continues thereafter for a period of time (e.g., at least one month or longer after the immunotherapy or checkpoint inhibitor therapy regimen).

[0070] In various embodiments, administration of a composition comprising laprazole peptide or a derivative thereof increases or restores the efficacy of immune checkpoint inhibitor therapy. For example, in some embodiments, a subject with cancer has previously been unresponsive to or has become resistant to immune checkpoint inhibitors. In some embodiments, for example, the cancer is refractory to or poorly responsive to immunotherapies such as anti-CTLA-4, anti-PD-1, or anti-PD-L1 and / or PD-L2 agents. In some embodiments, a cancer subject has progressed after or during treatment with anti-CTLA-4, anti-PD-1, or anti-PD-L1 and / or PD-L2 agents (including, for example, one or more of ipilimumab, trimemumab, pembrolizumab, and nivolumab), or has not shown a response to such treatment after at least about 4 weeks, or at least about 8 weeks, or at least about 12 weeks of treatment.

[0071] Cancer can be any cancer that can be treated with immune checkpoint inhibitor therapy, including primary cancer, metastatic cancer, or blood cancer, and can originate from any tissue. For example, in one implementation, the cancer originates in the skin, colon, breast, or prostate, and is therefore composed of cells that originally originated from the skin, colon, breast, or prostate, respectively.

[0072] In some implementations, the cancer is progressive, locally advanced, or metastatic. In some implementations, the cancer is metastatic melanoma and may be recurrent. In some implementations, the metastatic melanoma is stage III or IV and may be stage IVA, IVB, or IVC. Metastasis may be local or distant.

[0073] In some embodiments, the solid tumor is a sarcoma or carcinoma. In some embodiments, the solid tumor is a recurrent or refractory solid tumor. In some embodiments, the recurrent or refractory solid tumor is a sarcoma or carcinoma. In some embodiments, the solid tumor is a metastatic solid tumor. In some embodiments, the metastatic solid tumor is a sarcoma or carcinoma.

[0074] In some implementations, the cancer is a blood cancer. In some implementations, the blood cancer is leukemia (e.g., AML), lymphoma, myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell malignancy, or B-cell malignancy.

[0075] Other aspects and embodiments of the invention will become apparent from the following examples.

[0076] Example

[0077] Example 1: An in vivo model of leaky gut syndrome associated with NASH liver pathology

[0078] Decreased intestinal integrity (“leaky gut”) may occur during the progression of NAFLD and NASH. An improved competitive ELISA capable of continuous serum dextran measurement in small amounts of serum was used to measure intestinal integrity, and administration of laprazole acetate in this in vivo model was demonstrated to improve intestinal integrity.

[0079] 77 DIAMOND TM Mice were placed on a normal diet (NDNW) or a Western diet (WDSW) and were up to 8, 20, 28, 36, and 40 weeks of age, and then administered 4 kDa FITC-glucan at 600 mg / kg body weight via tube feeding. Four hours after administration, at least 20 μL of serum was collected via a tail vein incision. Serum glucan concentrations were quantified using a competitive ELISA measuring the small glucan polymers (conjugated or unconjugated). Significantly higher serum glucan levels were observed in the Western diet group compared to the normal diet group. Figure 1 As shown.

[0080] Subsequent experiments evaluated the effects of orally administered laprazole acetate on mice on a Western diet. The study design was as follows: Figure 2 As described in the study. Specifically, eight groups of mice were placed on a WDSW diet for 16 weeks and administered laprazole peptide (by drinking water or oral tube feeding), a pioglitazone comparator, or a mediator during weeks 8–16. Improvement in leaky gut syndrome was assessed as follows: mice were given unconjugated dextran at 600 mg / kg via oral tube feeding at baseline and at the end of the study. Serum was collected 4 hours later, and serum dextran concentration was measured.

[0081] Figure 3The results showed that administration of laprazole peptide improved intestinal integrity in a mouse model, as measured by serum dextran concentration. Figure 3 The results showed that various doses of laprazole acetate successfully reduced serum glucan concentrations in mice on a Western diet.

[0082] Example 2: Multiple administrations of laprazole peptide are effective at low concentrations.

[0083] The aim of this study was to determine the therapeutic effect of fractionated administration of laprazole peptide at low concentrations. In fact, this study validated the inverse dose-response observed with laprazole peptide, which is due to the local accumulation of inactive laprazole peptide fragments caused by excessive administration. Therefore, this study supports the theory that low doses of laprazole peptide exert their optimal effect by avoiding the accumulation of competing inactive fragments.

[0084] In an isolated ischemic jejunum model for measuring TER (transepithelial resistance), various concentrations (0.1 μM, 0.5 μM, or 1 μM) of laprazole peptide were administered in fractional doses every 45 minutes. Figure 10 As indicated by the arrows along the x-axis, fractional doses are administered at 45, 90, 135, and 180 minutes. Figure 10 Multiple administrations of low concentrations (e.g., 0.1 μM) of latazotide were shown to be more effective than a single 1 μM dose of latazotide during a recovery period of at least 240 minutes.

[0085] Specifically, Yorkshire crossbred pigs aged 6–8 weeks were anesthetized, followed by midline laparotomy to create a series of 10 cm intestinal loops (jejunum) starting from the proximal ileum via intestinal ligation. The mesenteric vascular system was ligated to select loops for treatment for 45 minutes, while other loops served as non-ischemic controls. The loops were then excised, and mucosal tissue was dissected from the muscle layer in oxygenated (95% O2 / 5% CO2) Ringer's solution in preparation for in vitro incubation. The tissues were then mounted on a Ussing perfusion chamber, and all tissues were allowed to acclimatize for 30 minutes to establish baseline measurements. The tissues were treated with different concentrations of laprazole peptide at varying time intervals, and transepithelial electrical resistance (TEER) was measured for up to 240 minutes.

[0086] Therefore, the results of this experiment support the conclusion that releasing a small amount of laprazole peptide over a prolonged period of time improves the effect of the activator.

[0087] Example 3: Sustained-release tablet formulation

[0088] The following examples illustrate the preparation of enteric-coated tablets for sustained release of laprazole peptide into the jejunum and ileum.

[0089] Table 1: Composition of the core tablets (% by weight)

[0090] Components F23 Laprazole peptide 1 Sodium carboxymethyl cellulose 48.5 magnesium stearate 0.5 HPMC 50

[0091] Table 2: Ingredients of the core tablets (mg / 3000mg)

[0092]

[0093]

[0094] Table 3: Composition of enteric coating solution (% by weight)

[0095] Components F23 Eudragit L100 6 Triethyl citrate 0.6 ethanol 93.4

[0096] Table 4: Ingredients of Enteric Coating Solution (g / 250g)

[0097] Components F23 Eudragit L100 15 Triethyl citrate 1.5 ethanol 233.5

[0098] The following procedure was used to prepare the core tablets. API, HPMC, sodium carboxymethyl cellulose, and magnesium stearate were weighed in a mortar and mixed thoroughly. 100 mg of the mixture was weighed to prepare the core tablets. The core tablets were divided into three parts: (1) tablets without further coating, coded F23-1; (2) tablets with an enteric coating, coded F23-2; and (3) tablets coated with a layer of HPMC followed by another enteric coating layer, coded F23-3.

[0099] The enteric coating is prepared by the following procedure: Eudragit L100 and triethyl citrate are weighed into a glass bottle according to the enteric coating ingredient list. Ethanol is added to dissolve the powder. The tablets are immersed in the enteric coating solution, then dried with nitrogen (N2). This step is repeated until the weight increases by approximately 10%.

[0100] Table 5: Dissolution Results

[0101]

[0102]

[0103]

[0104] in conclusion:

[0105] Sodium HPMC and CMC can prolong the release time of laprazole peptide.

[0106] L100 enteric coating can prevent the release of laprazole peptide in SGF dissolution medium.

[0107] F23-2 meets the requirements that the total API released in SGF after 2 hours does not exceed 5% and the total API released in SIF (pH 5.5) after 2 hours does not exceed 20%. See also Figure 5 .

[0108] Example 4: Core Particles and Enteric Coating Permitted Controlled-Release Formulation

[0109] The purpose of this experiment was to compare the release distribution and dissolution results of two controlled-release laprazole peptide microparticle formulations containing different components and enteric coatings.

[0110] Core particles are produced using a wet granulation method. Core particle A consists of the following components: laprazole peptide and hydroxypropyl methylcellulose (HPMC). 101 microcrystalline cellulose (MCC) and magnesium stearate. Core particle B consists of the following: laprazole peptide, VA64 101 microcrystalline cellulose (MCC) and magnesium stearate. The composition is shown in Table 1 below.

[0111] Table 6: Composition of core particles (% by weight)

[0112] Components A B Laprazole peptide 1 1 HPMC 3 Kollidon VA64 5 MCC 95.5 93.5 magnesium stearate 0.5 0.5

[0113] This wet granulation method begins by weighing and mixing each component, then gradually adding dihydrate (DI) to the mixture to form a dough. The dough is then extruded through a 1.0 mm dome sieve at 45 rpm using an extruder (Multi-Gran, Fuji Paudal, model: MG-55). The extrudate is collected and fed into a marumerizer (tabletop marumerizer, Fuji Paudal, model: QJ-230T-1) and run at 1,800 rpm / 1,300 rpm for 2 minutes / 1 minute to produce granules. The granules are dried for 3 hours, and the moisture content is then tested using a moisture analyzer (Mettler Toledo, model: HR73). Afterward, the dried granules are passed through an 18-mesh sieve (Fisher Scientific, 1.00 mm) and a 25-mesh sieve (Fisher Scientific, 710 μm).

[0114] Two enteric-coated solutions were prepared to compare their release distribution when coated onto core particles A and B. This was achieved by weighing... S100 enteric coating solutions were prepared by adding triethyl citrate, then ethanol and talc. The compositions of the two enteric coatings are described in Table 2.

[0115] Table 7: Composition of enteric coating (% by weight)

[0116]

[0117]

[0118] The particles were then immersed in an S100 enteric coating solution and subsequently dried with N2. This process was repeated until a specific weight gain was achieved, as shown in Table 3.

[0119] Table 8: S100 weight increase of core particles A and B respectively

[0120] name Enteric-coated solution % weight increase A S100 50 B-1 S100 30 B-2 S100 90

[0121] FS30D enteric-coated solution is prepared by shaking Plasacryl while stirring. FS30D and water were added to PlasACRYL for preparation. The composition of the FS30D enteric coating is described in Table 2. The granules were then sprayed with the FS30D enteric coating while stirring, followed by drying with N2. This step was repeated until a specific weight gain was achieved, as shown in Table 9.

[0122] Table 9: Weight Increase of FS30D for Core Particles A and B

[0123] name Enteric-coated solution % weight increase B-3 FS30D 10 B-4 FS30D 23 B-5 FS30D 50 B-6 FS30D 90

[0124] Then, in vitro dissolution tests were performed on each of the coated core particles under the conditions described in Table 10.

[0125] Table 10: In vitro dissolution method setup

[0126]

[0127]

[0128] The process begins by weighing the granules into capsules, which are then loaded into a rotating basket. A specific volume of pH 1.0 SGF dissolution medium is added to the container and heated to a high temperature of 37.0 ± 0.5 °C. The rotating basket containing the granules is then placed inside the container. The autosampler is set to collect approximately 1 mL of sample at the time points listed in Table 5. After 120 minutes, dissolution is stopped by lifting the rotating basket and the medium is discarded. Once a specific volume of preheated pH 5.5 SIF dissolution medium is transferred to the container (with stirring), the rotating basket is lowered again. At the sampling time specified in Table 5, the autosampler collects 1 mL of solution sample. After 120 minutes, dissolution is stopped again by lifting the rotating basket and the medium is discarded. A specific volume of preheated pH 7.0 SIF dissolution medium is transferred to the container (with stirring), and the rotating basket is lowered once more. At the sampling time specified in Table 10, the autosampler collects 1 mL of solution sample.

[0129] The dissolution test results for various core particles and enteric coating / enteric coating thicknesses are described in Table 11 and... Figures 6A-6F middle.

[0130] Table 11: Dissolution results of core particles A and B

[0131]

[0132]

[0133]

[0134]

[0135] Table 12 summarizes the formulation and its various properties (including dissolution profiles).

[0136] Table 12: Summary of Formulation Characteristics

[0137]

[0138] Compared to S100 enteric coating, FS30D enteric coating exhibited more favorable API (i.e., laprazole peptide) release characteristics. Furthermore, it was found that increasing the weight of the enteric coating reduced API release; however, excessive coating resulted in slow API release at higher pH levels. B-4 (23% weight increase) struck a balance, allowing the formulation to maintain slow release at low pH (<5.5) and rapid release at high pH (>7).

[0139] Example 5: In vivo release distribution of controlled-release laprazole peptide microparticle formulation

[0140] The purpose of this experiment was to establish the in vivo release distribution of lalazopeptide in B-4 delayed and extended release formulations.

[0141] Because the human digestive system is similar to that of a pig, the piglet was used as a model in this experiment. Figure 7 The diagram shows that probe 1 points towards the duodenum (8-10 cm from the pylorus); probe 2 points about 20 cm from probe 1; probe 3 points about 50 cm from probe 1; and probe 4 points towards the cecum-ileum junction.

[0142] Specifically, boars weighing 12-18 kg at the start of the study were used, aged 6-8 weeks. Ultrafiltration (UF) probes were surgically placed in the intestine. Pigs were not fed or given water for at least 12 hours prior to anesthesia and surgery. The ultrafiltration probes were implanted into the intestinal wall via a surgical midline abdominal incision. The ultrafiltration probe tubes were passed through the skin and attached with sutures. Collection tubes were attached to the outer end of each probe tube for sampling gastrointestinal fluid. The first probe was placed 8-10 cm from the pylorus (terminal duodenum), and the second probe was placed 20 cm distal to the first probe (in the jejunum). The third probe was placed 50 cm distal to the first probe (in the jejunum), and the fourth probe was placed at the cecum-ileum junction. Animals were fasted for at least 12 hours before each administration and for 4 hours after each administration event. Water intake was withheld for 1 hour before and 2 hours after administration. Administration was given in 120 ml of water. Gastrointestinal fluid samples were taken from filtered samples obtained from the collection tubes of each probe before administration and at 0–1, 1–2, 2–3, 3–4, and 4–5 hours after administration. The concentration of laprazole peptide in the gastrointestinal fluid samples was determined using UPLC-MS / MS.

[0143] For comparison, Figure 8 The in vivo release distribution of the delayed-release laprazole peptide formulation was described, while Figure 9 The in vivo release distribution of delayed and extended release B-4 formulations is shown. Figure 8 The delayed-release laprazole peptide formulation used in this study is formulated for the treatment of celiac disease and contains two types of beads, both of which are gastroresistant (i.e., they are not released in simulated gastric juice (SGF)). Once the beads reach the duodenum, the first type of bead is released within 60 minutes in simulated intestinal juice (SIF) with a pH greater than 5, and the second type of bead is released after approximately 30 minutes and approximately 90 minutes (in the SIF), targeting the jejunum.

[0144] 1mg dose Figure 8 The delayed-release formulation is released only in the duodenum and jejunum (20 cm), while Figure 9 The delayed and prolonged release formulations of B-4 released only small amounts in the duodenum, but released larger amounts in the jejunum (20 cm) and jejunum (50 cm). These results are supported by the in vitro release profiles of B-4, such as... Figure 6E As described in the text, the formulation is released with a delayed release until it reaches the jejunum and from there it exhibits a slow release from the jejunum to the ileum (after 180 minutes).

[0145] Example 6: Storage stability study of a 10g batch of extended-release B-4 formulation. (Appearance, content, and impurities)

[0146] The appearance, content and impurities of B-4 laprazole peptide granules (batch number #339-2-83) were tested using the laprazole peptide analytical method at T0 and T=3 months. The results are shown in Tables 13 and 14 below.

[0147] Table 13. Results of appearance and content of B-4 laprazole peptide particles.

[0148]

[0149] Table 14. Results of impurities in B-4 laprazole peptide particles.

[0150]

[0151] Equivalent solution

[0152] Although the invention has been described in conjunction with its specific embodiments, it should be understood that further modifications are possible and this application is intended to cover any variations, uses, or adaptations of the invention in general according to the principles of the invention, including known or conventional practices that, while not part of this disclosure, fall within the field of the invention and variations that are essential features described above and that fall within the scope of the appended claims.

[0153] Using only conventional experiments, those skilled in the art will recognize or be able to identify numerous equivalents of the particular embodiments specifically described herein. Such equivalents are intended to be covered within the scope of the following claims.

[0154] By incorporating via reference

[0155] All patents and publications cited in this article are incorporated herein in their entirety by reference. sequence list <110> 9 METERS BIOPHARMA, INC. <120> Laprazole peptide preparations <130> AJ3171PT2201 <150> US 62 / 888,052 <151> 2019-08-16 <150> US 63 / 009,768 <151> 2020-04-14 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polymers <400> 1 Gly Gly Val Leu Val Gln Pro Gly 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polymers <400> 2 Gly Val Leu Val Gln Pro Gly 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polymers <400> 3 Val Leu Val Gln Pro Gly 1 5

Claims

1. A composition comprising a bead cluster containing an effective amount of a peptide or a salt thereof, said peptide being a laprazole peptide or d-lalazole peptide, said peptide or salt thereof being contained within a biodegradable or erosive polymer matrix, said polymer matrix comprising microcrystalline cellulose and a copolymer of vinylpyrrolidine and vinyl acetate as a binder, said polymer matrix providing sustained release of said peptide or salt thereof in simulated intestinal fluid having a pH of 7.0 for at least 120 minutes. The beads further comprise an enteric coating comprising a copolymer of methyl acrylate, methyl methacrylate, and methacrylic acid, the enteric coating being resistant to dissolution in simulated gastric or intestinal fluids having a pH of 5.5 or lower, wherein the ratio of free carbonyl groups to ester groups in the copolymer is 1:10; wherein the composition provides less than 25% release of the peptide or its salt after 2 hours in the simulated gastric or intestinal fluids; and wherein the enteric coating constitutes 20% to 30% by weight of the composition. The d-lalazine peptide is D at every position other than Gly. Amino acids.

2. The composition of claim 1, wherein the composition provides sustained release of the peptide or a salt thereof in simulated intestinal fluid for at least 180 minutes.

3. The composition of claim 2, wherein the composition provides sustained release of the peptide or a salt thereof in simulated intestinal fluid for at least 210 minutes.

4. The composition of claim 3, wherein the composition provides sustained release of the peptide or a salt thereof in simulated intestinal fluid for at least 240 minutes.

5. The composition of claim 1, wherein the composition comprises at least 0.25 mg of the peptide or a salt thereof.

6. The composition of claim 5, wherein the composition comprises at least 0.5 mg of the peptide or a salt thereof.

7. The composition of claim 6, wherein the composition comprises at least 1 mg of the peptide or a salt thereof.

8. The composition of claim 7, wherein the composition comprises at least 2 mg of the peptide or a salt thereof.

9. The composition of claim 1, wherein the composition releases the peptide or a salt thereof in the jejunum and ileum of a human patient.

10. The composition of claim 9, wherein the composition does not release the peptide or its salt in the duodenum.

11. The composition of claim 9, wherein the composition does not release the peptide or its salt in the colon.

12. The composition of claim 1, wherein the enteric coating is 20% to 25% by weight of the composition.

13. The composition of claim 1, wherein the polymer matrix degrades or erodes in a pH-dependent manner.

14. The composition of claim 1, wherein the composition comprises a binder, a filler, or a plasticizer, and the binder, filler, or plasticizer comprises a fatty acid salt.

15. The composition of claim 14, wherein the fatty acid salt is selected from C8 to C18 fatty acid salts.

16. The composition of claim 14, wherein the fatty acid salt is a stearate.

17. The composition of claim 1, wherein the enteric coating comprises a plasticizer.

18. The composition of claim 17, wherein the plasticizer is triethyl citrate.

19. The composition of claim 1, wherein the beads comprise a surface coating or a sealing coating.

20. The composition of claim 19, wherein the topcoat or sealant comprises a plasticizer.

21. The composition of claim 20, wherein the plasticizer is an acrylic polymer.

22. The composition of claim 1, wherein the composition is a capsule for oral delivery comprising a bead cluster comprising 0.25 to 2 mg of laprazole peptide or d-laprazole peptide or a salt thereof contained within the erosive polymer matrix.

23. The composition of claim 22, wherein the composition further comprises a sealing layer or a topcoat.

24. Use of the composition of any one of claims 1 to 23 in the preparation of a medicament for treating non-alcoholic fatty liver disease (NAFLD).

25. Use of the composition of any one of claims 1 to 23 in the preparation of a medicament for treating non-alcoholic steatohepatitis (NASH).

26. The use as described in claim 24 or 25, wherein the composition is delivered from the proximal jejunum to the distal ileum.

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