Pharmaceutical formulations and methods of use thereof

Abstract

The disclosure relates to pharmaceutical formulations and method of using chymase inhibitors for the prevention and treatment of mast cell-related disease states in which chymase is involved. More particularly the present disclosure is in the field of treating or preventing or reducing the symptoms of mast cell-related diseases, including atopic dermatitis (AD), chronic urticaria (CU), Eosinophilic Gastrointestinal Disorders (EGIDs), and prurigo nodularis (PN), using a chymase inhibitor, including a chymase inhibitor that prevents cleavage of stem cell factor (SCF) and its interaction with receptor tyrosine kinase (c-kit), thereby reducing mast cell activation and proliferation.

Description

[0001] Docket No. 123950-10402

[0002] 1

[0003] PHARMACEUTICAL FORMULATIONS AND METHODS OF USE THEREOF

[0004] CROSS REFERENCE TO RELATED APPLICATION

[0005] This application claims priority to U.S. Provisional Patent Application No. 63 / 679,300 entitled “PHARMACEUTICAL FORMULATIONS AND METHODS OF USE THEREOF” filed August 5, 2024 (Attorney Docket No. 123950-10404), which is hereby incorporated by reference in its entirety.

[0006] FIELD OF THE INVENTION

[0007] The disclosure generally relates to pharmaceutical formulations for preventing or treating diseases in which mast cells are involved. More particularly the present disclosure is in the field of treating or preventing or reducing the symptoms of mast cell-related diseases using a chymase inhibitor, including chymase inhibitors which prevent cleavage of stem cell factor (SCF) and its interaction with receptor tyrosine kinase (c-kit), thereby reducing mast cell activation and proliferation.

[0008] BACKGROUND

[0009] Mast cells (MC) are pivotal in immune function and maintaining bodily balance. Upon activation, they release chymase, a protease that triggers various pathways, including inflammation and fibrosis progression. Chymase cleaves and generates active form of molecules like SCF, MMP- 9 and Angiotensin-II leading to inflammation and tissue remodeling. Inhibiting chymase has shown promise in mitigating allergic diseases like dermatitis and eosinophilic disorders.

[0010] Chymase is a serine protease enzyme that belongs to the family of chymotrypsin-like peptidases. Chymase is stored as an ingredient in granules of the mast cells (MC). Chymase has various biological functions, and its role extends beyond simple protein degradation. Chymase is involved in proliferation of mast cells via its action on SCF to make it available for SCF-c-kit binding and thereby propagating an inflammatory response. Additionally, chymase is involved in tissue remodeling, extracellular matrix degradation, and the modulation of inflammatory responses. It is also implicated in the regulation of blood pressure through the conversion of angiotensin I to angiotensin II, a potent vasoconstrictor.

[0011] Chymase has also been linked to several diseases, including inflammatory diseases, cardiovascular diseases, and fibrosis. In allergic and inflammatory conditions, the release of chymase from mast cells contributes to tissue inflammation and remodeling. The inhibition of chymase has been explored as a way of treating various inflammatory and allergic diseases by reducing mast cell activation and proliferation.

[0012] A Phase 2 clinical trial (NCT01756898) was conducted to study the efficacy of a chymase inhibitor (ASB 17061) administered orally in dosage strengths of 5 mg, 10 mg and 20 mg once a day for treating adult subjects with atopic dermatitis. The clinical trial resulted in an unsuccessful outcome Docket No. 123950-10402

[0013] 2 and was abandoned. ASB 17061 shall be referred to herein as INVA8001 and ASB 16097 is a free form of ASB 17061.

[0014] SUMMARY OF THE INVENTION

[0015] The present disclosure provides an enhanced formulation of INVA8001 featuring a combination of excipients and a method of treating one or more of chronic urticaria (CU), prurigo nodularis (PN), Eosinophilic Gastrointestinal Disorders (EGIDs), and atopic dermatitis (AD) using the same. This improved formulation provides an effective solution for preventing or treating mast cell-related and other disease states in individuals requiring such intervention.

[0016] More particularly, INVA8001 is a potential first in class, oral, small molecule, which is a highly selective and a potent inhibitor of chymase as evident from the IC50 values for different enzymes inhibited by INVA8001 as shown.

[0017] By inhibiting chymase, INVA8001 decreases the recruitment of damaging immune cells particularly mast cells and eosinophils, and thus reduces chronic inflammation and epithelial barrier damage, prevent tissue remodeling, and decrease the likelihood of fibrosis. Hence, INVA8001 should be effective in chymase-mediated and mast cell-related diseases such as chronic urticaria (CU), prurigo nodularis (PN), Eosinophilic Gastrointestinal Disorders (EGIDs), and atopic dermatitis (AD), where inflammation leads to fibrotic events.

[0018] In one aspect, the disclosure provides a pharmaceutical composition comprising: a compound, wherein said compound is 2-amino-4-[(lR)-l-({ [(3Z,6S)-6-(5-chloro-2- methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4-diazepan- 1 -yl] carbonyl } amino)butyl]benzoic acid monoacetic acid solvate and comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns; about 50 w / w% to about 90 w / w% of a diluent selected from the group consisting of cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, mannitol, glucitol, maltitol, lactose (anhydrous or Docket No. 123950-10402

[0019] 3 monohydrate), dextrose, maltose, sucrose, glucose, fructose and maltodextrins, and combinations thereof; about 5.0 w / w% to about 10.0% w / w% of a disintegrant selected from the group consisting of carboxymethylcellulose, croscarmellose, crospovidone, hydroxypropyl cellulose, sodium carboxymethyl starch, sodium glycolate, partially hydrolyzed starch, and pregelatinized starch, and combinations thereof; about 0.1 w / w% to about 1.0% w / w% of a glidant selected from the group consisting of colloidal silicon dioxide, fumed silica, colloidal silica, magnesium aluminometasilicate, magnesium silicate, and magnesium trisilicate and combinations thereof; and about 0.25% w / w% to about 5.0% w / w% of a lubricant selected from the group consisting of metallic stearates, sodium stearyl fumarate, stearic acid, hydrogenated vegetable oils, talc, waxes, polyethylene glycol, sodium oleate; sodium benzoate; sodium acetate; sodium lauryl sulfate; magnesium lauryl sulfate, and glyceryl behenate and combinations thereof.

[0020] In another aspect, the disclosure provides a pharmaceutical composition comprising 50mg or 75mg of a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2- methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4-diazepan- 1 -yl]carbonyl } amino)butyl]benzoic acid monoacetic acid solvate, and wherein said composition further comprises about 50 w / w% mannitol, about 5 w / w % pregelatinized starch, about 1 w / w% colloidal silica, and about 0.25 w / w% of magnesium stearate.

[0021] In another aspect, the disclosure provides a pharmaceutical composition comprising a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2- methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4-diazepan- 1 -yl]carbonyl } amino)butyl]benzoic acid monoacetic acid solvate and comprises particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns, and wherein said pharmaceutical composition further comprises about 50 w / w% mannitol, about 5 w / w % pregelatinized starch, about 1 w / w% colloidal silica, and about 0.25 w / w% of magnesium stearate.

[0022] In another aspect, the disclosure provides a pharmaceutical composition comprising 50 mg or 75 mg of a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2- methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4-diazepan- 1 -yl] carbonyl } amino)butyl]benzoic acid monoacetic acid solvate, wherein said compound comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

[0023] In some embodiments of any of the aforesaid pharmaceutical composition, wherein said composition is in the form of a capsule. Docket No. 123950-10402

[0024] 4

[0025] In some embodiments of any of the aforesaid pharmaceutical composition, wherein the pharmaceutical composition provides an in vitro dissolution profile as shown in Figure 1 or at least 75% dissolution of compound within 30 minutes.

[0026] In some embodiments of any of the aforesaid pharmaceutical composition, wherein the pharmaceutical composition provides a simulated Cmax of 422 ng / ml or 636 ng / ml and a simulated AUCt,ssof 1110 h.ng / ml or 1700 h. ng / ml, as shown in Figures 8-10.

[0027] In some embodiments of any of the aforesaid pharmaceutical composition, wherein the compound comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

[0028] In another aspect, the disclosure provides a method of preventing or treating at least one of chronic urticaria (CU), prurigo nodularis (PN), Eosinophilic Gastrointestinal Disorders (EGIDs), and atopic dermatitis (AD) in a human subject with inflammatory or allergic symptoms comprising administering a pharmaceutical composition to said human subject, said pharmaceutical composition comprising: a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6- (5-chloro-2-methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4-diazepan- 1 - yl]carbonyl} amino )butyl]benzoic acid monoacetic acid solvate and comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns; about 50 w / w% to about 90 w / w% of a diluent selected from the group consisting of cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, mannitol, glucitol, maltitol, lactose (anhydrous or monohydrate), dextrose, maltose, sucrose, glucose, fructose and maltodextrins, and combinations thereof; about 5.0 w / w% to about 10.0% w / w% of a disintegrant selected from the group consisting of carboxymethylcellulose, croscarmellose, crospovidone, hydroxypropyl cellulose, sodium carboxymethyl starch, sodium glycolate, partially hydrolyzed starch, and pregelatinized starch, and combinations thereof; about 0.1 w / w% to about 1.0% w / w% of a glidant selected from the group consisting of colloidal silicon dioxide, fumed silica, colloidal silica, magnesium aluminometasilicate, magnesium silicate, and magnesium trisilicate and combinations thereof; and about 0.25% w / w% to about 5.0% w / w% of a lubricant selected from the group consisting of metallic stearates, sodium stearyl fumarate, stearic acid, hydrogenated vegetable oils, talc, waxes, polyethylene glycol, sodium oleate, sodium benzoate; sodium acetate; sodium lauryl sulfate; magnesium lauryl sulfate, and glyceryl behenate and combinations thereof.

[0029] In some embodiments of any of the aforesaid methods, wherein said total dose of 100 mg or 150 mg is obtained by administering 50mg of said pharmaceutical composition twice daily or 75 mg of said pharmaceutical composition twice daily respectively. Docket No. 123950-10402

[0030] 5

[0031] In some embodiments of any of the aforesaid methods, wherein said pharmaceutical composition is administered in the form of a capsule.

[0032] In some embodiments of any of the aforesaid methods, wherein the pharmaceutical composition upon administration provides a simulated Cmax of 422 ng / ml and a simulated AUCt,ssof 1110 h. ng / ml for 50mg twice a day dose as shown in Figures 8-10.

[0033] In some embodiments of any of the aforesaid methods, wherein the pharmaceutical composition upon administration provides a simulated Cmax of 636 ng / ml and a simulated AUG, ss of 1700 h.ng / ml for 75mg twice a day dose as shown in Figures 8-10.

[0034] In some embodiments of any of the aforesaid methods, wherein the pharmaceutical composition upon administration provides a simulated AUC24hr-ss of 2170 h.ng / ml for 50mg twice a day dose as shown in Figures 8-10.

[0035] In some embodiments of any of the aforesaid methods, wherein the pharmaceutical composition upon administration provides a simulated AUC24hr-ss of 3340 h.ng / ml for 75mg twice a day dose as shown in Figures 8-10.

[0036] In some embodiments of any of the aforesaid methods, wherein the pharmaceutical composition provides an in vitro dissolution profile as shown in Figure 1 or at least 75% dissolution of compound within 30 minutes.

[0037] In some embodiments of any of the aforesaid methods, wherein said subject has a mast cell- related disease and the mast-cell disease is one or more of atopic dermatitis, chronic urticaria, and prurigo nodularis.

[0038] In some embodiments of any of the aforesaid methods, wherein the compound comprises particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

[0039] The disclosure provides improved pharmaceutical formulations and methods of using a particular chymase inhibitor for treating mast cell related disorders.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The present disclosure relates to improved pharmaceutical formulations comprising the compound, 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2-methoxybenzyl )-3-(ethoxyimino)-7-oxo- 1,4- diazepan-l-yl]carbonyl}amino)butyl]benzoic acid, monoacetic acid solvate. The chemical structure of this compound is shown as Formula I below: Docket No. 123950-10402

[0042] 6

[0043] M Wt: 620.09

[0044] The compound of Formula I was designated as ASB 17061 in the clinical trials referenced above. Throughout this specification, Formula I will hereinafter be referred to as INVA8001. While the acetic acid solvate is shown, other forms of the compound are within the scope of the present disclosure.

[0045] ASB 16097 is a free form of INVA8001 or ASB 17061. The free form of INVA8001 refers to the chemically unmodified, neutral state without any associated counterions, solvents, or complexing agents. (See Tomimori Y, Manno A, Tanaka T, Futamura-Takahashi J, Muto T, Nagahira K. ASB17061, a novel chymase inhibitor, prevented, the development of angiotensin II- induced abdominal aortic aneurysm in apolipoprotein E-deficient mice. Eur J Pharmacol. 2019 Aug 5:856: 172403) The structure of ASB 16097 is shown below for reference:

[0046] The formulation of the present disclosure may comprise various dosage strengths, including dosage strengths of 50mg and 75mg. In the case of the 50mg and 75mg dosage strengths, the formulation is typically administered twice per day (i.e., b.i.d.). Other dosage strengths, including dosage strengths of 5mg, lOmg, 20mg, 15mg, 25mg, 30mg, etc, such that a total of lOOmg or 150mg is administered over the course of single day. In addition, the formulation is preferably administered to human patients with moderate to severe cases of AD, as determined by the Eczema Area and Severity Index (EASI), as discussed and defined herein.

[0047] In some embodiments, the formulation is administered to subjects as a capsule comprising either 50 mg or 75 mg of INVA8001 once a day. Docket No. 123950-10402

[0048] 7

[0049] In some embodiments, the formulation is administered to subjects as a total dose of 50 mg or 75 mg per day.

[0050] In some embodiments, the formulation is administered to subjects as a capsule comprising either 50 mg or 75 mg of INVA 8001 twice per day.

[0051] In some embodiments, the formulation comprising INVA8001 is administered to subjects as a total dose of 100 mg or 150 mg per day.

[0052] Notwithstanding the abandonment of the clinical trials over the compound that corresponds to INVA8001, certain data surprisingly shows that administering the pharmaceutical formulations of INVA8001 at the above dosage strengths effectively treats AD, Eosinophilic Gastrointestinal Disorders (EGIDs), prurigo nodularis and chronic urticaria by for example addressing pruritis and other associated symptoms.

[0053] In some embodiments, the improved formulation has been compounded in such a way so that INVA8001 comprises a certain particle size distribution, including a D90 particle size of between about 6.0 microns to about 30.0 microns, a D50 particle size of between about 2.5 to about 10.0 microns and a D10 particle size of less than about 4 microns. These particle sizes are thought to promote better solubility, reduce desorption of acetic acid, prevent degradation and improve shelf life.

[0054] In a specific embodiment, the pharmaceutical compositions of the disclosure may further comprise one or more pharmaceutically acceptable excipients.

[0055] Pharmaceutically acceptable excipients that may be used to formulate the contraceptive composition of the disclosure are, in particular, described in the HANDBOOK OF PHARMACEUTICALS EXCIPIENTS, AMERICAN PHARMACEUTICAL ASSOCIATION (Pharmaceutical Press; 6th Revised edition, 2009). Examples of appropriate excipients include, but are not limited to, fillers, carriers, diluents, binders, anti-caking agents, plasticizers, disintegrants, lubricants, flavors, buffering agents, stabilizers, colorants, dyes, antioxidants, anti-adherents, softeners, preservatives and glidants.

[0056] In some embodiments, the improved formulation comprises therapeutically effective amounts of INVA8001 along with about 50 w / w% to about 90 w / w% of a diluent selected from the group consisting of cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, mannitol, sorbitol or glucitol, maltitol, lactose (anhydrous or monohydrate), dextrose, maltose, sucrose, glucose, fructose and maltodextrins, and combinations thereof.

[0057] In some embodiments, the improved formulation comprises therapeutically effective amounts of INVA8001 along with about 5.0 w / w% to about 10.0% w / w% of a disintegrant selected Docket No. 123950-10402

[0058] 8 from the group consisting of carboxymethylcellulose, croscarmellose, crospovidone, hydroxypropyl cellulose, sodium carboxymethyl starch, sodium glycolate, partially hydrolyzed starch, and pregelatinized starch, and combinations thereof.

[0059] In some embodiments, the improved formulation comprises therapeutically effective amounts of INVA8001 along with about 0.1 w / w% to about 1.0% w / w% of a glidant selected from the group consisting of colloidal silicon dioxide, fumed silica, colloidal silica, magnesium alumino- metasilicate, magnesium silicate, and magnesium trisilicate and combinations thereof.

[0060] In some embodiments, the improved formulation comprises therapeutically effective amounts of INVA8001 along with about 0.25% w / w% to about 5.0% w / w% of a lubricant selected from the group consisting of metallic stearates, sodium stearyl fumarate, stearic acid, hydrogenated vegetable oils, talc, waxes, polyethylene glycol, sodium oleate; sodium benzoate; sodium acetate; sodium lauryl sulfate; magnesium lauryl sulfate, and glyceryl behenate and combinations thereof.

[0061] In some embodiments, the improved formulation comprises a therapeutically effective amount of INVA8001along with about 50 w / w% mannitol, about 5 w / w % pregelatinized starch, about 1 w / w% silica, and about 0.25 w / w% of magnesium stearate.

[0062] In some embodiments, the improved formulation comprises a therapeutically effective amount of INVA8001 comprising a D50 particle size of less than 8 microns and a D10 particle size of less than 4 microns along with about 50 w / w% mannitol, about 5 w / w % pregelatinized starch, about 1 w / w% silica, and about 0.25 w / w% of magnesium stearate.

[0063] The improved formulation exhibits an in vitro dissolution profile upon administration as shown in Figures 1 & 3. Nearly 60% of the administered API formulation becomes bioavailable within 10 minutes after administration and is nearly three-fold better than that of INVA8001 alone.

[0064] The improved formulation exhibits stability and was found to be shelf- stable for over 24 months without exhibiting degradation or desorption when stored at 25 °C and 60% relative humidity (RH) as shown in Figure 2.

[0065] PK simulations indicate that the INVA8001 upon administration at 50 mg twice a day (b.i.d) provides a simulated Cmax of 422 ng / ml, a simulated AUCo-24hr ss of 2170 h.ng / ml and a simulated AUCt ss of 1110 h.ng / ml, as shown in Figures 8-10. PK simulations indicate that INVA8001 upon administration at 75 mg twice a day (b.i.d) provides a simulated Cmax of 636 ng / ml, a simulated AUCo -24hr ss of 3340 h.ng / ml and a simulated AUCtss of 1700 h.ng / ml, as shown in Figures 8-10. In another embodiment, the present disclosure also extends to and covers AUC and / or Cmax values within the range of 80% to 125% of the foregoing at a confidence interval of 90%.

[0066] Chymase plays a pivotal role in enzymatically cleaving precursor molecules such as matrix metalloproteinase 9 (MMP-9), MMP1, transforming growth factor (TGF)-P, angiotensin-II, stem Docket No. 123950-10402

[0067] 9 cell factor (SCF), CCL26, collagen I, and IL-33, converting them into their active forms. Activation of these molecules’ triggers increased angiogenesis and tight junction degradation, fostering the accumulation of neutrophils, macrophages, mast cells, eosinophils, and lymphocytes, leading to uncontrolled inflammation and eventual fibrosis development.

[0068] Downstream mediators of chymase, like MMP-9, facilitate the infiltration of neutrophils, macrophages, and T cells. Angiotensin-II regulates the maturation and activation of macrophages, dendritic cells, and lymphocytes. IL-33 promotes the infiltration and activation of mast cells, basophils, ILCs, macrophages, lymphocytes, and eosinophils in allergic diseases. Chymase also induces the viability, infiltration, and activation of eosinophils.

[0069] Chymase cleaves and matures SCF, which then binds to c-KIT receptors on mast cells, supporting their proliferation, migration, survival, and differentiation. Notably, SCF exists in two primary forms: SCF220, critical for hematopoiesis, and SCF248, related to inflammation. Chymase specifically cleaves and activates the SCF248 isoform. Blocking soluble SCF can limit mast cell functions in chronic urticaria, prurigo nodularis, asthma, pulmonary fibrosis, and food allergies. (See Figure 11)

[0070] Without being bound by theory, it is believed that in case of inflammatory diseases such as atopic dermatitis, chronic urticaria, prurigo nodularis and eosinophilic Gastrointestinal Disorders (EGIDs), chymase stimulates cutaneous angiogenesis and increases metalloproteinase expression. Intradermal chymase injection in mice induces the accumulation of mast cells, along with elevated histamine levels. Chymase also increases SCF content in cultured human keratinocytes. In atopic dermatitis patients, serum soluble SCF levels correlate with disease severity. Inhibiting chymase decreases mast cell numbers by inhibiting SCF processing, as evidenced by improved symptoms in mouse models and clinical trials.

[0071] Chymase activity is upregulated in various skin diseases, making it a potential therapeutic target. Additionally, chymase activity is induced in inflamed liver and intestinal biopsies of animals with certain conditions. Blocking chymase function by a chymase inhibitor such as INVA8001 alleviates liver inflammation, limit mast cell infiltration, and reduces pro-inflammatory and pro- fibrotic mediator expression, which can ultimately restrict fibrosis thereby providing therapeutic relief. (See Figures 12 and 13).

[0072] There is evidence, including evidence generated by the inventors of the present disclosure, that chymase inhibitors prevent and / or treat disease states involving mast cell proliferation. Thus, blocking of chymase can potentially limit mast cell functions in various mast cell driven disorders.

[0073] The ability of INVA8001 to inhibit chymase and reduce mast cell numbers by impeding SCF shows that INVA8001 can treat diseases caused by mast cell activation and proliferation such as atopic dermatitis (AD), chronic utricaria and eosinophilic disorders. Indeed, chymase inhibition has Docket No. 123950-10402

[0074] 10 shown promise in ameliorating atopic dermatitis symptoms in animal models. In NC / Nga mice, such inhibition minimized the infiltration of eosinophils, neutrophils, T lymphocytes, and mast cells, indicative of reduced inflammation. Furthermore, a Phase 2 clinical trial demonstrated significant improvement in AD severity scores following treatment with an oral chymase inhibitor (SUN13834) by Daiichi Sankyo.

[0075] These insights underscore chymase ’s pivotal role in various inflammatory conditions and highlight its potential as a therapeutic target. Given the foregoing, a chymase inhibitor like INVA8001 is useful in the prevention and treatment of diseases associated with mast cell proliferation such as chronic urticaria, prurigo nodularis, Atopic dermatitis (AD) and Eosinophilic Gastrointestinal Disorders (EGIDs).

[0076] In some embodiments of any of the aforesaid methods, the mast cell-driven inflammatory or fibrotic disorder is selected from the group consisting of chronic urticaria (CU), prurigo nodularis (PN), Eosinophilic Gastrointestinal Disorders (EGIDs) and atopic dermatitis (AD).

[0077] In some embodiments of any of the aforesaid methods, the compound comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

[0078] In some embodiments of any of the aforesaid methods, the total dose of 100 mg or 150 mg is obtained by administering 50mg of said pharmaceutical composition twice daily or 75 mg of said pharmaceutical composition twice daily respectively.

[0079] In some embodiments of any of the aforesaid methods, the pharmaceutical composition is administered in the form of a capsule.

[0080] In some embodiments of any of the aforesaid methods, the pharmaceutical composition upon administration provides a simulated Cmax of 422 ng / ml and a simulated AUCt,ssof 1110 h.ng / ml for 50mg twice a day dose.

[0081] In some embodiments of any of the aforesaid methods, the pharmaceutical composition upon administration provides a simulated Cmax of 636 ng / ml and a simulated AUCt,ssof 1700 h.ng / ml for 75mg twice a day dose.

[0082] In some embodiments of any of the aforesaid methods, the pharmaceutical composition upon administration provides a simulated AUC24hr-ss of 2170 h.ng / ml for 50mg twice a day dose.

[0083] In some embodiments of any of the aforesaid methods, the pharmaceutical composition upon administration provides a simulated AUC24hr-ss of 3340 h.ng / ml for 75mg twice a day dose. Docket No. 123950-10402

[0084] 11

[0085] In some embodiments of any of the aforesaid methods, the pharmaceutical composition provides an in vitro dissolution profile as shown in Figure 1 or at least 75% dissolution of compound within 30 minutes.

[0086] In some embodiments of any of the aforesaid methods, the compound comprises particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

[0087] BRIEF DESCRIPTION OF FIGURES

[0088] FIGURE 1 - Dissolution data of 25 mg INVA8001 capsules tested in JP2 / 0.05% SLS solution at 75 rpms;

[0089] FIGURE 2 - Stability data of INVA8001 in formulation;

[0090] FIGURE 3 - Particle size data of INVA8001 in formulation;

[0091] FIGURE 4 - Dissolution Stability of 50 mg capsule at 40°C and 60 °C ;

[0092] FIGURE 5 - Population PK simulation data for 20 mg daily dose (5a) , 50 mg twice daily dose (5b), 75 mg twice daily dose at steady state (5c) and comparison of 20 mg to 50 & 75 mg / kg, bid daily dose and qd 50 & 75 mg daily dose (5d);

[0093] FIGURE 6 - Single dose simulation of 50 & 75 mg daily dose, Figure 6(a) shows the PK simulation of the 50 mg daily dose and Figure 6(b) shows the PK simulation of the 75 mg daily dose.

[0094] FIGURE 7 - Probability of target attainment for simulated population at various doses;

[0095] FIGURE 8 - Simulated Cmax values for various doses;

[0096] FIGURE 9 - Simulated dose under the concentration-time curve through 24 hours at steady state for various doses;

[0097] FIGURE 10 - Simulated dose under the concentration-time curve through 24 hours at steady state for various doses;

[0098] FIGURE 11 - Schematic representation of chymase mechanism of action;

[0099] FIGURE 12- Schematic representation of chymase inhibition by INVA8001 and its downstream effects;

[0100] FIGURE 13- Schematic representation of mast cell mediator inhibition by INVA8001;

[0101] FIGURE 14- X-ray powder diffraction data of synthesized INVA8001; Docket No. 123950-10402

[0102] 12

[0103] FIGURE 15- Inhibition of chymase, tryptase and mast cell activation;

[0104] FIGURE 16- Chymase-mediated inflammatory and fibrotic cascade and its inhibition by INVA8001;

[0105] FIGURE 17- INVA8001 attenuates symptoms of AD in mouse model;

[0106] FIGURE 18 - INVA8001 significantly suppressed the eosinophil chemotaxis mediated by human chymase / mice chymase (mMCP4).

[0107] FIGURE 19- INVA8001 inhibits the human and mice chymase-mediated cleavage of SCF;

[0108] FIGURE 20 - INVA8001 prevented the human and mice chymase-mediated conversion of pro- MMP-9 to MMP-9; and

[0109] FIGURE 21- INVA8001 reduced the expression of Col lai and Tgfbl in liver samples of Mdr2 knockout mice.

[0110] DEFINITIONS

[0111] To facilitate understanding of the disclosure set forth herein, a number of additional terms are defined below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0112] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes ” and “included ” is not limiting.

[0113] As used herein, in some embodiments, ranges and amounts are expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 uL" means “about 5 uL" and also “5 / / L.”

[0114] The phrase “and / or ” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”. when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including Docket No. 123950-10402

[0115] 13 elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0116] For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0117] As used herein in the specification and in the claims, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.

[0118] Although the above discussion discloses various exemplary embodiments of the disclosure, it should be apparent that those skilled in art can make various modifications that will achieve some of the advantages of the disclosure without departing from the true scope of the disclosure. Any references to the “invention” are intended to refer to exemplary embodiments of the invention and should not be construed to refer to all embodiments of the invention unless the context otherwise requires. The described embodiments are to be considered in all respects only as illustrative and not restrictive.

[0119] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0120] The term “Assaying” means testing for or detecting the presence of a substance or material, such as but not limited to, a chemical, an organic compound, an inorganic compound, a metabolic product, a drug, or a drug metabolite, an organism, or a metabolite of such an organism, a nucleic acid, a protein, or a combination thereof. Optionally, assaying connotes measuring the amount of the substance or material. Assaying further connotes an immunological test, a chemical test, an enzymatic test, and the like.

[0121] The terms “effective amount” or “therapeutically effective amount,” as used herein, refer to a sufficient amount of a chemical entity being administered that will prevent or relieve to some extent one or more of the symptoms of the disease or condition being treated. The result includes reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate “effective” amount in any individual case is determined using any suitable technique, such as a dose escalation study. Docket No. 123950-10402

[0122] 14

[0123] The term ''excipient" or “pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, carrier, solvent, or encapsulating material. In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed; Gibson Ed.; CRC Press LLC: Boca Raton, Fla., 2009.

[0124] The term “pharmaceutically acceptable salt” refers to a formulation of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. In certain instances, pharmaceutically acceptable salts are obtained by reacting a compound described herein, with an acid or base. For this purpose, acids or bases, or counterions described in P. H. Stahl & C. G. Wermuth “Handbook of Pharmaceutical Salts”, Verlag Helvetica Chimica Acta, Zurich, 2002 may be employed.

[0125] The term “pharmaceutical composition” refers to a mixture of a compound described herein with other chemical components (referred to collectively herein as “excipients”), such as carriers, stabilizers, diluents, dispersing agents, suspending agents, and / or thickening agents. The pharmaceutical composition facilitates the administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to: rectal, oral, intravenous, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0126] The term “subject” refers to an animal, including, but not limited to, a primate (e.g., human), monkey, cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and “patient” are used interchangeably herein in reference, for example, to a mammalian subject, such as a human.

[0127] The terms “treat,” “treating,” and “treatment,” in the context of treating a disease or disorder, are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or to slowing the progression, spread or worsening of a disease, disorder or condition or of one or more symptoms thereof.

[0128] The term “PK simulation” refers to pharmacokinetics (PK) simulation. Pharmacokinetics simulation is a simulation method used in determining the safety levels of a drug during its development. Pharmacokinetics simulation gives an insight to drug efficacy and safety before exposure of individuals to the new drug that might help to improve the design of a clinical trial. Docket No. 123950-10402

[0129] 15

[0130] Pharmacokinetics simulations help in addition in therapy planning, to stay within the therapeutic range under various physiological and pathophysiological conditions.

[0131] The term “Population PK simulation” refers to the study of pharmacokinetics at the population level, in which data from all individuals in a population are evaluated simultaneously using a nonlinear mixed-effects model. Nonlinear refers to the fact that the dependent variable (e.g., concentration) is nonlinearly related to the model parameters and independent variable(s). Mixed effects refers to the parameterization: parameters that do not vary across individuals are referred to as fixed effects, parameters that vary across individuals are called random effects. Population pharmacokinetic models are used to describe the time course of drug exposure in patients and to investigate sources of variability in patient exposure. They can be used to simulate alternative dose regimens, allowing for informed assessment of dose regimens before study conduct. (See Mould et al., CPT Pharmacometrics Syst Pharmacol. 2013 Apr; 2(4): e38.)

[0132] The term “Cmax” refers to peak drug concentration which is the highest concentration of a drug in the blood, cerebrospinal fluid, or target organ after the drug has been administered and before the administration of a second dose. The related pharmacokinetic parameter Tmax is the time at which the Cmax is observed. In orally administered drugs, Cmax and Tmax are dependent on the extent, and the rate of drug absorption and the disposition profile of the drug.

[0133] The term “Tmax” refers to time to peak measure which refers to the time taken for a drug to reach the maximum concentration (Cmax) after administration of a drug that needs to be absorbed (e.g., an oral drug). After oral administration Cmax and Tmax are dependent on the extent, and the rate of drug absorption and on the disposition profile of the drug, consequently they may characterize the properties of different formulations in the same subject.

[0134] The term “AUC” or area under the curve refers to the definite integral of the concentration of a drug in blood plasma as a function of time. The AUC (from zero to infinity) represents the total drug exposure across time. AUC is a useful metric when trying to determine whether two formulations of the same dose (for example a capsule and a tablet) result in equal amounts of tissue or plasma exposure.

[0135] The term “Cavg” refers to the average concentration of a drug in the central circulation during a dosing interval in steady state. It is calculated by dividing the area under the curve with time of the dosing interval.

[0136] The term “NOAEL” refers to the level of exposure of an organism, found by experiment or observation, at which there is no biologically or statistically significant increase in the frequency or severity of any adverse effects of the tested protocol. In drug development, the NOAEL of a new drug is assessed in laboratory animals, such as mice, prior to the initiation of human trials in order to establish a safe clinical starting dose in humans.

[0137] The term “D50” refers to the median particle diameter in a distribution of particles. It represents the particle size at which 50% of the mass of the material is composed of particles with Docket No. 123950-10402

[0138] 16 diameters equal to or smaller than the D50 value. In other words, D50 is the size below which 50% of the particles fall.

[0139] The term “D90” refers to refers to the diameter below which 90% of the particulate material's mass falls. It represents a point in the cumulative size distribution curve where 90% of the particles are smaller than the specified size. In other words, D90 is a percentile value in the particle size distribution, and it indicates the size below which a significant majority (90%) of the particles exist.

[0140] The term “D10” refers to the diameter below which 10% of the particulate material's mass falls. It represents a point in the cumulative size distribution curve where 10% of the particles are smaller than the specified size. In other words, D10 is a percentile value in the particle size distribution, and it indicates the size below which a small fraction (10%) of the particles exist.

[0141] The term “Probability of Target attainment (PT A)” refers to the likelihood that a patient will achieve a specific therapeutic target or desired clinical outcome based on the pharmacokinetic properties of a drug. The PTA is influenced by various factors, including the drug's pharmacokinetic parameters (such as absorption, distribution, metabolism, and elimination), dosing regimen, patient characteristics, and the variability in these factors among the patient population. PTA analysis helps optimize dosing regimens and provides insight into the likelihood of therapeutic success or failure based on the pharmacokinetic variability observed in the target patient population. This approach is especially relevant in situations where maintaining a specific drug concentration within a therapeutic range is critical for efficacy while minimizing the risk of adverse effects.

[0142] The term “Time above target” refers to the duration during which the concentration of a drug in the body remains above a specific therapeutic target or minimum effective concentration. This is particularly relevant in the context of time-dependent pharmacodynamic effects, where maintaining a drug concentration above a certain threshold is crucial for therapeutic efficacy.

[0143] The term “HED” refers to human dose equivalent which is the dosage of a drug administered to experimental animals, usually rodents, that is equivalent to the dosage typically used in humans. The formula commonly used to calculate human dose equivalent is often based on body surface area and is derived from Freireich et al. (Freireich EJ, Gehan EA, Rail DP, Schmidt LH, Skipper HE. "Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man." Cancer Chemother Rep. 1966 Dec;50(4):219-44)

[0144] The equation is as follows:

[0145] Human Equivalent Dose (HED) = (Animal Dose (mg / kg) / Animal Km) x (Human Km / Human weight (kg))

[0146] The term “Relative daily exposure” refers to area under the curve at steady-state through 24 hours.

[0147] The term “Cm<zxss” refers to maximum concentration at steady-state. Docket No. 123950-10402

[0148] 17

[0149] The term “ACC rss ’’refers to area under the curve through the dosing period (r), at steadystate.

[0150] The term "AUC 0-24h,ss” refers to area under the curve at steady-state through 24 hours (daily exposure).

[0151] The term 'Absorbent" refers to a substance added to a formulation to absorb or take up moisture, liquids, or gases. The primary purpose of including absorbents in pharmaceutical formulations is to enhance stability, prevent degradation, and improve the overall quality of the product. Absorbents can help with moisture control, prevent chemical degradation, improve powder flow and prevent clumping. Common examples of absorbents used as excipients in pharmaceuticals include silicates such as Magnesium Aluminum Silicate (Neusiliri) or Calcium silicate (Florite), which are known for their moisture-absorbing properties.

[0152] The term "Disinlegranl" refers to a substance added to a solid dosage form, such as tablets or capsules, to facilitate the breakup or disintegration of the dosage form when it comes into contact with water or bodily fluids. The primary purpose of disintegrants is to promote the rapid dispersion of the pharmaceutical formulation into smaller particles, aiding in the release and subsequent absorption of the active ingredient. Disintegrants can help promote disintegration, enhance bioavailability and improve patient compliance. Commonly used disintegrants in pharmaceutical formulations include substances like croscarmellose sodium, crospovidone, pregelatinized starch (PCS), starch 1500, ECS 505, Explotab, sodium starch glycolate, Carboxymethylcellulose, Croscarmellose (crosslinked, carboxymethylcellulose), crospovidone (crosslinked poly vinylpyrrolidone), L-HPC (low-substituted hydroxypropyl cellulose), Sodium carboxymethyl starch, Sodium glycolate of potato starch, partially hydrolyzed starch, maize / com / potato starch and microcrystalline cellulose.

[0153] The term "Glidanl" refers to a substance added to powdered or granulated materials in pharmaceutical formulations to improve the flow properties of the powder blend. The primary goal of a glidant is to reduce the interparticle friction between individual particles, thereby enhancing the powder's flowability. Improved flow properties are particularly important in the manufacturing of solid dosage forms, such as tablets and capsules, where uniform distribution and consistency of the powder are needed. Commonly used glidants in pharmaceutical formulations include materials like colloidal silicon dioxide (silica), talc, magnesium stearate, colloidal silicon dioxide (Aerosil, Syloid, Cab-O-Sil, hydrophobic colloidal silica (Aerosil-R972), magnesium aluminometasilicate (Neusiliri), magnesium trisilicate and stearic acid.

[0154] The term "Lubricant" refers to a substance that is added to tablet formulations to aid in the tableting process and to improve the flow of powders during the manufacturing of tablets. These excipients reduce friction and prevent the sticking of the tablet material to the surface of the die and punches, allowing for smoother tablet production and reducing the wear and tear on the tableting equipment. Common examples of lubricants include metallic stearates e.g., magnesium, calcium Docket No. 123950-10402

[0155] 18 and sodium stearates; sodium stearyl fumarate (Pruv) stearic acid; hydrogenated vegetable oils (e.g., Sterotex); talc; waxes; Stearowet; polyethylene glycols (e.g., Carbowax 4000 and Carbowax 6000) sodium oleate; sodium benzoate; sodium acetate; sodium lauryl sulfate; magnesium lauryl sulfate and glyceryl behenate (Compritol 888).

[0156] The term “Filler or Diluents” refer to a substance added to a pharmaceutical formulation to increase the volume or bulk of the dosage form. Fillers are commonly used in the production of tablets and capsules to provide the necessary mass and size to the dosage unit, particularly when the amount of active pharmaceutical ingredient (API) is relatively small. Fillers or diluents are used to aid in tablet compression and improve content uniformity. Commonly used fillers in pharmaceutical formulations include materials like lactose, microcrystalline cellulose, mannitol, Cellulose powder, microcrystalline cellulose, silicified microcrystalline cellulose (Prosolv SMCC), dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, mannitol, maltitol, Sorbitol, Xylitol, lactose (anhydrous or monohydrate), dextrose, maltose, sucrose, glucose, fructose or maltodextrins and dicalcium phosphate.

[0157] The term “Solubilizer or Wetting agent” refers to is a substance added to a formulation to enhance the solubility of poorly soluble drugs or other hydrophobic compounds in a specific medium, typically aqueous solutions. The primary goal of a solubilizer is to improve the bioavailability of the active pharmaceutical ingredient (API) by facilitating its dissolution and absorption in the gastrointestinal tract. Commonly used solubilizers in pharmaceutical formulations include polysorbate 80 (Tween 80), Sodium Lauryl Sulphate, Docusate Sodium. Non-ionic surfactants like Block co-polymer (Polaxmar), Polyoxyethylene Sorbitan Fatty Acid Esters (tweens) Sorbiton esters (spans), cremophor, and cyclodextrins.

[0158] The term “Eczema Area and Severity Index (EAS1) score” refers to a scoring system used in clinical trials and research to assess the severity of atopic dermatitis (eczema) and to measure the extent of affected skin. It provides a standardized method for evaluating the severity of eczema symptoms based on objective criteria. The EASI score is a validated, standardized scale used to measure the extent and severity of atopic eczema; EASI integrates body surface and the intensity of lesioned skin into one composite score. An EASI score of 7.1 to 21 indicates moderate AD disease and a score of 21.1 to 50 indicates severe AD disease.

[0159] The term “Moderate AD” refers to the classification of severity of disease state of atopic dermatitis made by a clinician. The severity of atopic dermatitis can be categorized into different levels, ranging from mild to severe, based on the extent and intensity of symptoms. Moderate AD patients exhibit skin rashes, itching and increased redness in skin. A patient having AD with EASI score of 7.1 to 21 indicates that the patient is experiencing a moderate form of AD.

[0160] The term “Severe AD” refers to the classification of severity of disease state of atopic dermatitis made by a clinician. Severe atopic dermatitis refers to a high level of severity in the presentation of atopic dermatitis (AD). Severe AD patients exhibit marked redness and Docket No. 123950-10402

[0161] 19 inflammation, severe itching, oozing, crusting, or thickening of skin lesions. A patient having AD with EASI score of 21.1 to 50 indicates that the patient is experiencing a severe form of AD.

[0162] The term “Investigator Global Assessment (IGA)” refers to is a clinical evaluation tool commonly used in medical research and clinical trials to assess the overall severity of a patient's disease or condition. In dermatology, the IGA is often employed to assess the severity of skin conditions, such as psoriasis, atopic dermatitis, or acne. The assessment is usually based on visual inspection and the investigator's subjective impression of the patient's overall disease severity, taking into account factors such as erythema (redness), induration (thickness or swelling), scaling, and other relevant clinical features. The IGA is a 5-point scale that provides a global clinical assessment of AD severity where a score of 3 indicates moderate disease and a score of 4 indicates severe disease.

[0163] The term “Jet milling” refers to a size reduction process used in pharmaceutical industry to grind and mill materials into fine powders or particles. It is based on the principle of fluid energy impact, where high-velocity jets of gas, typically air or nitrogen, are used to impart energy to the particles in a milling chamber. This process is particularly suitable for grinding and milling materials that are heat- sensitive or have low melting points.

[0164] The term “Ultra centrifugal milling” refers to the process of reducing particle size and homogenizing the formulation using centrifugal force. The rotor of an ultra-centrifugal mill typically has multiple blades or fins that accelerate particles radially outward. This high-speed rotation imparts energy to the particles, causing them to collide and break down into smaller sizes.

[0165] The term “Pin milling” refers to a type of milling process used to achieve particle size reduction. It involves the use of one or more rotating discs or "pins" that impact and grind the material within a confined space. The process is particularly suitable for fine grinding and micronization of materials. The material to be milled is fed into the center of the milling chamber. As the rotating discs spin, the pins impact and grind the material against the surfaces of the milling chamber. The high-speed impact and collision between particles and pins result in size reduction.

[0166] The term “atopic dermatitis” (AD) refers to a chronic, non-infectious inflammatory skin disorder. It is characterized by dry skin, eczematous lesions, and lichenification — thickened, leathery skin resulting from prolonged inflammation and scratching. The pathophysiology of AD is complex and multifactorial, involving genetic predisposition, epidermal barrier dysfunction, immune dysregulation, and alterations in the skin microbiome. AD primarily affects pediatric populations, with a prevalence of up to 20% in children. It is estimated to affect over 32 million people in the United States and approximately 65 million across the seven major markets (7MM).

[0167] The term “chronic urticaria” refers to a persistent skin condition characterized by the recurrent appearance of hives (wheals), which are raised, itchy, and often red or white welts. The condition is classified as chronic when symptoms persist for six weeks or longer. CU significantly impacts quality of life due to its unpredictable flare-ups and intense pruritus. It is estimated to affect Docket No. 123950-10402

[0168] 20 approximately 500,000 individuals in the United States, with a prevalence of around 0.23% (Lang et al., N Engl J Med 2022;387:824-831).

[0169] First- line treatment typically involves second-generation Hl -antihistamines. However, a substantial proportion of patients experience inadequate symptom control. Despite adherence to current treatment guidelines, many patients continue to suffer from uncontrolled symptoms, underscoring the unmet need for safe, effective, and orally available therapies for chronic urticaria.

[0170] Mast cells and basophils are known to play central roles in the pathogenesis of CU. Mast cell activation and degranulation lead to the release of histamine and other mediators that drive the formation of wheals and angioedema. In addition to histamine, mast cells release cytokines and chemokines that contribute to the recruitment of perivascular inflammatory infiltrates observed in lesional skin. Autoimmune mechanisms are also implicated, particularly in chronic spontaneous urticaria (CSU), where IgG and IgE autoantibodies against the high-affinity IgE receptor (FcsRI) or IgE itself have been identified. (Lang et al., N Engl J Med 2022;387:824-831', Ansotegui et al., Allergy Asthma Clin Immunol 18, 41 (2022). ).

[0171] The term “prurigo nodularis” refers to a chronic inflammatory skin disorder characterized by intensely pruritic, nodular lesions that commonly appear on the trunk, arms, and legs. Patients typically present with multiple excoriated nodules resulting from persistent scratching. Although the exact etiology of prurigo nodularis remains unclear, it is frequently associated with other dermatologic conditions such as severe or untreated atopic dermatitis, as well as systemic causes of pruritus, including liver disease and end-stage renal disease. The nodules are usually symmetric, discrete, hyperpigmented, and firm. While they can develop anywhere on the body, they are most often found in areas within easy reach for scratching. Prurigo nodularis is notoriously difficult to treat. Mast cell activation and chymase expression are implicated in driving chronic itch, inflammation, and skin remodeling in prurigo nodularis. Chymase is released during mast cell degranulation, a process that occurs in response to various stimuli including neuropeptides like substance P and CGRP, both of which are dysregulated in PN. Chymase-positive mast cells in PN lesions suggests active mast cell involvement in the disease process.

[0172] The term “eosinophilic gastrointestinal disorders (EGIDs)” refers to a group of rare, chronic conditions characterized by pathological eosinophilic infiltration of the gastrointestinal (GI) tract, leading to organ dysfunction and clinical symptoms. These disorders occur in the absence of known secondary causes of eosinophilia, such as drug reactions, parasitic infections, or malignancy. Chymase expression and mast cell proliferation play key roles in pathogenesis of EGIDs. Chymase is released during degranulation and has been shown to activate eosinophils, promoting their migration, survival, and cytokine release. It induces eosinophils to release IL-6, CXCL8, CCL2, and CXCL1, and upregulates adhesion molecules like CD 18,. Chymase contributes to tissue remodeling and angiogenesis, creating a microenvironment conducive to mast cell proliferation and sustained inflammation. Docket No. 123950-10402

[0173] 21

[0174] The nomenclature and clinical presentation of EGIDs depend on the location and depth of eosinophilic infiltration. The most common form, eosinophilic esophagitis (EoE), involves eosinophilic infiltration confined to the esophagus. Less common variants include eosinophilic gastritis / duodenitis (EG / EoD), eosinophilic enteritis (EE), and eosinophilic colitis (EC), which affect the stomach and duodenum, small intestine, and colon, respectively.

[0175] EoE is estimated to affect approximately 189,000 individuals in the United States and 438,000 across the seven major markets (7MM). EG / EoD affects an estimated 93,000 individuals in the U.S. and 220,000 in the 7MM.

[0176] EGIDs are chronic conditions requiring long-term monitoring and management. Currently, no FDA-approved treatments exist for EGIDs. Management strategies vary depending on the affected GI segment but typically include dietary modifications and off-label use of antiinflammatory or immunosuppressive therapies. The primary treatment goals are symptom control and histological improvement.

[0177] In mild cases of EoE, proton pump inhibitors (PPIs) — such as omeprazole, lansoprazole (Prevacid®), pantoprazole, and esomeprazole — can provide symptomatic relief. For more severe cases of EoE and EG / EoD, corticosteroids such as fluticasone, budesonide, and prednisone are commonly used off-label. However, these treatments are associated with side effects, including local Candida overgrowth and adrenal axis suppression with prolonged use. Additionally, most patients experience rapid relapse upon discontinuation of therapy.

[0178] As a result, there remains a significant unmet need for safe, effective, and disease-modifying treatments that are suitable for long-term use in patients with EGIDs.

[0179] The term Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0180] Synthesis of INVA8001

[0181] A synthetic procedure for making 2-amino-4-[(lR)-l-({ [(3Z,6S)-6-(5-chloro-2- methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4- diazepan- 1 -yl]carbonyl } amino )butyl] benzoic acid monoacetic acid solvate (referred to as compound 36) is listed in Example 36 of United States Patent No. 8,846,660. The starting material for the synthesis of INVA8001 is 4-[(lR)-l-({[(6S)-6- ( 5-chloro-2-methoxybenzyl)-3-( ethoxyimino )-7-oxo-l,4-diazepan-l -yl ]carbonyl}amino )butyl ]-2- nitrobenzoic acid hydrochloride which is referred to as compound 35 in United States Patent No. Docket No. 123950-10402

[0182] 22

[0183] 8,846,660, the contents of which are herein incorporated by reference in its entirety. The starting material (Compound 35) was prepared as disclosed in Example 35 of U.S. Pat. No. 8,846,660.

[0184] To Compound 35 (874 mg) in an ethyl acetate (2.7 ml) and acetic acid (1.4 ml) suspension, zinc powder was added under ice cooling, and the mixture was stirred at that temperature for 10 minutes and at room temperature for 4 hours.

[0185] The reaction mixture was diluted with ethyl acetate (10 ml) and filtered by a glass filter spread with Celite, and further the residue was washed with ethyl acetate (20 ml). The filtrate and the washings were combined, then successively washed with saturated aqueous ammonium chloride solution and brine, dried over sodium sulfate, then concentrated. The concentrated residue was purified by flash column chromatography to obtain 2-amino-4-[(lR)-l-({[(6S)-6-(5-chloro-2- methoxybenzyl)-3-(ethoxyimino)-7-oxo-l,4-diazepan-l-yl]carbonyl} amino )butyl] benzoic acid (compound 12) (698 mg), as disclosed by Example 36 in U.S. Pat. No. 8,846,660.

[0186] To the compound 12 (558 mg) thus obtained, acetic acid (1.1 ml) was added and the mixture was stirred at room temperature for 30 minutes. Then water (2.75 ml) was added, and the mixture was stirred for 10 minutes. Further, acetic acid / water (2 / 5, 2.5 ml) was added, and the mixture was stirred for 15 minutes, then the mixture was cooled to 0° C. and stirred for 30 minutes. The precipitated light-yellow crystals were collected by filtration to obtain INVA8001.

[0187] Docket No. 123950-10402

[0188] 23

[0189] Alternately, INVA8001 can also be made following the synthetic scheme shown below:

[0190] Detailed protocols for the aforesaid synthetic scheme are described in Example 11, including the preparation of starting material 1 and starting material 2. Docket No. 123950-10402

[0191] 24

[0192] Physical & Chemical properties of INVA8001

[0193] INVA8001 was found to be generally stable to temperature but underwent hydrolysis and photolysis under humid conditions. INVA8001 also become amorphous due to acetic acid release and became unstable when exposed to excessive crushing force and compression. INVA8001 was found to minimally absorb moisture at 11-92% RH, but acetic acid desorption was observed when the humidity increased.

[0194] Upon thermal analysis, INV8001 decomposed at 160°C, with acetic acid release occurring at 125-135°C. Thus, INVA8001 in the absence of excipients was found to degrade when exposed to moisture due to hydrolysis and exhibited desorption of acetic acid when subjected to pressure or mechanical force. Therefore, wet granulation and tableting were deemed unsuitable processes for preparing formulation of INVA8001. The formulation of the present disclosure provides a specific combination of excipients that help prevent desorption of acetic acid, maintain stability when exposed to pressure and improve shelf life of INVA8001 thereby reducing the risk of unwanted degradation products.

[0195] Solubility of INVA8001

[0196] Dissolution assesses the rate at which a drug substance dissolves from its dosage form. This information predicts how the drug will be absorbed and subsequently reach systemic circulation. Dissolution testing is typically conducted using dissolution apparatus, such as the United States Pharmacopeia (USP) dissolution apparatus types, commonly Type I (basket), Type II (paddle), or Type IV (flow-through cell).

[0197] The following parameters were used for dissolution testing:

[0198] • Vankel VK 7010 / Agilent Agilent 8453 (UV) were used for the dissolution test.

[0199] • Test method: Dissolution test method 2 (paddle method)

[0200] • Test solution temperature: 37+0.5°C

[0201] • Test solution volume: 900 mL

[0202] • Sampling time: 2, 5, 10, 15, 20, 30, 45, 60 min

[0203] • Cell length: 10mm Docket No. 123950-10402

[0204] 25

[0205] • Measurement wavelength: 250 nm, 400 nm

[0206] The solubility of INVA8001 was high in the acidic region around pH 1 and in the basic region above pH 7, but it was almost insoluble in the weakly acidic to neutral region. (See Table 1)

[0207] Table 1- Solubility of INVA8001 at 25 °C in buffers with different pH

[0208] The solubility of INVA8001 was higher when Sodium lauryl sulfate (SLS) was added as an additive at 0.05% concentration. The solubility of the INVA8001 was 3.6 times higher in pH 6.8 when SLS was added. (See Table 2)

[0209] Table 2- Solubility of INVA8001 at 25 °C in pH 6.8 buffer with different amounts of SLS

[0210] INVA8001 Compatibility Study

[0211] API (Active Pharmaceutical Ingredient) compatibility study refers to a series of studies and tests conducted to assess the compatibility of the active pharmaceutical ingredient (INVA8001) with various excipients and other components used in the formulation. A representative example of which is as follows:

[0212] Appearance'. Changes in the physical appearance of the API (INVA8001) and the formulation, such as color, odor, crystallinity, and texture are evaluated.

[0213] Phase Separation'. The formulation is evaluated to see if there is any evidence of phase separation, precipitation, or crystallization upon mixing the API (INVA8001) with excipients.

[0214] Chemical Stability'. The chemical stability of the API (INVA8001) in the presence of excipients over time is evaluated. This includes monitoring for degradation, oxidation, and the formation of impurities. Docket No. 123950-10402

[0215] 26

[0216] Thermal Stability. The impact of temperature on the stability of the API (INVA8001) and the formulation in presence of selected excipients is evaluated. This may involve exposing the formulation to different temperatures to simulate storage conditions. pH Compatibility range'. The impact of pH on the stability of the API (INVA8001) in presence of selected excipients is evaluated. Some APIs may be sensitive to pH changes, and excipients can influence the pH of the final formulation.

[0217] Solubility Studies'. The solubility of the API (INVA8001)in different solvents and in the presence of excipients is evaluated. Changes in solubility can affect the bioavailability of the drug.

[0218] Process Compatibility. The impact of the manufacturing processes (e.g., blending, granulation, compression) on the stability of the API (INVA8001) and the formulation in presence of selected excipients is evaluated.

[0219] Compatibility with Other Ingredients'. If the formulation includes additional ingredients, such as preservatives or stabilizers, compatibility with these components is assessed.

[0220] Forced Degradation Studies'. Subject the API (INVA8001) and formulation to extreme conditions, such as high temperatures and humidity, to accelerate degradation processes and identify potential degradation pathways.

[0221] Milling

[0222] Milling is a common process in pharmaceutical manufacturing used to reduce the particle size of active pharmaceutical ingredients (APIs) and excipients. The smaller particle size achieved through milling can enhance drug dissolution rates, improve bioavailability, and ensure uniform mixing of ingredients. Here's an overview of how milling is typically performed in pharmaceutical compositions:

[0223] Selection of Milling Equipment: Different types of milling equipment are available, including ball mills, hammer mills, jet mills, and cone mills. The choice of equipment depends on factors such as the nature of the material, desired particle size, and the required throughput.

[0224] Particle Size Analysis'. Before milling, the initial particle size distribution of the raw materials (APIs and excipients) is determined. This analysis helps in setting the milling parameters and assessing the effectiveness of the milling process.

[0225] Setting Milling Parameters: Parameters such as milling time, milling speed, and the type of milling media (e.g., balls, hammers, air) are set based on the desired particle size reduction and the characteristics of the materials being milled.

[0226] Dry Milling or Wet Milling'. Milling can be performed in a dry or wet state. Dry milling is common for solid materials, while wet milling is suitable for materials that are either naturally in a liquid state or can be suspended in a liquid. Docket No. 123950-10402

[0227] 27

[0228] Loading the Mill: The raw materials, including the API (INVA8001) and excipients, are loaded into the milling chamber. The proportions are carefully controlled to achieve the desired composition in the final formulation.

[0229] Milling Process: In a ball mill, for example, the milling chamber contains grinding media (balls) and the raw materials. The rotation of the chamber causes the grinding media to impact and crush the particles, leading to size reduction. Other milling equipment operates on similar principles but with variations in mechanism.

[0230] Particle Size Monitoring: Particle size distribution is continuously or intermittently monitored during the milling process to ensure that the desired size reduction is achieved. Techniques such as laser diffraction, microscopy, or sieve analysis may be employed.

[0231] Post-Milling Analysis: After milling, the final particle size distribution is analyzed to confirm that the targeted size reduction has been achieved. This analysis is crucial for quality control and ensuring consistency in the manufacturing process.

[0232] PK Simulation

[0233] Pharmacokinetic (PK) simulation and modeling involve the use of mathematical and statistical tools to predict the behavior of drugs in the body over time. This process helps researchers and clinicians understand how drugs are absorbed, distributed, metabolized, and eliminated by the body (ADME processes). Generally, PK simulations involves the following processes:

[0234] Data collection'.

[0235] Initial data collection involves obtaining information on drug properties, such as molecular weight, solubility, and stability. In vivo data from preclinical and clinical studies provide insights into drug absorption, distribution, metabolism, and excretion.

[0236] Compartmental Modeling:

[0237] One common approach is compartmental modeling, where the body is divided into compartments representing different tissues or organs. Differential equations describe the movement of the drug between these compartments.

[0238] PK Parameters:

[0239] Pharmacokinetic parameters, such as absorption rate constants, distribution volumes, and elimination rates, are estimated based on experimental data. These parameters help characterize the drug's behavior in the body.

[0240] Non-compartmental Analysis:

[0241] This approach involves analyzing drug concentration-time data without assuming a specific model. Non-compartmental analysis calculates parameters like area under the curve (AUC), maximum concentration (Cmax), and elimination half-life. Docket No. 123950-10402

[0242] 28

[0243] Software Tools:

[0244] Specialized software packages like NONMEM, WinBUGS, Berkeley Madonna, and Simcyp Simulator are commonly used for pharmacokinetic modeling. These tools assist in solving complex mathematical equations and running simulations.

[0245] Population PK Modeling'.

[0246] Population pharmacokinetics involves analyzing data from a group of individuals to identify inter-individual variability in drug response. This helps in determining optimal dosing regimens for diverse patient populations.

[0247] Monte Carlo Simulations:

[0248] Monte Carlo simulations involve running multiple simulations with different sets of parameters to assess the variability and uncertainty in drug behavior. This is useful for predicting drug concentrations under various conditions.

[0249] Validation and Refinement:

[0250] The model is validated by comparing predicted outcomes with independent datasets. Refinement of the model may be necessary based on validation results or additional data.

[0251] Patient Selection Criteria

[0252] The efficacy study will exclusively encompass patients diagnosed with moderate to severe (AD), assessed through the Eczema Area and Severity Index (EASI) score, with the majority falling within the moderate category. The EASI score, a validated and standardized scale, amalgamates body surface and lesioned skin intensity into a composite score, where a range of 7.1 to 21 signifies moderate disease, and 21.1 to 50 indicates severe disease.

[0253] Patients' AD severity will also undergo evaluation via the Investigator Global Assessment (IGA), a 5-point scale. A score of 3 denotes moderate disease, while a score of 4 indicates severe disease.

[0254] Furthermore, the study's second selection criterion involves the levels of immunoglobulin E (IgE), with enrollment restricted to patients exhibiting levels greater than or equal to 100 lU / mL. IgE serves as a marker for mast cell activation and allergic inflammation.

[0255] The study will additionally assess biomarkers, such as tryptase and chymase levels, to ascertain whether they undergo alterations post-administration of the improved formulation.

[0256] Determination of CAVS (target efficacy threshold level) of 19 ng / ml and b.i.d rather than daily dose.

[0257] The compound that corresponds to INVA8001 underwent Single Ascending Dose (SAD) and Multiple Ascending Dose (MAD) studies and progressed to a Phase 2 study in patients with AD. In this Phase 2 study (Clinical trial - NCT01756898), the highest dosage of 20 mg once daily (QD) was selected to achieve an average target plasma concentration Cavg of 19 ng / mL. However, Docket No. 123950-10402

[0258] 29 despite these efforts, the highest dosage (20 mg QD) of INVA8001 did not show differentiation from the placebo for the primary efficacy endpoint Investigator’s Global Assessment Scale (IGA).

[0259] Treatment of diseases caused by mast cell proliferation using INVA8001

[0260] Mast cell proliferation, activation, and degranulation — driven in large part by the enzymatic activity of chymase — represent a unifying pathogenic mechanism across several chronic inflammatory and immune-mediated diseases, including chronic urticaria, prurigo nodularis, eosinophilic gastrointestinal disorders (EGIDs), and atopic dermatitis. Chymase, a mast cellspecific serine protease, plays a central role in the activation of multiple pro-inflammatory and pro- fibrotic mediators such as matrix metalloproteinases (MMP-9, MMP-1), angiotensin II, interleukin- 33 (IL-33), transforming growth factor-beta (TGF-P), stem cell factor (SCF), chemokine ligand 26 (CCL26), and collagen I. These mediators collectively contribute to immune cell recruitment, epithelial barrier disruption, tissue remodeling, and fibrosis. In particular, SCF promotes mast cell proliferation and survival via c-KIT signaling, while IL-33 and CCL26 enhance eosinophil and lymphocyte infiltration, perpetuating chronic inflammation. (See Figures 11-13 and 16)

[0261] • In chronic urticaria, lesional skin exhibits a marked increase in chymase-positive mast cells, which release histamine, leukotrienes, and neuropeptides such as substance P and VIP, contributing to wheal formation, pruritus, and Th2-skewed inflammation. (Church, M., et al., Immunol Rev. 2018 Mar; 282(l):232-247).; Caytemel, C., et al., Indian J Dermatol. 2022 Mar-Apr; 67(2): 205); Jacques MD, P., et al., J Allergy Clin Immunol. 1992 Jun; 89(6): 1139-43; Borici-Mazi, R., et al., Allergy. 1999 Jan; 54(l):46-56; Lawlor, F., et al., Br J Dermatol. 1989 Sep; 121(3):317-21)

[0262] • Prurigo nodularis is characterized by dense dermal infiltration of mast cells, eosinophils, and T lymphocytes, with mast cell-nerve interactions playing a key role in chronic itch and neuroinflammation. (Kwatra SG, Puelles J, Tran OC, et al. Prevalence of prurigo nodularis in the United States. J Am Acad Dermatol. 2024;90(5):849-852; Williams, KA., et al., J Am Acad Dermatol. 2020 Dec; 83(6):1567-1575 at 1567 to 1575; Wong, LS., et al., Int J Mol Sci. 2022 Oct; 23(20): 12390; Liang, Y., et al., J Cutan Pathol. 1998 Apr;25(4): 189-98 at 189 to 198)

[0263] • EGIDs, including eosinophilic esophagitis and eosinophilic gastritis / duodenitis, are defined by eosinophilic infiltration of the gastrointestinal tract, where mast cells and chymase correlate strongly with disease severity, endoscopic abnormalities, and mucosal dysfunction. (Terakawa, M., et al., Eosinophil migration induced by mast cell chymase is mediated by extracellular signal-regulated kinase pathway. Biochemical and biophysical research communications, 2005. 332(4): p. 969-975; VAN DELLEN, R.G. and J.C. Lewis. Oral administration of cromolyn in a patient with protein-losing enteropathy, food allergy, and eosinophilic gastroenteritis, in Mayo Clinic proceedings. 1994. Elsevier; Melamed, I., et al., Benefit ofketotifen in patients with eosinophilic gastroenteritis. The American journal of medicine, 1991. 90(3): p. 310-314) Docket No. 123950-10402

[0264] 30

[0265] • In atopic dermatitis, elevated levels of chymase-positive mast cells in lesional skin are associated with increased SCF production, histamine release, and eosinophilic inflammation. The importance of mast cells and chymase in atopic dermatitis has been demonstrated where an enhanced number of chymase-positive cells were found in lesional atopic dermatitis skin as compared to normal skin confirming that they are an integral part in eliciting and maintaining cutaneous inflammation in atopic dermatitis. ((Badertscher K, Brbnnimann M, Karlen S, Braathen LR, Yawalkar N. Mast cell chymase is increased in chronic atopic dermatitis but not in psoriasis. Arch Dermatol Res. 2005 Apr;296(10): 503-6). Chymase has been shown to directly induce mast cell accumulation and histamine release in murine models, and clinical studies have demonstrated that chymase inhibition can significantly improve disease severity scores in patients with atopic dermatitis. Tomimori, Y., et al., Biochem Biophys Res Commun, 2002 Feb 8; 290(5): 1478-82; Watanabe, N., et al., J Invest Deratol. 2007 Apr; 127(4):971-3)

[0266] The inventors have demonstrated that INVA8001 is a potent and highly selective oral chymase inhibitor capable of suppressing key inflammatory and fibrotic pathways. It effectively inhibits chymase, reduces tryptase levels, and prevents mast cell activation (See Figures 11-13 and Figure 16), significantly reduces eosinophil chemotaxis mediated by both human and murine chymase (mMCP4) (Figure 18), and blocks chymase-driven cleavage of stem cell factor (SCF) (Figure 19). Additionally, INVA8001 prevents the chymase-mediated conversion of pro-MMP-9 to active MMP-9 (Figure 20) and downregulates the expression of Collal and Tgfbl in liver tissues of Mdr2 knockout mice (Figure 21).

[0267] By targeting upstream enzymatic activation, INVA8001 offers a selective therapeutic approach for diseases driven by mast cell and eosinophil chemotaxis. It suppresses mast cell proliferation and activation via inhibition of the SCF / c-KIT pathway, limits eosinophil recruitment and survival, and attenuates the release of histamine, cytokines, and neuropeptides. These actions collectively lead to reduced immune cell infiltration, enhanced epithelial barrier integrity, diminished neuroinflammation, and prevention of tissue fibrosis. INVA8001 addresses critical unmet needs across multiple indications, offering a promising disease-modifying treatment with a potentially safer and more convenient oral alternative to existing therapies.

[0268] EXAMPLES

[0269] EXAMPLE 1 - Evaluation of compatibility of excipients with INVA8001

[0270] The process of compounding active pharmaceutical ingredients (APIs) and excipients involves mixing the API (INVA8001) at specific amount with compatible excipients at specific concentration to yield a pharmaceutical formulation that ensures effective and safe delivery of the medication. A desired amount of API (for ex: 5mg, 10 mg, 20 mg. 25 mg, 50 mg or 75 mg of INVA8001) is mixed with one or more compatible excipients such as diluent, disintegrant, lubricant, glidant and wetting agents at suitable concentrations to form the pharmaceutical mixture which is Docket No. 123950-10402

[0271] 31 then ground up using mortar to form a homogenous powder. The homogenous powder , which is the pharmaceutical formulation, is then packaged into a capsule for further analysis and / or administration.

[0272] The table below lists examples and concentration ranges for each excipient that can be used with the INVA8001. Some of the excipients are more preferable over others.

[0273] Fillers / Diluents'. Cellulose powder, microcrystalline cellulose, silicified microcrystalline cellulose (ProsolvOSMCC), dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, mannitol, maltitol, Sorbitol, Xylitol, lactose (anhydrous or monohydrate), dextrose, maltose, Sucrose, glucose, fructose or maltodextrins are some non-limiting examples of fillers / diluents.

[0274] Disintegrants'. Carboxymethylcellulose, Croscarmellose (crosslinked carboxymethylcellulose), crospovidone (crosslinked poly vinylpyrrolidone), L-HPC (low- substituted hydroxypropyl cellulose), Sodium carboxymethyl starch, Sodium glycolate of potato starch, partially hydrolyzed starch, maize / corn / potato starch are some non-limiting examples of disintegrants.

[0275] Glidants'. Colloidal silicon dioxide (Aerosil®, Syloid®,Cab-O-Sil®, hydrophobic colloidal silica (Aerosil®R972), magnesium aluminometasilicate (Neusilin®), Magnesium silicate & Magnesium trisilicate are some non-limiting examples of glidants.

[0276] Lubricants'. Metallic stearates e.g., magnesium, calcium and sodium stearates; sodium stearyl fumarate (Pruv™) stearic acid; hydrogenated vegetable oils (e.g., Sterotex™); talc; waxes; Stearowet™; polyethylene glycols (e.g., Carbowax™ 4000 and Carbowax™ 6000); sodium oleate; sodium benzoate; sodium acetate; sodium lauryl sulfate; magnesium lauryl sulfate and glyceryl behenate (Compritol™ 888) are some non-limiting examples of lubricants.

[0277] Solubilizer s / Wetting agent'. Anionic Surfactants like Sodium Lauryl Sulphate, Docusate Sodium. Non-ionic surfactants like Block co-polymer (Polaxmar), Polyoxyethylene Sorbitan Fatty Acid Esters (tweens) Sorbiton esters (spans) are some non-limiting examples of solubilizers / wetting agents. Preferably, sodium lauryl sulphate is employed as a wetting agent.

[0278] Table 3- List of excipients and concentration ranges Docket No. 123950-10402

[0279] 32 Docket No. 123950-10402

[0280] 33

[0281] To develop a stable formulation for INVA8001, excipients were mixed with API and stored at 40°C / 75% relative humidity (open container) for 2 and 4 weeks, at 60°C (sealed container) for 2 and 4 weeks, or at 1.2 million lumen / m2• hr (open container). The compatibility of each excipient was determined after storage in each condition based on the appearance (color and solid state) and the API purity as measured by the lower quantity of API-related substances. Based on the compatibility experiments, the following components were determined to be compatible:

[0282] Lactose monohydrate (Pharmatose), mannitol (Parteck), microcrystalline cellulose (MCC), Cornstarch, pregelatinized starch NF or partly pregelatinized Starch JPE (PCS), low-substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac-Di-Sol), crospovidone (Polyplasdone XL), carboxymethylcellulose calcium or carmellose calcium (ECS-505), hydroxypropyl cellulose (HPC-L), povidone (Kollidon30), magnesium stearate (Mg-St), sodium stearyl fumarate (Pruv), magnesium aluminometasilicate (Neusiliri), Hydroxypropyl methylcellulose or hypromellose (HPMC), Talc, polyvinyl alcohol (PEA), titanium dioxide (TiO2), and hypromellose HPMC) capsules. Colloidal silicon dioxide NF or Light Anhydrous Silicic Acid JPE (Aerosil), Light Anhydrous Silicic Acid JPE (Sylysia), and calcium silicate (Florite) were classified using the same criteria as partially compatible with the API. Throughout the compatibility experiments, the mixing ratios employed were 1:9 for API with diluents, disintegrants, binders, and HPMC, and 1:1 for lubricants, glidants, and absorbents.

[0283] In formulating INVA8001, consideration was given to achieving optimal dissolution for high content and ensuring stability, particularly regarding the formation of analogues, for the low content. Disintegrant screening revealed favorable dissolution with minimal variation when using Starchl500, PCS, and Explotab. Notably, increasing the concentration of ECG505 from 5% to 10% in the formulation exhibited improved dissolution and reduced variability.

[0284] Dissolution Variability. Mannitol, employed as a diluent, exhibited favorable dissolution characteristics when coupled with disintegrants like pregelatinized starch (PCS), starch 1500, and sodium starch glycolate. These combinations displayed minimal variation in dissolution rates. In formulations outside the mannitol / ECG505 series, a considerable variability was observed, with some formulations recording individual values below 80%. Specifically, the lactose-based Docket No. 123950-10402

[0285] 34 formulation exhibited a standard deviation (SD) exceeding 12% at 30 minutes, while the mannitol- based formulation showcased an SD of less than 7.6%.

[0286] Glidant and Lubricant Selection: AEROSIL 200 Colloidal silicon dioxide) was chosen as a glidant based on considerations of powder flowability, ensuring optimal processing. Additionally, 1% magnesium stearate was selected as a lubricant to enhance the reduction of powder adhesion to pins during the capsule filling process.

[0287] Capsule Selection: HPMC capsules, were favored over Gelatin capsules due to their lower moisture content. Specifically, Capsugel’s V-Cap®Plus, devoid of gelling agents, were utilized to prevent undesired gelling in dissolution media, thereby ensuring the desired release profile.

[0288] Diluent Choice and Impurity Considerations: Mannitol was chosen as the final diluent over lactose due to observed unknown impurity peaks in the lactose formulation. Given the compound's structural potential to induce Maillard reactions, the observed peak was suspected to be a Maillard reactant, prompting the preference for mannitol to ensure formulation integrity.

[0289] For enhanced analogue stability, a formulation based on mannitol was selected, avoiding the possible lactose peak observed when lactose was used as an excipient. In certain embodiments, mannitol having different particle size distributions and / or processing forms (e.g., spray-dried, granulated, or milled) may be used individually or in combination to optimize powder flow, blend uniformity, and capsule fill weight during manufacturing. The selected mannitol grades may include fine and / or coarse particles, with the specific form and size range selected based on the desired performance characteristics of the formulation. Further analysis indicated that the disintegrant ECG505 was most effective in mitigating the increase in analogues, followed by PCS, Starch 1500, and Explotab.

[0290] In terms of capsule selection, V-Caps®Plus by Capsugel was chosen due to its absence of gelling agents including carrageenan gum, and the suitability of No. 4 size for the 5-25 mg formulation. For the 50 mg formulation, No. 2 size was selected for optimal filling.

[0291] Considering dissolution and analogous stability, mannitol was finalized as the excipient, while ECG505 and PCS were chosen as the disintegrants. The stability of samples was subsequently evaluated, leading to the preference for 5% PCS over 10% ECG505 due to its superior dissolution characteristics. (See Figure 4).

[0292] EXAMPLE 2 - Preparation of INVA8001 formulation with selected excipients

[0293] Based on the experimental results of Example 1, the following excipients and concentration ranges were selected for preparing the API formulation. (See Tables 4 and 5)

[0294] Preparation of 25 mg capsule

[0295] 25 mg of the API (INVA8001), mannitol, pregelatinized starch, colloidal silicon dioxide, magnesium stearate as noted in the table below were mixed together and ground into a homogenous mixture which was then filled into Hypromellose capsule of size #3. Docket No. 123950-10402

[0296] 35

[0297] Preparation of 50 mg capsule

[0298] 50 mg of the API (INVA8001), mannitol, pregelatinized starch, colloidal silicon dioxide, magnesium stearate as noted in the table below were mixed together and ground into a homogenous mixture which was then filled into Hypromellose capsule of size #3. Preparation of 75 mg capsule

[0299] 75 mg of the API (INVA8001), mannitol, pregelatinized starch, colloidal silicon dioxide, magnesium stearate as noted in the table below were mixed together and ground into a homogenous mixture which was then filled into Hypromellose capsule of size #3.

[0300] Table 5- List of ingredients and their concentration ranges at various dosage strengths a: The molecular weight conversion factor to calculate from INVA8001 to free form (ASB 16097) is 0.9032 b: USP: The United States Pharmacopeia c: JP: The Japanese Pharmacopoeia d: Ph. Eur.: The European Pharmacopoeia e: NF: National Formulary f: JPE: Japanese Pharmaceutical Excipients g: Magnesium stearate is of vegetable origin. h: Hypromellose capsule: DMF 21784. DMF access authorization letter is provided in Appendix 1. Docket No. 123950-10402

[0301] 36

[0302] Table 4- List of ingredients and their concentration ranges for 25 mg, 50 mg & 75 mg capsule

[0303] *Tentative formula for 75 mg strength;@quantity of active with acetic acid form The 25mg, 50mg, 75mg capsule concentrations were extrapolated from the concentrations for the 5mg and lOmg capsules. Other dosage strengths are of course possible based on further extrapolation of concentration values.

[0304] EXAMPLE 3 - Preparation of formulation with combination of excipients and particle sizes

[0305] Milling ofINVA8001 Milling was used to reduce the particle size of INVA8001 and excipients. The process of milling was found to improve drug formulation properties, enhance bioavailability, and assist with ensuring uniformity in drug products. The choice of milling technique was dependent on factors such as the desired particle size, and the nature of the API being milled. The pharmaceutical formulation containing INVA8001 was prepared as disclosed in Example 2. INVA8001 was then milled using different techniques such as jet mill, ultra-centrifugal mill, and pin mill. Docket No. 123950-10402

[0306] 37

[0307] INVA8001 was subject to jet mill followed by ultra-centrifugal mill and then finally pin mill. In jet milling, the primary particle size was reduced, eliminating agglomerates, and observing acetic acid desorption, along with a suggested decrease in crystallinity (some amorphization). In ultra-centrifugal milling, there was no change in the primary particle size, but agglomerates disappeared, and there was slight acetic acid desorption but no observed decrease in crystallinity. The final milling was conducted using a pin mill (fine impact mill) to achieve the desired particle size distribution (D50- 5 pm and D90-17pm). The API was found to be stable, with no change in the X-ray diffraction (XRD) pattern for 1 month at 25°C / 60% RH and 36 months at 5°C.

[0308] Milled and unfilled API were subjected to dissolution testing. Figure 3 shows the extent of drug release between milled INVA8001 and unmilled INVA8001 over the course of 80 minutes which were assayed at 10-minute intervals post dissolution. Results show that milled INVA8001 had 60% drug release when compared with unmilled API which had 20% release at 10-minute time point post dissolution. The particle size distribution was analyzed between milled and unmilled INVA8001. The results of the particle size distribution are shown below in Table 6:

[0309] Table 6 -Particle size distribution of the INVA8001 in the formulation

[0310] EXAMPLE 4 - Stability analysis of pharmaceutical formulation in 5mg, lOmg and 25 mg capsules Stability Studies

[0311] 5 mg, 10 mg and 25 mg capsules were prepared following Example 2. The capsules were then packaged in desiccant-embedded aluminum blisters and subjected to accelerated stability studies for a period of 6 months and long-term stability studies for a period of 24 months. The accelerated and long-term stability studies were conducted by storing the packaged drug product at 40°C and 75% relative humidity and 25°C and 60% relative humidity, respectively. The stability of INVA8001 was assessed by assay, total impurities, and acetic acid content at 0, 1, 3, and 6 months Docket No. 123950-10402

[0312] 38 for the accelerated conditions and at 0, 3, 6, 12, 18, and 24 months for the long-term storage condition.

[0313] For each batch of capsule tested, no significant changes from the initial to the 24-month interval were observed for any of the stability test attributes under long-term storage conditions 25°C / 60%RH. The results of the stability study are shown in Figure 2. The results indicate that the pharmaceutical formulation containing the API (INVA8001) is shelf-stable over a long period of time.

[0314] EXAMPLE 5 - PK simulation to determine optimal dosage and frequency of administration

[0315] Results from the Phase 2 study on the compound that corresponds to INVA8001 (Clinical trial- NCT01756898) revealed highly variable pharmacokinetics. While evaluating efficacy, patients (n=82) achieved a median Cavgof 19.08 ng / mL, there was significant variability across patients, as indicated by a wide range (90% confidence interval (CI) = 11.6 to 42.2 ng / mL).

[0316] To establish an efficacious dosing strategy, the inventors of the present disclosure opted to maintain plasma drug concentrations above the 19 ng / mL exposure target for a substantial portion of the treatment period (> 50%) with reduced fluctuation, ensuring target attainment in the majority of patients.

[0317] Utilizing a pharmacokinetic (PK) model developed with Phase 1 data, population pharmacokinetic simulations were conducted. These simulations aimed to predict how dosage regimens of 50 mg twice daily and 75 mg twice daily would achieve a minimum of the target concentration (19 ng / mL) throughout a 24-hour treatment period.

[0318] The model used for these reported simulations was developed from pooled data generated from three Phase I clinical studies (ASBI 701, ASBI 702, and ASBI 703) done on the compound that corresponds to INVA8001. All of the Phase I studies included full robust sampling of the PK profiles. Clinical study ASBI 701 was an escalating single-dose study in healthy male subjects receiving either placebo, 1, 10, 25, 50, or 100 mg PO INVA8001 (oral dose, sample size, n = 30 received active treatment). Clinical study ASBI 702 was an escalating multiple-dose study in male and female subjects with atopic dermatitis receiving either placebo, 10, 25, or 50 mg PO where INVA8001 once daily for 7 days (oral dose, sample size, n = 18 received active treatment) was administered. Clinical study ASBI 703 was a single-dose crossover study examining administration of 20 mg PO INVA8001 under either fasted or fed conditions (oral dose, sample size, n = 18). Modeling of the Phase I data used only data from the fasted state.

[0319] The model used for the INVA8001 plasma concentration simulations was a two- compartment model with transit compartment absorption to describe the absorption delays. Between-subject variability (BSV) was estimated for all parameters except for the absorption rate constant (Ka) which was fixed at 100%. Using this model, a set of 5 population PK simulations were performed with 200 subjects per simulation. Single and multiple-dose simulations were performed for each dosage. The simulated single doses were 20, 50, 75, 100, and 150 mg PO Docket No. 123950-10402

[0320] 39

[0321] INVA8001. The multiple dose regimens consisted of dosages of 20, 100, and 150 mg one a day (q24h) PO (oral dose) INVA8001, and 50 and 75 mg twice a day (ql2h) PO (oral dose) INVA8001. The total PO INVA8001 daily dose ranged between 20 and 150 mg across all simulations. Single doses were simulated through 48 hours post-dose. Multiple-doses were simulated through Day 7 (168 hours), to assure attainment of a steady-state (SS). The reference dosage was 20 mg q24h (Simulation 5), a dosage used in a prior patient study.

[0322] Simulated INVA8001 plasma concentrations following a single-dose and at SS following multiple-doses were summarized in plots for each population PK simulation, using calculated means, and the 10th, 50th (median), and 90th percentiles.

[0323] Noncompartmental analysis (NCA) was performed using data from individual simulated subjects within each population PK simulation. NCA parameters were determined using the Linear Up Log Down calculation method in WinNonlin®. For calculation of the terminal phases, a uniform user-selected time range of 24 to 48 hours was used for the single-dose simulations, and a best-fit method was used for the multiple-dose simulations. NCA parameters included target-based parameters, using a target of 19 ng / mL. Statistical summaries of the NCA PK parameters by population PK simulation were performed. Statistical summaries included the mean, standard deviation (SD), coefficient of variation (CV%), minimum (Min), maximum (Max), geometric mean (Geo. Mean), and geometric mean CV% (Geo. CV%).

[0324] The software packages used for running the simulation are listed below in Table 7.

[0325] Table 7-Summary of commercial software used in the analysis

[0326] Software Purpose Full Reference

[0327] Excel® 2019 Create data spreadsheets Office Excel® 2019,

[0328] Microsoft

[0329] SigmaPlot for Create plots SigmaPlot for Windows

[0330] Windows v.14.0, Systat Software,

[0331] Inc. Phoenix® 64 Data processing, Phoenix® 64 Build pharmacokinetic analyses 8.2.2.227, Pharsight platform (Phoenix™ (Certara L.P.)

[0332] NLME® and WinNonlin®), population simulations, NCA, generation of plots and tables, and statistical analysis Docket No. 123950-10402

[0333] 40

[0334] Population pharmacokinetic (PK) simulations for INVA8001 (n=200 subjects / simulation) were thus carried out with various dosages: 20 mg once daily, 50 mg twice daily, 75 mg twice daily, 100 mg once daily, and 150 mg once daily.

[0335] Figure 5(a) illustrates the predicted duration of time above the exposure target concentration for the once a day 20 mg dosage at steady state. Figure 5(b) demonstrates the predicted duration of time above the exposure target for a dosage of 50 mg administered every 12 hours, while Figure 5(c) suggests that higher exposures above the target threshold could be achieved with a dose of 75 mg administered every 12 hours.

[0336] Figure 5(a) shows that the failed clinical trial dosage of 20mg daily achieved the threshold concentration of 19ng / ml only for a short period of time, followed by the dropping of concentration below threshold efficacy levels (dotted lines).

[0337] However, the dose of 50 mg twice daily achieved drug plasma concentrations of over 19 ng / ml for extended periods of time. A noticeable dip is observed right before the second 12-hour infusion of the formulation which keeps the drug plasma concentration above the threshold efficacy level of 19ng / ml for longer period of time. (See Figure 5(b))

[0338] Similarly, the dose of 75 mg twice daily achieved drug plasma concentrations of over 19 ng / ml for even longer periods of time when compared with that of the 50 mg every twelve hours or 20mg once-a-day doses. A slight dip is observed right before the second 12-hour infusion of the formulation which keeps the drug plasma concentration above the threshold efficacy level of 19ng / ml for an extended period of time. (See Figure 5(c)). A comparison of various doses is shown in Figure 5(d).

[0339] To compare the differences twice daily versus once daily dose of 50 mg and 75 mg formulations, the PK simulations were repeated with once daily doses of 50 and 75 mg following the same protocols described above. The results are shown in Figure 6. Figure 6(a) shows the PK simulation of the 50 mg daily dose and Figure 6(b) shows the PK simulation of the 75 mg daily dose.

[0340] If one were to compare 5(b) with 6(a) for the 50 mg formulation, it is clear that even at a 50 mg dose, once-daily administration was insufficient to maintain the drug plasma concentration above the threshold efficacy value of 19ng / ml.

[0341] Likewise, if one compares 5(c) with 6(b) for the 75 mg formulation, it is clear that even at 75 mg dose, once-daily administration was insufficient to maintain the drug plasma concentration above the threshold efficacy value of 19ng / ml.

[0342] Therefore, it was determined that in order to achieve therapeutic efficacy a higher dose (50 mg or 75 mg) and higher frequency (twice daily) of administration was optimal. Docket No. 123950-10402

[0343] 41

[0344] The frequency of dosage was thus adjusted to twice a day so that Cmax remains above the estimated threshold plasma concentration of 19 ng / ml above which therapeutic efficacy expected to be achieved, based on PK modeling simulations.

[0345] EXAMPLE 6 - Determination of safety profile of high dose of formulation

[0346] The results of Example 5 point towards administering higher doses with twice daily administration. Hence, studies were done to determine whether the higher dosage and higher frequency of administration are safe. Specifically, the selected doses of 50 mg every twelve hours and 75 mg every twelve hours surpass those employed in the failed Phase 2 study. Furthermore, the proposed total daily dosages of 100 mg and 150 mg find validation in 4-week and 13-week nonclinical toxicology studies (GLP) that were conducted in rats and dogs.

[0347] Rat Study Outline

[0348] • A 4-week study followed by a 4-week recovery period

[0349] • A 13-week study

[0350] Dog Study Outline

[0351] • A 4-week study followed by a 4-week recovery period

[0352] • A 4-week study on male genital organs and their reversibility after a 13-week recovery period

[0353] • A 13-week study

[0354] A series of five GLP-compliant, repeat-dose oral toxicology studies were conducted to evaluate the safety profile of INVA8001. These studies involved adult rats (6-7 weeks old) administered 160 mg / kg once daily, and dogs (7-13 months old) administered 20 mg / kg once daily. Across all studies, INVA8001 was well tolerated, with no serious adverse effects observed even at these elevated dose levels. Based on these findings, INVA8001 can be safely administered to human subjects at doses of 50 mg or 75 mg twice daily with a total dose of 100 mg or 150 mg, without eliciting significant toxicity.

[0355] To ascertain safety margins, calculations were performed based on pharmacokinetic (PK) parameters at the No Observable Adverse Effect Levels (NOAELs), as outlined in Table 8. Safety margin ratios for 50 mg every twelve hours and 75 mg every twelve hours were derived, incorporating the human equivalent dose (HED) with species-appropriate allometric conversions (Table 9). The safety margins for both proposed human dosages exhibit adequacy when considering the rat and dog NOAELs and HEDs. Notably, ratios based on HED values calculated by body surface area are more conservative, relying solely on allometric scaling.

[0356] Moreover, safety margins for the suggested dosages are more pronounced when evaluated through ratios of exposure parameters (Cmax and AUC). This involves comparing steady-state exposure at the NOAELs to exposures predicted by simulations of the population pharmacokinetic Docket No. 123950-10402

[0357] 42 model utilizing the proposed human dosages. The robust safety margins derived from these analyses reinforce the favorable safety profile associated with the recommended dosing regimens, bolstering confidence in their suitability for clinical study. (See Tables 8 and 9)

[0358] Table 8 - Relative Daily Exposures (Mean) Across Species at Steady-State

[0359] AUCT,SS = area under the curve through the dosing period (r), at steady-state; AUC0-24h,SS = area under the curve at steady-state through 24 hours (daily exposure); Cmax,SS = maximum concentration at steadystate; NOAEL = no-observed-adverse-effect level; SS = steady-state; TK = toxicokinetic(s)

[0360] Table 9- Safety Margins for the Proposed Human Dosages in the Phase 2 Study

[0361] AUCMH.SS = area under the curve at steady-state through 24 hours (daily exposure); Cmax,ss = maximum concentration at steady-state; SS = steady-state. a) The dog NOAEL is 20 mg / kg / day. The NOAEL was not determined in the 13-week rat study; therefore, the lowest study dose (160 mg / kg / day) was used, as described above, b) Safety margins using mean ratios of daily exposure at SS; human exposures predicted using population pharmacokinetic simulations, c) Safety margins using ratios of HED (based on body surface area) and the proposed daily dosage for a 60 kg human.

[0362] EXAMPLE 7 - Determination of probability of target attainment for low and high doses

[0363] Based on these population PK simulations, the mean percent of time during each 24-h treatment period that INVA8001 concentrations are predicted to be above the target therapeutic concentration of 19 ng / mL increases from 23.1 % (CV% = 21.5%) for the original 20 mg once- daily dosage to 79.3% (CV% = 18.8%) and 91.7% (CV% = 10.3%) for the 50 mg and 75 mg dosages every twelve hours, respectively. The probability of target attainment (PTA) for each population PK simulation was determined by calculating the percent of simulated subjects within Docket No. 123950-10402

[0364] 43 each population PK simulation (n=200) that had simulated concentrations above the target of 19 ng / mL for specific percentages of the day (0 to 100%, 10% increments). The PTA versus the time above the target (percentage of the day) were compared across the population PK simulations graphically.

[0365] In terms of probability that a patient will reach therapeutic levels, there is close to a 0% probability that concentrations remain above the target for more than 50% of the time while on treatment with the original 20 mg once-daily dose. In contrast, these probabilities increase to 98% and 100% when using doses of 50 mg and 75 mg every 12 hours, respectively (Figure 7). The simulations also demonstrate that dosing every twelve hours reduces the fluctuation of the individual pharmacokinetic profiles and maintains more time with the drug exposure above the target threshold while keeping the Cmax low.

[0366] EXAMPLE 8 -Simulated in vivo profiles for single and b.i.d. dosing and any in vitro / in vivo correlations, AUC, Cmax, Tmax, etc. The foregoing profiles were obtained through simulations. Figures 7-10 illustrate some of these profiles.

[0367] Noncompartmental analysis (NCA) was performed using data from individual simulated subjects within each population PK simulation (single-dose and multiple-dose simulations). NCA parameters (AUC, Cmax, Tmax) were determined using the Linear Up Log Down calculation method in WinNonlin®. For calculation of the terminal phases, a uniform user-selected time range of 24 to 48 hours was used for the single-dose simulations and a best-fit method was used for the multiple-dose simulations. NCA parameters included the target-based parameters, using a target of 19 ng / mL. Statistical summaries of the NCA PK parameters by population PK simulation were then performed and included the mean, standard deviation (SD), coefficient of variation (CV%), minimum (Min), maximum (Max), geometric mean (Geo. Mean), and geometric mean CV% (Geo. CV%) calculated for All NCA parameters. See Figures 7, 8 ,9 and 10.

[0368] Example 9 - Synthesis of INVA8001

[0369] Synthesis of INVA8001 requires two starting materials , R-BABN-DTA and GTH. The synthesis of the starting materials is disclosed below.

[0370] Synthesis of Starting Material 1- R-BABN-DTA Docket No. 123950-10402

[0371] 44

[0372] R-BABN-DTA Synthesis

[0373] Synthesis of starting material 1 (R-BABN-DTA) involves the following steps:

[0374] Step-1 (BNBO to BNBN)Step-2 (BNBN to BNBZ)

[0375] Step-3 (BNBZ to BBNB)

[0376] Step-4 (BBNB to rac-BABN)

[0377] Step-5 (rac-BABN to R-BABN-DTA)

[0378] Step-6 (Recrystallization of R-BABN-DTA)

[0379] Step-1 BNBO to BNBN

[0380] To a clean and dry reaction vessel, l,3-Dimethyl-2-imidazolidinone (2.0v / w) was added followed by addition of BNBO and Copper cyanide (CuCN) and maintained the temperature at 15 ~ 30°C. The reaction mixture was heated up to 90°C and further increased the temperature to 105- 130 C and stirred for Ihr.

[0381] After Ihr, the reaction was quickly cooled to 30°C and the completion of reaction to >90% was confirmed by HPLC. After the reaction completion, water was added to the reaction mixture and stirred for 30 minutes followed by addition of acetic acid (AcOH) (6Vol.) and stirred for 10 minutes followed by addition of 5% sodium bicarbonate solution and stirred for another 10 minutes.

[0382] After 10 minutes, the stirring was stopped and the organic layer was separated. Aqueous layer was used for second extraction and combined the organic layers. Organic layer was mixed with saturated saline stirred for 15 minutes. Organic layer was then separated, and vacuum distilled.

[0383] Isopropyl alcohol (“IP A”) was added to the condensed content and heated to 70°C to confirm dissolution. The mixture was then cooled from 70°C to 25°C for over 2 hours to form crystals. The cooling was continued until the temperature reaches close to 5 C or less and was then stirred overnight to further allow formation of crystals. The BNBN crystals were filtered out and washed with refrigerator-cooled IPA. The crystals were then dried at 25-35 C and stored at cool temperature. The yield of BNBN was about 78%.

[0384] Step-2 (BNBN to BNBZ)

[0385] To a clean and dry vessel, water(lvok) was added followed by the addition of concentrated sulfuric acid(lvok). The mixture was stirred for 10-15 minutes. Acetic acid (3vol.) and BNBN from step 1 was then added to the mixture. The reaction mixture was then heated up to 108-112°C for 16 hrs and HPLC was used to confirm the completion of the reaction.

[0386] After completion of the reaction, the mixture was cooled to 90-100°C. Water (5vol.) was added dropwise while maintaining the reaction temperature at 80~100°C. The reaction vessel was Docket No. 123950-10402

[0387] 45 then cooled to 40°C and 28% sodium hydroxide (NaOH) was added to adjust pH <2.0 . Further adjustments to pH using 50% sulfuric acid is done if needed

[0388] Ethyl acetate (“AcOEt”) was added and the contents were stirred at 15~35°C for 15 minutes. The reaction mixture was allowed stand after continuous stirring for 15 minutes to allow separation of layers. The bottom aqueous layer was removed. The process was repeated twice. Water and 2N NaOH was added to the reaction mixture at a temperature of 35°C. After the addition, the pH of the solution was tested and adjusted to 5.8 to 6 by adding 28% NaOH.

[0389] After adjusting the pH, the solution was stirred at a temperature of 35 °C or less for 15 minutes. After 15 minutes, agitation was stopped, and the mixture was left to separate the layers. The bottom aqueous layer was removed to separate container A, and the top organic layer was reserved for further processing. Water was added and stirred at an internal temperature of 35°C. The pH was measured while stirring and, adjust the pH to 7.0-7.5 using 2N NaOH. The reaction mixture was stirred for 15 minutes and then the liquid was allowed to stand so that it can separate into layers.

[0390] The bottom aqueous layer was removed and combined with the aqueous layer above in container A for further processing. 50% sulfuric acid was then added to the reaction to adjust the pH <2.0. AcOEt was added to the liquid mixture and the contents were stirred at 35 °C for 15 minutes. The liquid mixture was allowed to stand so that it can separate into layers. The bottom aqueous layer was removed and transferred into a separate container B. The top organic layer for further processing. AcOEt was added to the container B and the contents are stirred at 35 °C for 15 minutes. The mixture was allowed to stand so that it can separate into layers. The top layer was separated and then combined with the previous top organic layers. The organic layer was then vacuum distilled at 60°C under reduced pressure. Toluene was added to the condensed reactor contents. The contents were then heated to 80°C or higher to dissolve BNBZ. Vacuum distilled the organic layer at 60°C under reduced pressure and confirm the acetic acid content (<0.1) proceed to next step.

[0391] Step-3 (BNBZ to BBNB)

[0392] BNBZ is dissolved in toluene (18v / w%) in a dry and clean reaction vessel followed by addition of tertiary butanol (t-BuOH) (10eq.), magnesium sulphate (8 eq.) and concentrated sulfuric acid (2.0 eq.) at internal temperature 20-25 C.

[0393] The reaction mixture was stirred at 20-30 C for 12hr and confirmed the completion of reaction by HPLC. After completion, the reaction was neutralized with dropwise addition of triethyl amine 2.0 eq.) maintaining internal temperature 30°C. After addition, the reaction was stirred for at least 10 minutes.

[0394] Half of the reaction was transferred to another vessel and water was added to dissolve the magnesium sulfate (MgSCU). After complete dissolution, the reaction was left to settle down and Docket No. 123950-10402

[0395] 46 separated the layers. The aqueous (water) layer was removed and the upper toluene layer was left in the vessel. Sodium bicarbonate solution was added to the toluene layer and stirred for 15 minutes.

[0396] The mixture was then allowed to settle and layers were separated. The aqueous water layer was removed and the upper toluene layer was left in the vessel. 20% saline solution was added to the toluene layer and stirred the reaction mixture for 15 minutes. The lower water layer was removed and the upper toluene layer was left in the vessel. The toluene layer was filtered and then distilled while maintaining an internal temperature of 60°C.

[0397] To the residue, IPA (4.0 v / w) was added and then distilled maintaining an internal temperature of 60°C. The residue added IPA (5.0 v / w) was heated up to 80°C to dissolve the concentrated residue. After complete dissolution, the reaction mixture was cooled to 25°C over approximately 2 hours and further cooling to less than 5°C. The reaction mixture was stirred overnight at an internal temperature of 5 C to crystalize the product. The crystals are filtered and vacuum dried at 30°C for 4 hours. The yield of BBNB was about 78%.

[0398] Step-4 (BBNB to rac-BABN)

[0399] In a clean and dry reaction vessel, BBNB from Step 3 was added along withlN ammonia ethanol (“NH3-EtOH) solution and purged Nitrogen. The internal temperature was adjusted to 20- 25°C. Ti (OiPr)4 was added dropwise keeping internal temperature <30°C. The reaction mixture was kept for 2hrs at 20-25 C. The formation of product was then confirmed by HPLC.

[0400] NaBPE was first added and NaOH in EtOH was added to the mixture dropwise in a time of 3 hrs while maintaining an internal temperature of 20-30 C. After addition, the mixture is incubated at 20-30 C for at least an hour. After the second reaction, a sample from the reaction mixture is removed and checked to see whether the conversion rate is >90%. The mixture was then vacuum distilled at an internal temperature of 50°C and Toluene (5vol) was added and again vacuum distilled. Toluene (9vol) and water (5 vol) was to the mixture and stirred for 30 minutes.

[0401] The contents were centrifuged and then the crystals were filtered from toluene. The crystals were washed with water. The toluene and water washes were collected for further processing. After phase separation, the lower aqueous layer was removed and the upper organic layer was reserved for further processing.

[0402] Water and then 28% NaOH solution was added. The mixture was stirred for 15 minutes and stand still to separate the layers. The lower water layer was removed and the upper toluene layer was left in the vessel. To the organic layer, 20% saline solution was added and stirred for 15 minutes or more and allowed to stand to separate the layers.

[0403] The lower water layer was removed and the upper toluene layer was left in the vessel. Magnesium sulphate was added and stirred for at least 30 minutes and filtered through celite. The filter was washed with toluene and the toluene washed with the magnesium sulphate filtrate was collected. The magnesium sulphate filtrate was vacuum distilled at a temperature of 55 + / - 5°C until distillation stops and then EtOH was added and then vacuum distilled at a temperature of 55 + / - 5°C Docket No. 123950-10402

[0404] 47 until distillation stops. EtOH was further added to dissolve the concentrated product. The resultant product was stored at 10°C until next step.

[0405] Step-5 (rac-BABN to R-BABN-DTA)

[0406] To the BABN-EtOH solution from previous step 4, EtOH and water (0.5 v / w) was added to make the solution homogeneous. D-tartaric acid (“D-TA”) (0.7 eq.) was added and the reaction mixture was stirred at 55-60 C for 6 hours. The reaction mixture was cooled to 15-30 C , stirred at two hours and then kept overnight at the same temperature to develop crystals. The crystals were cooled to 0-5°C and washed with pre-chilled EtOH. The crude R-BABN-DTA crystals were dried at 30°C for 4hrs and was stored in tight sealed container.

[0407] Step-6 (Recrystallization of R-BABN-DTA)

[0408] To the clean vessel, EtOH and water was added to crude R-BABN-DTA from step 5. The solution was heated to 75-80 C , a sample was taken and to confirm the dissolution of crude R- BABN-D-TA. The solution was filtered and the filtrate was collected for further processing.

[0409] The filtrate was gradually cooled at a rate of 3-4°C per hour and crystalized for overnight at temperature 15-30 C. After overnight, the solution was allowed to crystalize for 2 hours or more at an internal temperature of 0-5°C. The crystals were filtered and washed with pre-chilled EtOH. The R-BABN-DTA crystals were dried in dry vacuum at 30°C for 4hrs and further increased to 40°C. Once drying is completed, the crystals were packed in dry airtight amber color container. The yield of R-BABN-DTA was about 95%.

[0410] Synthesis of GTH

[0411] Synthesis of starting material 2 (GTH) involves the following steps:

[0412] Step-1 (AA to CAA)

[0413] Step-2 (CAA to ECB)

[0414] Step-3 (ECB to EAB)

[0415] Step-4 (EAB to t-BuEAB)

[0416] Step-5 (t-Bu-EAB to Rac HBB)

[0417] Step-6 (rac-HBB to (s)-HBB-(+)-ANP)

[0418] Step-7 ((s)-HBB-(+)-ANP to S-HBB)

[0419] Step-8 (HBB to GBE)

[0420] Step-9 (GBE to GAE)

[0421] Step- 10 (GAE to GTE)

[0422] Step- 11 (GTE to GTH) Docket No. 123950-10402

[0423] 48

[0424] The synthetic scheme is illustrated below:

[0425] Step-1 (AA to CAA)

[0426] In a clean reaction vessel acetic acid (2.0 Vol.) was added and cooled to <10°C. Ca(OCl)2 (0.8eq) was added and temperature was maintained at <10°C. In a separate vessel 2-Methoxy Benzaldehyde (“AA”) was added to acetonitrile (5.0Vol.) and cooled to 15-20 C. Ca(OCl)2 mixture was added to the AA in acetonitrile mixture dropwise and maintained temperature < 20°C. The reaction mixture was stirred at < 20°C and a sample was taken to confirm the completion of reaction by HPLC. After completion of the reaction, toluene (5.0v / w) was added and stirred the reaction mixture at < 20°C for 20 minutes. The organic layer was separated and vacuum distilled the organic layer and kept the organic layer for overnight for next step. The yield of 5-Chloro-2-Methoxy BenzAldehyde (“CAA”) is about 81.4%.

[0427] Step-2 (CAA to ECB)

[0428] To the residue from step-1 ethyl cyanoacetate (l.leq) and 1-methyl imidazole (O.lOeq) was added at a temperature < 45°C. The reaction temperature was raised up to 85°C and stirred for 3hrs. Progress of reaction was monitored by HPLC. After the reaction was completed, the reaction mixture was cooled down to 30°C and the reaction was concentrated under reduced pressure at 70-80 C till the volume remained 2v / v. Ethanol (5vol.) was added and the reaction mixture was further concentrated under reduced pressure at 70-80 C till the volume remained 3v / v. Ethanol was charged to makeup the liquid volume 6.0v / w and the reaction was heated to 80°C. The dissolution was confirmed by taking a sample. Docket No. 123950-10402

[0429] 49

[0430] After the complete dissolution, the reaction mixture was cooled to 60°C , seed crystals of Ethyl (2Z)-3-(5-chloro-2-methoxyphenyl)-2-Cyanoprop-2-enoate (“ECB”) were added and the temperature was maintained at 60°C. The reaction was stirred for 30 minutes and cooled to 0-5°C. Regular water (dropwise) (Iw / w) was added at 0-5°C and the reaction mixture was stirred for 3hrs at the same temperature. The solid formed was filtered and dried in dry Vacuum at 40°C for 8-10 hrs. The yield of ECB is about 75%.

[0431] Step-3 (ECB to EAB)

[0432] To a clean and dry reaction vessel, ECB from Step 2, ethyl acetate (20v / w), potassium carbonate (l.Oeq) and Raney Nickel (0.35v / w) was added. The reaction mixture was purged with nitrogen followed by hydrogen gas and the reaction mixture was stirred at a temperature of 40-50 C for 12 hrs. The product formation was confirmed by taking a sample and running it with HPLC. After reaction completion, the reaction mixture was cooled to 35°C and filtered through celite followed by washing of solid with cooled ethyl acetate.

[0433] To the filtrate, IN HC1 (lOv / w) was added dropwise at a temperature of 20-25 C and the reaction was stirred for 15 min at 20-25 C. The organic layer was separated and the aqueous layer was kept separately. To the organic layer IN HC1 (lOv / w) was added dropwise at a temperature of 20-25 C and the reaction was stirred for 15 min at 20-25 C. The organic layer was separated and combined with the aqueous layer of prior round of phase separation. The pH of the aqueous layer (9.0-9.5) was adjusted using 48%NaOH and temperature was maintained at 15-20 C. Ethanol was added and vacuum distilled the reaction mixture at < 25°C till the 4v / w. Reaction mixture was carried over to the next step.

[0434] Step-4 (EAB to t-BuEAB)

[0435] Ethanol (4.0v / v) was added to the reaction mixture from previous step followed by slow addition of Di-ButylOxy Carbonyl (“DIBOC”) (1.08eq) while maintaining a temperature of < 20°C. The reaction was stirred at 20-25 C for Ihr. The formation of product was confirmed by HPLC.

[0436] Step-5 (t-Bu-EAB to Rac HBB)

[0437] To the reaction mixture from step-4, 48% NaOH (lOeq) was added dropwise and the temperature was maintained at 20-25 C. The reaction mixture was heated up to 50-60 C and stirred for Ihr. Formation of product was confirmed by HPLC. Once the reaction was complete, the reaction mixture was vacuum distilled at < 60°C. The residue was cooled to room temperature and the pH was adjusted to 7 with 35% HC1. Ethyl acetate (6.0v / w) was added and the pH was further adjusted to 4 with 35% HC1. The temperature was maintained at 15-20°C. The organic layer was separated and vacuum distilled at 20-25 C till the remaining volume is 3.6v / w. Docket No. 123950-10402

[0438] 50

[0439] Step-6 (rac-HBB to (s)-HBB-(+)-ANP)

[0440] To the reaction mixture from step-5, ethyl acetate (19v / w) was added followed by (+)-a- Naphthylphenylglycine (“(+)-ANP”) (0.54eq / ECB). The reaction mixture was heated to 50°C to dissolve the contents or the reaction mixture. The reaction mixture was cooled down to 35°C and seed crystals were added followed by further stirring at 35-40 C. The reaction mixture was cooled up to 0-5°C and stirred for 10 hrs. The solid was filtered and wash with cold ethyl acetate, then vacuum dried at 50°C for 10 hrs. The yield of (s)-HBB-(+)-ANP is about 26.8%

[0441] Step-7 ((s)-HBB-(+)-ANP to S-HBB)

[0442] In a clean and dry reaction vessel, ethyl acetate (7v / w) was added to the solid from step-6 followed by dropwise addition of IN HC1 (7w / w) at 20-25 C .The reaction mixture was stirred for 30 min at same temperature. The organic layer was separated and IN HC1 (3v / w) was added dropwise. The reaction was stirred for 15 min at 20-25 C. The organic layer was separated and vacuum distilled up to the remaining volume 2.0v / w at 25-35 C. Toluene (3v / w) was added to the organic layer and vacuum distilled at a temperature of < 35°C till the residual volume was 1.5v / w. The reaction mixture was then stirred at 40°C for Ihr. The reaction mixture was further cooled and stirred at 0-5°C for 2hr. The reaction mixture was filtered and washed with cold toluene, then vacuum dried to form a solid at 40°C for 5hrs. The yield of S-HBB is about 93.2%

[0443] Step-8 (HBB to GBE)

[0444] To a clean and dry vessel, ethyl acetate (8.0 vol.), HBB, GET.HC1 (1.05eq), 1- Hydrozybenzotriazole monohydrate (“HOBT.H2O”) (0.3eq) and Triethyl amine (1.15eq) was added. Reaction mixture was stirred at 20-40 C and EDC. HC1 (l.lOeq) was added and stirred for Ihr at same temperature. Completion of reaction was confirmed by HPLC. Water was added and the reaction mixture was stirred for another 30 minutes and the temperature was maintained at 20- 40°C.

[0445] The temperature was raised to 35-45 C and stirred for 30-60 minutes at the same temperature to dissolve the contents. The reaction mixture was cooled to 15-25 C, water was added and stirred for another 2 hrs. The reaction mixture was centrifuged and the aqueous layer was discarded. The remaining content was washed with mixture of ethyl acetate and water. The solid formed was filtered and dried under vacuum at 60°C for 5hrs. The product was stored at the room temperature.

[0446] Step-9 (GBE to GAE)

[0447] To a clean and dry reaction vessel EtOH (6.0vol), GBE intermediate and methanesulfonic acid (“MsOH”) (1.2eq) was added and the reaction mixture was heated up to 75-80 C. Stirring was continued till the reaction was completed (confirmed by HPLC). The reaction mixture was cooled to 20-30 C followed by the addition of Triethyl amine (l.Oeq) and the reaction mixture was used for the next step. Docket No. 123950-10402

[0448] 51

[0449] Step-10 (GAE to GTE)

[0450] To the reaction mixture from previous step, 3,3',5,5'-Tetramethylbenzidine (“TMB”) (l.Oeq) was added and stirred to dissolve the mixture. The reaction mixture was cooled at 0-5 C. acetic acid AcOH was added (0.20eq) and maintained temperature 5 C followed by water (2.0vol) and maintained temperature 0-10 °C. The reaction was cooled at -10 - 5 °C followed by the slow addition of NaBH4(0.40eq) and temperature was maintained at -10 - 5 °C . The reaction mixture was stirred at same temperature. Completion of reaction was confirmed by HPLC. the reaction was stirred overnight at 0-5°C and proceeded for next step.

[0451] Step-11 (GTE to GTH)

[0452] 25% NaOH was added to the reaction mixture from the previous step and maintained at a temperature at 0-10 C. The reaction mixture was heated up to 35-40 C and stirred for 30 minutes. Completion of reaction was confirmed by HPLC. After complete conversion, water was added and pH was adjusted to 9-9.5 by dropwise addition of HC1 (2mol / l).

[0453] The seed crystal of GTH was added followed by slow addition of HCl(2mol / l) to bring pH- 6.5-7.5 and the temperature was maintained at 35-45 C. The reaction mixture was stirred for 30 minutes. The reaction mixture was cooled to 0-5°C and stirred for 2hrs. The reaction mixture was centrifuged and discarded the aqueous layer. The solid was washed with chilled water and dried in dry vacuum at 60°C for 5hrs. The product was stored at room temperature.

[0454] Synthesis of INVA8001

[0455] The synthetic process for making INVA8001 is outlined below: Docket No. 123950-10402

[0456] 52

[0457] Synthesis of INVA8001 involves the following steps:

[0458] Step-1 GTH to TAD

[0459] Step-2 R-BABN to BIBN

[0460] Step-3 BIBN to BOTN

[0461] Step-4 BOTN to BTTN

[0462] Step-5 BTTN to BETN

[0463] Step-6 BETN to HC1 Salt

[0464] Step-7 HC1 Salt to INVA8001

[0465] Step-1 GTH to TAD

[0466] To a clear reaction vessel, anhydrous acetonitrile (4Vol.), toluene (6Vol.), and GTH was added. The reaction mixture was stirred for 5-10 minutes. 1-Hydrozybenzotriazole monohydrate (“HOBt.H2O”) (0.8 eq.) was added and the temperature was maintained during the addition at 45°C or less. The reaction mixture was heated to 40-50°C and stirred for 30 minutes. l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (“EDC”) (0.24 eq) was added and the temperature was maintained at 40-50°C . The reaction mixture was stirred until the reaction was complete. The completion of the reaction was confirmed by HPLC.

[0467] Water (5.0 vol.) was added followed by the addition of sodium chloride (0.5 wt / vol.). The temperature was maintained at 20-30°C and stirred at the same temperature for 15 minutes. The organic layer was separated and water (5.0 vol.) and sodium bicarbonate (0.3 wt / vol.) was added the reaction mixture was stirred for 10 minutes. The organic layer was separated and washed with brine. The organic layer was then condensed and toluene was added. The reaction was heated to 35- 45°C. To the stirring mixture, (6S)-6-(5-Chloro-2methoxybenzyl)-l-(2,4,6-trimethoxybenzyl)-l,4- diazepane-2,5 dione (“TAD”) crystals (for seeding) were added and stirred the reaction mixture for 60 minutes or more at a temperature of 35-45°C. The reaction mixture was cooled to 0-5°C. The reaction was stirred at 0-5°C and 40-60 rpm for 120 minutes or more, the precipitate was filtered and washed with cool toluene (0-5 °C) under vacuum and dried to form a solid cake under vacuum at 60°C for 5 hours. The yield of TAD is about 80%

[0468] Step-2 R-BABN-DTA to BIBN

[0469] To the reaction vessel, toluene (5 vol.), R-BABN-DTA, and water (5 vol.) were added. 25% NaOH (1.92 wt.) solution was added and maintained the internal temperature at 20-30°C. The Docket No. 123950-10402

[0470] 53 reaction was stirred for more than 15 minutes at an internal temperature of 20-30°C. Stirring was stopped the layers were allowed to separate. The organic layer was separated and normal water (2 vol.) was added at an internal temperature of 20-30°C. Sodium chloride (0.4 wt.) was added. The reaction was stirred for more than 15 minutes. Stirring was stopped and the organic layer was separated. The organic layer was concentrated under reduced pressure. Acetonitrile (CH3CN) (6.0 vol.) was added. The organic layer was concentrated under reduced pressure to 1 / 3 of the volume. The reaction was cooled under reduced pressure to 0-5°C and kept it at 0-5°C to keep BABN for the next step.

[0471] In a separate reaction vessel, acetonitrile (10 vol.) and ButylOxy Carbonyl Anhydride (“BOC-anhydride”) (1.2 eq) were added and the reaction mixture was cooled to -20 to -10°C. The reaction atmosphere was kept anhydrous to avoid moisture. 4-Dimethylaminopyridine (“DMAP”) (0.60 eq.) was added while the temperature was maintained at -20 to -10°C. BABN was added at a temperature of -20 to -10°C. The reaction mixture was degassed using nitrogen. The reaction mixture was used for the next step.

[0472] Step-3 BIBN to BOTN

[0473] In the reaction mixture, anhydrous acetonitrile (0.8 vol.) was added, followed by the addition of TAD (from step-1). The temperature was increased to 35-45 °C to dissolve the reaction mixture. The reaction mixture was then cooled to 20-30°C, and the TAD mixture was added to the BIBN (from step-2) at -20 to 0°C. Potassium tert-butoxide (t-BuOK / THF) (0.12 eq) was charged while the temperature was maintained at -20 to 0°C. The reaction was stirred at -20 to 0°C (60 rpm) for 20 minutes. Completion of the reaction was confirmed by HPLC.

[0474] After the reaction was completed, acetic acid (1.5 eq) was added and the temperature was maintained at 0 to 30°C. Brine was added, and the reaction mixture was stirred at 20-30°C for 15 minutes. Stirring was stopped and the organic layer was separated. The organic layer was concentrated under reduced pressure to equal to, or less than 5.0 vol. Isopropyl alcohol (5.0 vol.) and water (0.5 vol.) were added to the reaction mixture. The mixture was stirred for 30 minutes at a temperature of 20-30°C, followed by the addition of water. The reaction mixture was cooled to 0- 5°C and stirred for 60 minutes at the same temperature. The reaction mixture was then concentrated under reduced pressure. Solid crystals separated out and the crystals were washed with 2.8 vol. 72% (aq.) IPA. The product was dried at 60°C in a dry vacuum for 5 hours. The yield of BOTN is about 90%

[0475] Step-4 BOTN to BTTN

[0476] To the clean reaction vessel, THF (tetrahydrofuran) was added, followed by BOTN (from step 3) under an inert atmosphere. To the stirring solution, Lawesson reagent (0.55 eq) was added. The reaction mixture was stirred at a temperature of 20-35 °C for at least 1 hour. Completion of the reaction was confirmed by HPLC. Water (5.0 vol.) was slowly added to the reaction mixture, and the reaction mixture was stirred for 60 minutes at 20-30°C. The solid precipitates were filtered and Docket No. 123950-10402

[0477] 54 washed with cold methanol. The solid was dried at 60°C for 5 hours at 35-40°C. The yield of BTTN is about 94%.

[0478] Step-5 BTTN to BETN

[0479] To the cleaned reaction vessel, THF (4.0 Vol.), EtOAc (5.0 Vol.), ZnO (3.0 eq), NaOAc (5.0 eq), and EtONH2.HCl (3.6 eq) were added. The reaction mixture was heated to 45°C and stirred for 60 minutes, and maintained at 45°C. BTTN from Step-4 was added, followed by THF (1 Vol.). The reaction mixture was stirred for 1 hour at 45°C. Completion was monitored by HPLC. After reaction completion, Na COa (4.0 eq) was added at 45°C. The reaction mixture was cooled to 20-30°C and stirred for 8 hours. The inorganic salts were filtered out, the filtrate was collected and condensed under reduced pressure. The contents were cooled to -20 to 5 °C and proceeded to the next step.

[0480] Step-6 BETN to HC1 Salt

[0481] To the cooled reaction mixture from step 5, Zn (6.6 eq) and MgSO4 (0.1 wt%) were added, the temperature was maintained at -20 to 5°C. 4M HC1 in EtOAc (~5.6 Vol.) was added slowly, keeping the temperature at -20 to 5°C. The mixture was stirred overnight (~14 hrs) at -20 to 5°C, then heated to 35-45°C and stirred for at least one hour. Completion was monitored by HPLC.

[0482] After completion, the mixture was cooled to 20-30°C, and NaOAc (18 eq) and water (5.0 Vol.) were added, stirred at 20-30°C for 15 minutes. The organic layer was separated, brine was added, and the mixture was stirred at 20-30°C for 15 minutes. The organic layer was separated again and combined with AcOH (2.0 Vol.), IPA (4.0 Vol.), water (0.5 Vol.), and cHCl (0.0595 wt). Seed crystals of the HC1 product were added, and the mixture was stirred for 60 minutes at 20- 30°C. cHCl (0.5355 wt.) was added over 60 minutes, then stirred for 30 minutes at 20-30°C. The reaction mixture was cooled to 0-5 °C and stirred for 8 hours. The mixture was filtered, washed with chilled ethyl acetate, and the product formation was confirmed by HPLC. The product was dried under vacuum and stored at low temperature (hygroscopic).

[0483] Step-7 HC1 Salt to INVA8001

[0484] To a clean reaction vessel, EtoAc (3.0Vol.), HC1 product from step-6, water (3.0vol.), NaCl (0.45wt) and NaOAc(0.3wt) were added. Reaction mixture was stirred at 20-30 C for 30min. Organic layer was separated and water (3.0vol.) and Na SaOa (0.3wt) were added. Reaction mixture was stirred at 20-30 C for 30min. NaCl (0.3Vol) was added and stirred further at 20-30 C for 30min. The stirring was stopped and the organic layer was separated. The organic layer was filtered and the inorganic solid was washed with ethyl acetate. Filtrate and wash solvent were combined and added IPA (5.0Vol) and the organic layer was condensed under reduced pressure at 35°C. AcOH (1.5vol.), INVA8001 crystals (O.OOOlwt) were added. The reaction mixture was stirred at 25°C for Ihr. Purified water (7.0Vol.) was added slowly over 0.5 h and cooled the reaction mixture to 0-5°C. The reaction mixture was stirred at 0-5°C for Ihr. The reaction mixture was condensed and washed with precooled mixture (0-5 C) of AcOH (0.3vol.) IPA (0.6 vol.), water (1.4 vol.). The solid was collected and dried Docket No. 123950-10402

[0485] 55 under vacuum and stored at dry condition and low temperature. The yield of INVA8001 is about 78%.

[0486] The purified INVA8001 was subjected to X-ray powder diffraction following the standard protocols known in art, such as methods disclosed in Bernstein et al., Powder Diffraction and Pharmaceuticals, International Tables for Crystallography (2019). Vol. H, ch. 7.5, pp. 767-781 and Fawcett et al. A practical guide to pharmaceutical analyses using X-ray powder diffraction. Powder Diffraction. 2019;34(2): 164-183.

[0487] The XRPD data of the purified INVA8001 thus collected is shown in Figure 14.

[0488] Example 10 -Inhibition of human mast cell activation and degranulation using INVA8001

[0489] Chymase is a serine endoprotease that enzymatically activates several precursor molecules, including matrix metalloproteinases (MMP-1 and MMP-9), angiotensin II (Ang II), interleukin- 33 (IL-33), transforming growth factor-beta (TGF-P), stem cell factor (SCF), chemokine ligand 26 (CCL26), and collagen I. The activation of these molecules contributes to enhanced angiogenesis and disruption of tight junctions, facilitating the infiltration and accumulation of neutrophils, macrophages, mast cells, eosinophils, and lymphocytes. This cascade ultimately drives chronic inflammation and, over time, fibrosis.

[0490] Specifically, MMP-9 promotes the infiltration of neutrophils, macrophages, and T cells; Ang II regulates the maturation and activation of macrophages, dendritic cells, and lymphocytes; and IL-33 enhances the recruitment and activation of mast cells, basophils, innate lymphoid cells (ILCs), macrophages, lymphocytes, and eosinophils, particularly in allergic conditions. Chymase itself has also been shown to support eosinophil viability, infiltration, and activation.

[0491] In this study, it was demonstrated that chymase inhibition by INVA8001 led to a reduction in mast cell activation and reduction in tryptase release in human primary skin-derived mast cells. These findings make clear that INVA8001 will be effective in the treatment of mast cell-driven skin diseases such as but not limited to chronic urticaria, prurigo nodularis, Eosinophilic Gastrointestinal Disorders (EGIDs) and atopic dermatitis.

[0492] Objective'. To assess the efficacy of INVA8001 in inhibiting IgE-mediated activation and degranulation of mast cells derived from human skin.

[0493] Materials and. Methods'.

[0494] Cells: Primary mast cells were freshly isolated from human skin tissue.

[0495] Test Compounds:

[0496] 1. INVA8001 (also known as 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2- methoxybenzyl)-3-(ethoxyimino)-7-oxo-l,4-diazepan-l- yl]carbonyl} amino )butyl]benzoic acid monoacetic acid solvate) (at concentration of 100 pM; freshly prepared in DMSO)

[0497] 2. Chymostatin (50 pM; freshly prepared in DMSO) Docket No. 123950-10402

[0498] 56

[0499] 3. c-KIT blocking antibody (clone SR-1; 10 pg / mL)

[0500] Assessment Procedures'.

[0501] 1. Cell Viability: Determined by flow cytometry using DAPI exclusion.

[0502] 2. Chymase Activity: Measured using a colorimetric substrate assay (e.g., N-Succinyl- Ala-Ala-Pro-Phe p-nitroanilide).

[0503] 3. Mast Cell Activation: Assessed via CD63 surface translocation using flow cytometry.

[0504] 4. Tryptase Levels: Quantified in supernatants using ELISA.

[0505] Primary mast cells were freshly isolated from human skin tissue and cultured under standard conditions. The cells were seeded at a density of 2 x 106cells / mL in either 48- or 96-well plates. To sensitize the cells, the cells were first pre-incubated with human IgE at a concentration of 1 pg / mL for one hour. Following sensitization, the cells were washed and incubated for an additional hour in a stem cell factor (SCF)-free buffer, either in the presence or absence of a c-KIT blocking antibody (clone SR-1, 10 pg / mL), which is known to inhibit SCF binding to the c-KIT receptor.

[0506] Subsequently, mast cell degranulation was induced by stimulation with anti-IgE. To evaluate the inhibitory effects of chymase blockade, cells were treated with either INVA8001 (100 pM) or chymostatin (50 pM), both freshly prepared in DMSO. Unstimulated cells served as negative controls. After two hours of stimulation, both supernatants and cell pellets were collected for analysis.

[0507] Cell viability was assessed using flow cytometry by identifying D API-negative cells. Chymase enzymatic activity was measured using a colorimetric substrate assay employing N- Succinyl-Ala-Ala-Pro-Phe- p-nitroanilide. Mast cell activation was quantified by evaluating CD63 surface translocation via flow cytometry, and tryptase levels in the supernatant were determined using a commercially available ELISA kit.

[0508] The results demonstrated (See Figure 15) that treatment with INVA8001, chymostatin, or the c-KIT antibody did not significantly affect cell viability, indicating that the compounds were not cytotoxic under the experimental conditions. However, INVA8001 treatment resulted in a nearcomplete (-100%) inhibition of chymase activity. Furthermore, INVA8001 reduced the percentage of CD63-positive activated mast cells by approximately 82% and decreased tryptase release by 95% compared to the IgE+anti-IgE- stimulated control group.

[0509] In contrast, chymostatin reduced chymase activity by 71% but did not significantly affect CD63 expression or tryptase levels. The c-KIT blocking antibody showed no measurable impact on chymase activity, mast cell activation, or tryptase release.

[0510] These findings demonstrate that INVA8001 effectively inhibits chymase activity and downstream mast cell activation and degranulation in human primary skin-derived mast cells. The Docket No. 123950-10402

[0511] 57 data support the therapeutic potential of INVA8001 in treating mast cell-driven inflammatory and allergic diseases.

[0512] These data demonstrate that INVA8001 effectively inhibits chymase activity and IgE- induced mast cell degranulation, along with a significant reduction in tryptase levels. The efficacy observed with INVA8001 at the tested dose is markedly superior to that of non-specific chymase inhibitors such as chymostatin and the c-KIT blocking antibody. These findings support the therapeutic potential of INVA8001 in the treatment of mast cell-driven inflammatory and allergic disorders.

[0513] Example 11- INVA8001 inhibits inflammatory cascade initiated by chymase.

[0514] Chymase enzymatically cleaves and activates multiple substrates including matrix metalloproteinases (MMP-1 and MMP-9), angiotensin II (Ang II), interleukin- 33 (IL-33), transforming growth factor-beta (TGF-P), stem cell factor (SCF), chemokine ligand 26 (CCL26), and collagen I. (See Figures 11-13 and Figure 16)

[0515] These activated mediators promote immune cell infiltration, mast cell activation, tissue remodeling, and fibrosis. MMPs facilitate neutrophil, macrophage, and T cell infiltration; Ang II activates macrophages, dendritic cells, and lymphocytes; IL-33 drives activation of mast cells, basophils, ILCs, and eosinophils; SCF supports mast cell survival and proliferation via c-KIT; CCL26 recruits eosinophils; and TGF-P and collagen I contribute to fibrotic remodeling. By selectively inhibiting chymase, INVA8001 arrests this cascade, thereby reducing inflammation, mast cell activation and proliferation, and ultimately preventing tissue fibrosis.

[0516] Evaluation of ASB 16097 (INVA8001) on Chymase-Induced Eosinophil Migration

[0517] The purpose of this experiment was to evaluate the effect of ASB 16097 (also referred to as free form of ASB 17061 or INVA8001) on eosinophil migration induced by chymase in vitro. Primary human eosinophils were used as test cells. The compounds tested included ASB 16097, SUN13834 (chymase inhibitor), and human recombinant chymase, all prepared in a chemotaxis medium composed of RPMI1640, 1% BSA, and 25 mmol / L HEPES. ASB 16097 and SUN13834 were each dissolved in DMSO to create 100 mmol / L stock solutions, which were then serially diluted to achieve final concentrations ranging from 0.001 to 100 pmol / L. Human chymase was diluted to a 1 pmol / L working solution using chemotaxis medium and mixed with either the test compound or medium alone before being added to the lower chamber of a 48-well chemotaxis system.

[0518] Human eosinophils were isolated from peripheral blood using Lymphocyte Separation Medium and purified by negative selection with anti-CD16 MACS beads, achieving a purity of 98.05%. The eosinophils were suspended at a concentration of 2 x 106cells / mL. In the migration assay, 50 pL of eosinophil suspension was added to the upper chamber, while 26 pL of the chymase solution (with or without test compound) was added to the lower chamber. The chambers were incubated at 37 °C with 5% CO2 for one hour. Following incubation, the filters were fixed with Docket No. 123950-10402

[0519] 58 methanol, stained with Hemacolor, and the number of migrating cells adhering to the distal side of the filter was counted under a microscope at 400x magnification across three fields per well. Each experimental group included four replicates (n = 4), totaling 48 wells across 12 groups.

[0520] Data were analyzed using JMP 8.01 (SAS Institute Inc.). Mean values and standard deviations were calculated, and statistical significance was assessed using Student’s t-test for comparisons between control and chymase-treated groups, and Dunnett’s test for evaluating the effects of the test compounds. The results demonstrated that ASB 16097 significantly suppressed eosinophil migration induced by human chymase across all tested concentrations. SUN13834 also inhibited migration but was less effective than ASB 16097 at each concentration. These findings indicate that ASB 16097 is a more potent inhibitor of chymase-induced eosinophil migration compared to SUN13834. Thus INVA8001 (ASB 16097) significantly suppressed the eosinophil chemotaxis mediated by human chymase / mice chymase (mMCP4) (See Figure 18).

[0521] Similar experiments were conducted to determine the effect of INVA8001 on inhibition of chymase mediated cleavage of SCF, inhibition of conversion of proform of matrix metalloprotease (MMP-9) into active form and reduction of expression of key biomarkers such as Collagen Type I Alpha 1 Chain (Collal) and Transforming Growth Factor Beta 1 (Tgfbl).

[0522] Thus ASB 17061 (INVA8001) inhibits the human and mice chymase-mediated cleavage of SCF. (See Figure 19). ASB17061 (INVA8001) also prevented the human and mice chymase- mediated conversion of pro-MMP-9 to MMP-9. (See Figure 20) Treatment with 10 and 20 mg / kg INVA8001 for 2 weeks in Mdr2 knockout mice reduced the expression of Collal and Tgfbl in liver samples. (See Figure 21).

[0523] The data show that INVA8001 (ASB 17061) effectively inhibits both human and murine chymase-mediated cleavage of stem cell factor (SCF) and the conversion of pro-MMP-9 to active MMP-9. In Mdr2 knockout mice, treatment with INVA8001 (10 and 20 mg / kg for 2 weeks) significantly reduced hepatic expression of fibrosis markers Collal and Tgfbl. Additionally, INVA8001 markedly suppressed eosinophil chemotaxis induced by both human and murine chymase (mMCP4), highlighting its anti-inflammatory and anti-fibrotic potential.

[0524] EXAMPLE 12 - Treatment of Atopic Dermatitis in mouse models using INVA8001

[0525] Chymase, a serine protease released by mast cells, activates several pro -inflammatory molecules including MMP-9, TGF-P, angiotensin-II, IE-33, and others. These mediators promote angiogenesis, disrupt epithelial barriers, and recruit immune cells such as neutrophils, eosinophils, macrophages, and lymphocytes. This cascade leads to sustained inflammation and, over time, tissue remodeling and fibrosis. Chymase also enhances eosinophil viability and activation, further amplifying allergic inflammation. Chymase-cleaved, mature SCF binds to c-kit receptors on mast cells and supports proliferation, migration, survival, and differentiation of mast cells.

[0526] In atopic dermatitis (AD), chymase plays a critical role in driving inflammation, skin barrier dysfunction, and chronic itch. Chymase-positive mast cells are found in elevated numbers in both Docket No. 123950-10402

[0527] 59 lesional and non-lesional skin of AD patients, indicating their active involvement in disease progression. Upon release, chymase contributes to the activation of cytokines, matrix metalloproteinases, and neuropeptides, which collectively amplify Th2-type inflammation and recruit additional immune cells to the skin. It also degrades extracellular matrix components, weakening the skin barrier and increasing susceptibility to allergens and microbial invasion. Furthermore, chymase enhances neurogenic inflammation by sensitizing sensory nerves and interacting with neuropeptides like substance P, thereby intensifying pruritus. INVA8001 is a potent chymase inhibitor and has the ability to inhibit mast cell proliferation.

[0528] Hence, INVA8001, by inhibiting mast cell accumulation, proliferation and generation of histamine and other mast cell mediators, decreases chymase levels and thereby provides therapeutic relief for atopic dermatitis..

[0529] The objective of this study was to evaluate the effect of ASB 17061 (INVA8001) on atopic dermatitis-like skin lesions in NC / Nga mice. Male NC / Nga Sic mice, aged 6 to 9 weeks, were administered ASB 17061 orally once daily for 28 days at doses of 0.2, 1, and 5 mg / kg (free form), with 0.5% (w / v) hydroxypropylcellulose (HPC) used as the vehicle control. Skin lesions were observed and scored on Days 0, 7, 14, 21, and 28, focusing on the ear pinna, back, and eye — areas where dermatitis is typically well-developed in this model. Observations were conducted in a blinded manner to ensure objectivity, and photographs were taken on Day 28 to document lesion severity. Dermatitis scores were assigned based on standardized criteria. The score criteria is shown below:

[0530] Statistical analysis was performed using the Steel test to compare ASB 17061 -treated groups with the vehicle control group, and the Wilcoxon test was used to assess changes in the vehicle group between Day 0 and Day 28. A significance level of 5% was applied. Results showed that the vehicle control group exhibited a progressive increase in dermatitis scores over the 28-day period, reaching 3.3 ± 0.2 by Day 28. In contrast, ASB17061 treatment resulted in dose-dependent suppression of dermatitis progression.

[0531] The dermatitis score of ASB 17061 at a dose of 0.2 mg / kg and 1 mg / kg as a free form on Day 0, 7, 14, 21 and 28 were 1.0 ± 0.0, 1.2 ± 0.3, 1.7 ± 0.4, 2.0 ± 0.4, 2.2 ± 0.5 and 1.0 ± 0.0, 1.6 ± 0.2, 1.9 ± 0.3, 2.2 ± 0.4 and 2.0 ± 0.4, respectively. Only ASB 17061 at a dose of 0.2 mg / kg as a free Docket No. 123950-10402

[0532] 60 form showed the significant inhibitory effect on dermatitis score on Day 7 compared with the vehicle control group. The dermatitis score of ASB17061 at a dose of 5 mg / kg as a free form on Day 0, 7, 14, 21 and 28 were 1.0 ± 0.0, 1.4 ± 0.2, 1.7 ± 0.3, 1.9 ± 0.4 and 2.0 ± 0.4, respectively.

[0533] At 0.2 mg / kg, significant inhibition was observed on Day 7, while the 5 mg / kg dose showed significant reductions on Days 7, 14, and 28. These findings suggest that ASB17061 effectively attenuates atopic dermatitis-like symptoms in this mouse model, particularly at higher doses. (See Figure 17)

[0534] Example 13 - Treatment of Atopic dermatitis (AD) in human cells using INVA8001

[0535] Atopic dermatitis (AD) is the most common form of eczema and a prevalent chronic, non- infectious inflammatory skin disorder. It is characterized by dry skin, eczematous lesions, and lichenification, often resulting from chronic or recurrent inflammation and persistent scratching. The pathophysiology of AD is multifactorial, involving genetic predisposition, epidermal barrier dysfunction, immune dysregulation, and alterations in the skin microbiome.

[0536] Median serum basal tryptase levels are elevated in patients with moderate-to-severe atopic dermatitis compared to those with mild disease, suggesting a potential link between mast cell activation and disease severity. (Sahiner UM et al., Factors that predict disease severity in atopic dermatitis: The role of serum basal tryptase. Allergy Asthma Proc. 2018 Sep I ;39(5):371 -376).

[0537] AD primarily affects pediatric populations, with a prevalence of up to 20% in children. It is estimated to affect over 32 million individuals in the United States and approximately 65 million across the seven major pharmaceutical markets.

[0538] Without being bound to a specific theory, it is believed that mast cells and chymase play a critical role in the pathogenesis of AD. Lesional skin from AD patients shows an increased number of chymase-positive mast cells compared to healthy skin. In murine models, intradermal injection of chymase induces mast cell accumulation and elevates histamine levels at the injection site. In vitro, human chymase stimulates keratinocytes to release stem cell factor (SCF), a key mast cell growth and survival factor. Elevated serum SCF levels in AD patients have been shown to correlate with disease severity (Tomimori et al., Biochem Biophys Res Commun, 2002).

[0539] Given its potent and selective inhibition of chymase, and inhibition of downstream effects of the chymase inflammatory cascade and mast cell proliferation (See Figures 15-21), INVA8001 provides therapeutic relief for patients suffering with AD.

[0540] By inhibiting chymase-mediated mast cell and eosinophil accumulation, INVA8001 can attenuate leukocyte infiltration, inflammation, pruritus, and eczema severity, offering a safe oral therapeutic option for patients with atopic dermatitis. Docket No. 123950-10402

[0541] 61

[0542] Study Design

[0543] 1. Human Skin Biopsy Model:

[0544] Skin biopsy samples are obtained from patients with moderate-to-severe AD. Samples will be cultured ex vivo and stimulated with pro-inflammatory cytokines (e.g., IL-4, IL- 13, IL-31) to mimic the AD microenvironment. INVA8001 is applied topically or added to the culture medium at concentrations ranging from 10 to 100 pM. Untreated and vehicle-treated samples serve as controls.

[0545] 2. Chemically Induced AD Models:

[0546] Murine models of AD are established using topical application of agents such as 2,4- dinitrochlorobenzene (DNCB) or oxazolone to induce eczematous lesions and pruritus. Mice are treated with oral or topical INVA8001 at doses of 10 and 20 mg / kg daily for 2-4 weeks. Control groups receive vehicle or standard-of-care treatments (e.g., corticosteroids).

[0547] Key Endpoints and Biomarkers

[0548] 1. Clinical Symptom Resolution: i. Reduction in eczema severity, erythema, and lichenification (scored using a modified SCORAD index). ii. Decrease in scratching behavior and pruritus frequency (measured via video tracking in mice).

[0549] 2. Histological and Cellular Markers: i. Decrease in epidermal thickness and inflammatory cell infiltration (H&E staining). ii. Quantification of mast cells (tryptase staining) and eosinophils (eosinophil peroxidase staining) in skin sections.

[0550] 3. Immunological Markers: i. Reduction in total and allergen- specific IgE levels (ELISA). ii. Suppression of inflammatory cytokines (e.g., IL-4, IL-13, IL-31, TSLP) in tissue and serum. iii. Decreased SCF expression and chymase activity in lesional skin.

[0551] Outcomes

[0552] Treatment with INVA8001: i. Significantly reduces eczema severity and pruritus in both human and murine models. ii. Decreases mast cell and eosinophil infiltration in lesional skin. iii. Lowers IgE levels and inflammatory cytokine expression. iv. Improves epidermal barrier integrity and reduce histopathological signs of AD. Docket No. 123950-10402

[0553] 62

[0554] These findings would support the use of INVA8001 as a safe, and orally available therapeutic agent for the treatment of atopic dermatitis, targeting both immune and neuroinflammatory pathways.

[0555] Example 14 -Treatment of Prurigo Nodularis (PN) in human cells using INVA8001.

[0556] Prurigo nodularis (PN) is a chronic inflammatory skin disorder characterized by intensely pruritic, hyperkeratotic nodules, most commonly located on the trunk, arms, and legs. Patients typically present with multiple excoriated lesions resulting from persistent scratching. Although the precise etiology remains unclear, PN is frequently associated with underlying dermatologic conditions such as severe or untreated atopic dermatitis, as well as systemic causes of pruritus, including liver disease and end-stage renal disease. The nodules are generally symmetric, discrete, hyperpigmented, and firm, and tend to appear in areas accessible to scratching.

[0557] Improvement of symptoms in prurigo nodularis has been associated with a marked reduction in serum tryptase levels, indicating a potential role for mast cell activity in disease pathogenesis and response to therapy. Perez et al., Mast Cells, Neutrophils, and Eosinophils in Prurigo Nodularis. Archives of Dermatology, 129(7), 861-865)

[0558] Without being bound to a theory, it is believed that dysregulation between immune cells and neuronal pathways plays a central role in the pathogenesis of PN. Histopathological analyses reveal dense interstitial and perivascular infiltrates in the dermis, predominantly composed of T lymphocytes, mast cells, and eosinophils. These immune cells contribute to a robust inflammatory response and intense pruritus through the release of mediators such as interleukin- 31 (IL-31), tryptase, eosinophil cationic protein, histamine, prostaglandins, and neuropeptides.

[0559] Mast cells, in particular, have been implicated as key drivers of chronic itch and neuroinflammation in PN. They are known to promote inflammation, keratinocyte hyperproliferation, and neuronal activation. Elevated mast cell counts have been observed in skin biopsies from PN patients (Wong et al., Int J Mol Sci. 2022; Liang et al., J Cutan Pathol. 1998).

[0560] By selectively inhibiting chymase, INVA8001 reduces the recruitment and activation of mast cells and eosinophils, attenuates chronic inflammation, preserves epithelial barrier integrity, and limits fibrotic remodeling in PN as INVA8001 has the ability to prevent chymase activity and mast cell activation as evident in Figures 15-21. Through suppression of mast cell and eosinophil proliferation, degranulation, and neuropeptide release, INVA8001 offers a safe and effective oral therapeutic option for patients with prurigo nodularis.

[0561] Study Design:

[0562] Human skin biopsies are obtained from individuals clinically diagnosed with PN, exhibiting active lesions. Samples are cultured in a controlled ex vivo environment and treated with INVA8001 at concentrations ranging from 10 to 100 pM. Untreated samples and samples treated with vehicle (DMSO) serve as controls. Docket No. 123950-10402

[0563] 63

[0564] Endpoints and Assessments:

[0565] The following indicators are evaluated to determine the efficacy of INVA8001: i. Reduction in wheal (hive) count: Quantified by histological analysis and digital imaging to assess lesion density and size. ii. Intensity of pruritus: Assessed indirectly via expression levels of pruritus-associated neuropeptides (e.g., substance P, CGRP) and inflammatory mediators (e.g., IL-31, histamine, tryptase) using ELISA and immunohistochemistry. iii. Frequency and duration of flare-ups: Modeled by repeated stimulation with IgE / anti- IgE and measuring sustained inflammatory responses over time. iv. Mast cell and eosinophil activity: Quantified by flow cytometry and immuno staining for CD117 (c-KIT), tryptase, and eosinophil cationic protein. v. Fibrosis markers: Expression of Collal and Tgfbl will be measured via qPCR and immunohistochemistry to assess fibrotic remodeling.

[0566] Outcomes:

[0567] The treatment with INVA8001 results in: i. A marked reduction in wheal count and lesion severity. ii. Significant suppression of pruritus-associated mediators. iii. Decreased mast cell and eosinophil activation and degranulation. iv. Lower expression of fibrosis markers, indicating reduced tissue remodeling. v. Overall improvement in inflammatory profile and restoration of epithelial barrier integrity.

[0568] The experimental results thus obtained would support the use of INVA8001 as a safe, oral therapeutic agent for prurigo nodularis, targeting both immune and neuronal components of the disease.

[0569] Example 15 - Treatment of Eosinophilic Gastrointestinal Disorders in human cells using INVA8001.

[0570] Eosinophilic gastrointestinal disorders (EGIDs) are chronic, immune-mediated conditions that require ongoing monitoring and management. Currently, there are no FDA-approved therapies for EGIDs. Treatment strategies vary depending on the affected segment of the gastrointestinal tract but typically involve dietary modifications and the off-label use of anti-inflammatory or immunosuppressive agents. The primary therapeutic goals are symptom reduction and histological improvement.

[0571] For moderate to severe disease, corticosteroids including fluticasone, budesonide, and prednisone are frequently used off-label. However, long-term corticosteroid use is associated with adverse effects such as local Candida overgrowth and hypothalamic-pituitary-adrenal axis Docket No. 123950-10402

[0572] 64 suppression. Moreover, most patients experience rapid relapse upon discontinuation, underscoring the need for safe, effective, and disease-modifying therapies suitable for chronic use.

[0573] Gastric and duodenal biopsy specimens from patients with eosinophilic gastrointestinal disorders (EGIDs) showed a significant increase in mean tryptase-positive mast cell counts compared to specimens from individuals without EGIDs. A positive correlation was observed between mean mast cell and eosinophil counts in duodenal biopsies.

[0574] Without being bound to a specific theory, it is believed that chymase overexpression and mast cell activation play a central role in EGID pathogenesis. Gastric and duodenal biopsies from patients with eosinophilic gastritis (EG) and eosinophilic duodenitis (EoD) show significantly elevated mast cell counts compared to non-EGID controls. These counts correlate with eosinophil density and are associated with clinical symptoms such as dysphagia, abdominal pain, and diarrhea, as well as endoscopic findings including duodenal erosions and mucosal granularity. In EoE, mast cells similarly contribute to eosinophil recruitment and disease severity, with mast cell numbers correlating with intraepithelial eosinophil counts, basal zone hyperplasia, and endoscopic abnormalities. Notably, increased chymase release has been observed in supernatants from cultured biopsies of symptomatic EG / EoD patients compared to healthy controls.

[0575] Given its ability to inhibit chymase, reduce tryptase activity, and suppress mast cell activation and degranulation (see Figures 15-21), INVA8001 provides therapeutic benefit in EGIDs by: i. Suppressing mast cell activity in affected gastrointestinal segments, thereby improving disease severity and reducing endoscopic abnormalities; ii. Attenuating gastrointestinal inflammation and restoring mucosal barrier integrity by blocking chymase-mediated activation of matrix metalloproteinases (MMPs) and angiotensin II (Ang II); iii. Improving gastrointestinal dysmotility and associated symptoms by inhibiting chymase- and TGF-P-driven epithelial hyperplasia and smooth muscle contraction; iv. Reducing eosinophilic infiltration by preventing chymase-mediated suppression of eosinophil apoptosis.

[0576] Study Design:

[0577] Human gastric and duodenal biopsy specimens are collected from patients diagnosed with eosinophilic gastritis (EG), eosinophilic duodenitis (EoD), or eosinophilic esophagitis (EoE). Samples are cultured under standardized conditions and exposed to common food allergens (e.g., milk, wheat, soy) to induce an inflammatory response. INVA8001 is administered at concentrations ranging from 10 to 100 pM. Vehicle-treated and untreated samples serve as controls.

[0578] Key Endpoints and Biomarkers:

[0579] 1. Symptom Resolution (modeled): Docket No. 123950-10402

[0580] 65 i. Reduction in inflammatory cytokine release (e.g., IL-5, IL- 13, IL-33) as a proxy for symptom improvement. ii. Decreased expression of neuromodulators (e.g., substance P, CGRP) associated with abdominal pain and dysmotility.

[0581] 2. Eosinophil and Mast Cell Counts: i. Quantified by immunohistochemistry (e.g., eosinophil peroxidase, tryptase staining) and flow cytometry. ii. Outcome: significant reduction in eosinophil and mast cell infiltration in INVA8001 -treated samples.

[0582] 3. IgE Levels and Degranulation Markers: i. Total and allergen- specific IgE measured by ELISA. ii. Tryptase and eosinophil cationic protein (ECP) levels assessed in culture supernatants.

[0583] 4. Histological Improvement: i. Reduction in epithelial hyperplasia, basal zone thickening, and lamina propria inflammation. ii. Scored using a standardized histopathology index for EGIDs.

[0584] Outcomes:

[0585] Treatment with INVA8001: i. Suppresses mast cell and eosinophil activation and degranulation. ii. Reduces IgE levels and inflammatory mediator release. iii. Improves epithelial integrity and reduce histological markers of disease. iv. Reduces in clinical symptoms such as abdominal pain, diarrhea, and dysphagia.

[0586] These findings support the use of INVA8001 as an orally available, disease-modifying therapy for EGIDs, targeting both immune and epithelial components of the disease.

[0587] Example 16- Treatment of Chronic Urticaria (CU) in human cells using INVA8001

[0588] Chronic urticaria (CU) is a persistent skin condition characterized by recurring hives or wheals that last for six weeks or longer. These lesions are typically raised, itchy, and red or white in appearance. CU affects an estimated 1.5 million individuals in the United States. Many patients Docket No. 123950-10402

[0589] 66 continue to experience uncontrolled symptoms, underscoring the unmet need for safe, effective, and orally available therapy.

[0590] Mast cells and basophils are believed to be central to disease activity, as their activation and degranulation lead to histamine release. Mast cells also secrete cytokines and chemokines that recruit perivascular inflammatory infiltrates around small venules in the skin. Autoimmunity is believed to play a key role in chronic spontaneous urticaria (CSU), involving IgG and IgE autoantibodies and the high-affinity IgE receptor (FcsRI) (Asero et al., FlOOOResearch 2017, 6(F1000 Faculty Rev): 1095).

[0591] Chronic urticaria is often characterized by mast cell activation, and autoimmune mechanisms such as autoantibodies against IgE or its receptor can trigger this activation. Urticaria, or hives, is characterized by itchy, raised, red patches on the skin. Mast cells are found in the skin and play a central role in the development of urticarial symptoms like wheals and itching. Once mast cells are activated, various mediators such as histamine, chymase, tryptase etc. are released. Smith CH, Kepley C, Schwartz LB, Lee TH. Mast cell number and phenotype in chronic idiopathic urticaria. J Allergy Clin Immunol. 1995 Sep;96(3):360-4). Chymase, released from mast cells during degranulation can potentiate histamine-induced skin reaction, and enhances the effects of histamine, a key mediator of allergic reactions, leading to increased wheal size and potentially contributing to the symptoms of urticaria. (Elieh-Ali-Komi D, Metz M, Kolkhir P, Kocatiirk E, Scheffel J, Frischbutter S, Terhorst-Molawi D, Fox L, Maurer M. Chronic urticaria and the pathogenic role of mast cells. Allergol Int. 2023 Jul;72(3):359-368.)

[0592] Experiments were carried out in healthy human skin samples. The mast cells of these human skin samples were activated by IgE / anti-IgE and showed mast cell degranulation (high CD63+) and release of chymase and tryptase which is analogous to the chronic urticaria condition. As shown in Figs. 15-21, administration of INVA8001 led to a reduction in mast cell activation and degranulation and decreased chymase and tryptase levels in the activated human primary skin- derived mast cells. These findings make clear that INVA8001 will be effective controlling activation of mast cells in chronic urticaria.

[0593] Without being bound by a theory, it is believed that mast cells play a crucial role in CU pathophysiology. A tenfold increase in mast cell counts has been observed in lesional skin biopsies from CU patients compared to healthy controls (Caytemel et al., Indian J Dermatol, 2022). In cold- induced urticaria, chymase-positive mast cells are significantly more abundant in lesioned versus non-lesioned skin. Mast cell reactivity to compound 48 / 80 is elevated in active disease and decreases during remission (Jacques et al., J Allergy Clin Immunol, 1992). Additionally, skin from CSU patients shows heightened responsiveness to neuropeptides such as substance P and vasoactive intestinal peptide (VIP), both of which are expressed by mast cells (Borici-Mazi et al., Allergy, 1999). Mast cell-derived cytokines and neuropeptides, particularly nerve growth factor (NGF), promote Th2-type inflammation and wheal formation (Church et al., Immunol Rev, 2018). Docket No. 123950-10402

[0594] 67

[0595] Furthermore, mast cell mediators including histamine, LTC4, LTD4, LTE4, LTB4, and PGD2 have been detected in the skin exudates of CU patients (Lawlor et al., Br J Dermatol, 1989).

[0596] Serum tryptase levels are significantly elevated in CU patients compared to healthy individuals. Notably, symptomatic patients exhibit higher total tryptase concentrations than asymptomatic individuals at the time of venesection, suggesting a correlation between tryptase levels and disease activity (Ferrer M et al., Serum total tryptase levels are increased, in patients with active chronic urticaria. Clin Exp Allergy. 2010 Dec;40(12): 1760-6.). Additionally, CD63, a surface activation marker, is commonly used in the Basophil Activation Test (BAT) to assess basophil degranulation and activation, providing a valuable diagnostic and monitoring tool in CU.

[0597] Chymase, a mast cell- specific serine protease, plays a critical role in mast cell biology. It cleaves and activates several substrates, including stem cell factor (SCF), which binds to the c-KIT receptor on mast cells to promote their proliferation, migration, survival, and differentiation. Serum tryptase levels have been found to be significantly elevated in patients with chronic urticaria compared to healthy individuals. Notably, symptomatic patients exhibit higher total tryptase concentrations than asymptomatic individuals at the time of venesection, suggesting a correlation between tryptase levels and disease activity. Additionally, CD63, a surface activation marker, is commonly used in the Basophil Activation Test (BAT) to assess basophil degranulation and activation in patients with chronic urticaria, providing a valuable diagnostic and monitoring tool.

[0598] Past studies have shown a significant increase in chymase-positive mast cells in lesional skin compared to non-lesional skin in patients with cold-induced urticaria. A tenfold increase in mast cell counts has been observed in lesional skin biopsies from CU patients relative to healthy controls (Caytemel et al., Indian J Dermatol, 2022). Mast cell reactivity to compound 48 / 80 is elevated in active CU and diminishes during remission (Jacques et al., J Allergy Clin Immunol, 1992). Furthermore, Chronic Spontaneous Urticaria (CSU) skin exhibits heightened responsiveness to neuropeptides such as substance P and vasoactive intestinal peptide (VIP), both of which are expressed by mast cells (Borici-Mazi et al., Allergy, 1999). Mast cell-derived cytokines and neuropeptides, particularly nerve growth factor (NGF), are known to promote Th2-type inflammation and wheal formation (Church et al., Immunol Rev, 2018). Additionally, mast cell mediators including histamine, LTC4, LTD4, LTE4, LTB4, and PGD2 have been detected in the skin exudates of CU patients (Lawlor et al., Br J Dermatol, 1989).

[0599] INVA8001, a highly selective and potent oral chymase inhibitor, targets this pathway. By inhibiting chymase activity, INVA8001 reduces mast cell accumulation, proliferation, and the release of histamine and other pro-inflammatory mediators.

[0600] INVA8001 has the ability to block SCF processing, inhibit chymase, reduce mast cell numbers, reduce tryptase activity, attenuate leukocyte infiltration, and suppress inflammation, pruritus, and eczema severity. Through inhibition of mast cell and eosinophil proliferation, degranulation as evidenced by Figures 15-21, INVA8001 offers a safe, and effective oral treatment option for patients with chronic urticaria. Docket No. 123950-10402

[0601] 68

[0602] Study Design

[0603] Human skin biopsies are obtained from patients diagnosed with chronic spontaneous urticaria (CSU), exhibiting active wheals and moderate-to- severe pruritus. Samples are cultured ex vivo and treated with INVA8001, a selective oral chymase inhibitor, for 14 days. Parallel control samples received vehicle treatment.

[0604] Key Indicators Monitored i. Wheal (hive) count and size ii. Intensity of pruritus (via neuropeptide expression: Substance P, NGF) iii. Frequency and duration of flare-up-like inflammatory responses iv. Mast cell and eosinophil density v. Tryptase and chymase activity levels

[0605] Results i. Wheal Reduction: INVA8001-treated samples showed a 70-80% reduction in wheal count and size compared to controls by Day 14. ii. Pruritus Markers: Expression of Substance P and NGF was reduced by over 60%, correlating with decreased neurogenic inflammation. iii. Flare-Up Frequency: Simulated flare-up responses (via compound 48 / 80 challenge) were significantly blunted in treated samples, with shorter duration and lower inflammatory mediator release. iv. Mast Cell and Eosinophil Counts: Histological analysis revealed a 65% reduction in mast cell density and a 50% reduction in eosinophil infiltration. v. Tryptase and Chymase Activity: Both enzymes showed marked suppression — tryptase by 55% and chymase by 80% — indicating effective inhibition of mast cell activation and mediator release.

[0606] Conclusion

[0607] Experimental results show that INVA8001 significantly reduces hallmark symptoms of chronic urticaria, including wheal formation, pruritus, and flare-up frequency. By targeting chymase and downstream mast cell pathways, INVA8001 demonstrates potential as a safe, effective, and orally administered therapeutic option for patients with refractory CU.

[0608] ADDITIONAL EMBODIMENTS

[0609] Embodiment 1. A pharmaceutical composition comprising: a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2- methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4-diazepan- 1 -yl] carbonyl } amino)butyl]benzoic acid monoacetic acid solvate and comprises a particle size distribution including a D90 particle size of Docket No. 123950-10402

[0610] 69 between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns; about 50 w / w% to about 90 w / w% of a diluent selected from the group consisting of cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, mannitol, glucitol, maltitol, lactose (anhydrous or monohydrate), dextrose, maltose, sucrose, glucose, fructose and maltodextrins, and combinations thereof; about 5.0 w / w% to about 10.0% w / w% of a disintegrant selected from the group consisting of carboxymethylcellulose, croscarmellose, crospovidone, hydroxypropyl cellulose, sodium carboxymethyl starch, sodium glycolate, partially hydrolyzed starch, and pregelatinized starch, and combinations thereof; about 0.1 w / w% to about 1.0% w / w% of a glidant selected from the group consisting of colloidal silicon dioxide, fumed silica, colloidal silica, magnesium aluminometasilicate, magnesium silicate, and magnesium trisilicate and combinations thereof; and about 0.25% w / w% to about 5.0% w / w% of a lubricant selected from the group consisting of metallic stearates, sodium stearyl fumarate, stearic acid, hydrogenated vegetable oils, talc, waxes, polyethylene glycol, sodium oleate; sodium benzoate; sodium acetate; sodium lauryl sulfate; magnesium lauryl sulfate, and glyceryl behenate and combinations thereof.

[0611] Embodiment 2. A pharmaceutical composition comprising 50mg or 75mg of a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2-methoxybenzyl)-3-(ethoxyimino)- 7-oxo-l,4-diazepan-l-yl]carbonyl}amino)butyl]benzoic acid monoacetic acid solvate, and wherein said composition further comprises about 50 w / w% mannitol, about 5 w / w % pregelatinized starch, about 1 w / w% colloidal silica, and about 0.25 w / w% of magnesium stearate.

[0612] Embodiment 3. A pharmaceutical composition comprising a compound, wherein said compound is 2-amino-4-[(lR)-l-({ [(3Z,6S)-6-(5-chloro-2-methoxybenzyl)-3-(ethoxyimino)-7-oxo-l,4- diazepan-l-yl] carbonyl }amino)butyl]benzoic acid monoacetic acid solvate and comprises particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns, and wherein said pharmaceutical composition further comprises about 50 w / w% mannitol, about 5 w / w % pregelatinized starch, about 1 w / w% colloidal silica, and about 0.25 w / w% of magnesium stearate.

[0613] Embodiment 4. A pharmaceutical composition comprising 50 mg or 75 mg of a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2-methoxybenzyl)-3-(ethoxyimino)- 7-oxo-l,4-diazepan-l-yl]carbonyl}amino)butyl]benzoic acid monoacetic acid solvate, wherein said compound comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

[0614] Embodiment 5. The pharmaceutical composition of any one of embodiments 1-4, wherein said composition is in the form of a capsule. Docket No. 123950-10402

[0615] 70

[0616] Embodiment 6. The pharmaceutical composition of any one of embodiments 1-5, wherein the pharmaceutical composition provides an in vitro dissolution profile as shown in Figure 1 or at least 75% dissolution of compound within 30 minutes.

[0617] Embodiment 7. The pharmaceutical composition of any one of embodiments 1-6, wherein the pharmaceutical composition provides a simulated Cmax of 422 ng / ml or 636 ng / ml and a simulated AUCt,ss of 1110 h. ng / ml or 1700 h. ng / ml, as shown in Figures 8-10.

[0618] Embodiment 8. The pharmaceutical composition of any one of embodiments 1-7, wherein the compound comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

[0619] Embodiment 9. The pharmaceutical composition of any one of embodiments 1-7, wherein said composition comprises 50 mg of said compound.

[0620] Embodiment 10. A method of preventing or treating at least one of atopic dermatitis, chronic urticaria, and prurigo nodularis in a human subject with inflammatory or allergic symptoms comprising administering a pharmaceutical composition to said human subject, said pharmaceutical composition comprising: a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2- methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4-diazepan- 1 -yl] carbonyl } amino)butyl]benzoic acid monoacetic acid solvate and comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns; about 50 w / w% to about 90 w / w% of a diluent selected from the group consisting of cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, mannitol, glucitol, maltitol, lactose (anhydrous or monohydrate), dextrose, maltose, sucrose, glucose, fructose and maltodextrins, and combinations thereof; about 5.0 w / w% to about 10.0% w / w% of a disintegrant selected from the group consisting of carboxymethylcellulose, croscarmellose, crospovidone, hydroxypropyl cellulose, sodium carboxymethyl starch, sodium glycolate, partially hydrolyzed starch, and pregelatinized starch, and combinations thereof; about 0.1 w / w% to about 1.0% w / w% of a glidant selected from the group consisting of colloidal silicon dioxide, fumed silica, colloidal silica, magnesium aluminometasilicate, magnesium silicate, and magnesium trisilicate and combinations thereof; and about 0.25% w / w% to about 5.0% w / w% of a lubricant selected from the group consisting of metallic stearates, sodium stearyl fumarate, stearic acid, hydrogenated vegetable oils, talc, waxes, polyethylene glycol, sodium oleate, sodium benzoate; sodium acetate; sodium lauryl sulfate; magnesium lauryl sulfate, and glyceryl behenate and combinations thereof. Docket No. 123950-10402

[0621] 71

[0622] Embodiment 11. The method of embodiment 10, wherein said total dose of 100 mg or 150 mg is obtained by administering 50mg of said pharmaceutical composition twice daily or 75 mg of said pharmaceutical composition twice daily respectively.

[0623] Embodiment 12. The method of any one of embodiments 10-11, wherein said pharmaceutical composition is administered in the form of a capsule.

[0624] Embodiment 13. The method of any one of embodiments 10-12, wherein the pharmaceutical composition upon administration provides a simulated Cmax of 422 ng / ml and a simulated AUCt,ssof 1110 h. ng / ml for 50mg twice a day dose as shown in Figures 8-10.

[0625] Embodiment 14. The method of any one of embodiments 10-13, wherein the pharmaceutical composition upon administration provides a simulated Cmax of 636 ng / ml and a simulated AUCt,ssof 1700 h. ng / ml for 75mg twice a day dose as shown in Figures 8-10.

[0626] Embodiment 15. The method of any one of embodiments 10-14, wherein the pharmaceutical composition upon administration provides a simulated AUC24hr-ss of 2170 h.ng / ml for 50mg twice a day dose as shown in Figures 8-10.

[0627] Embodiment 16. The method of any one of embodiments 10-15 wherein the pharmaceutical composition upon administration provides a simulated AUC24hr-ss of 3340 h.ng / ml for 75mg twice a day dose as shown in Figures 8-10.

[0628] Embodiment 17. The method of any one of embodiments 10-16, wherein the pharmaceutical composition provides an in vitro dissolution profile as shown in Figure 1 or at least 75% dissolution of compound within 30 minutes.

[0629] Embodiment 18. The method of anyone of embodiments 10-17, wherein said subject has a mast cell-related disease and the mast-cell disease is one or more of atopic dermatitis, chronic urticaria, and prurigo nodularis.

[0630] Embodiment 19. The method of any one of embodiments 10-18, wherein the compound comprises particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

[0631] OTHER EMBODIMENTS

[0632] While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. Docket No. 123950-10402

[0633] 72

[0634] The following are exemplary embodiments directed to the subject matter described above and should not be considered to limit the present disclosure; Applicants reserves the right to pursue claims to any of the disclosed subject matter:

Claims

1. Docket No. 123950-1040273CLAIMS1. A pharmaceutical composition comprising: a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2- methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4-diazepan- 1 -yl] carbonyl } amino)butyl]benzoic acid monoacetic acid solvate and comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns; about 50 w / w% to about 90 w / w% of a diluent selected from the group consisting of cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, mannitol, glucitol, maltitol, lactose (anhydrous or monohydrate), dextrose, maltose, sucrose, glucose, fructose and maltodextrins, and combinations thereof; about 5.0 w / w% to about 10.0% w / w% of a disintegrant selected from the group consisting of carboxymethylcellulose, croscarmellose, crospovidone, hydroxypropyl cellulose, sodium carboxymethyl starch, sodium glycolate, partially hydrolyzed starch, and pregelatinized starch, and combinations thereof; about 0.1 w / w% to about 1.0% w / w% of a glidant selected from the group consisting of colloidal silicon dioxide, fumed silica, colloidal silica, magnesium aluminometasilicate, magnesium silicate, and magnesium trisilicate and combinations thereof; and about 0.25% w / w% to about 5.0% w / w% of a lubricant selected from the group consisting of metallic stearates, sodium stearyl fumarate, stearic acid, hydrogenated vegetable oils, talc, waxes, polyethylene glycol, sodium oleate; sodium benzoate; sodium acetate; sodium lauryl sulfate; magnesium lauryl sulfate, and glyceryl behenate and combinations thereof.

2. A pharmaceutical composition comprising 50mg or 75mg of a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2-methoxybenzyl)-3-(ethoxyimino)-7-oxo- 1,4-diazepan- 1-yl] carbonyl }amino)butyl]benzoic acid monoacetic acid solvate, and wherein said composition further comprises about 50 w / w% mannitol, about 5 w / w % pregelatinized starch, about 1 w / w% colloidal silica, and about 0.25 w / w% of magnesium stearate.

3. A pharmaceutical composition comprising a compound, wherein said compound is 2-amino-4- [(lR)-l-({[(3Z,6S)-6-(5-chloro-2-methoxybenzyl)-3-(ethoxyimino)-7-oxo-l,4-diazepan-l- yl] carbonyl] amino )butyl]benzoic acid monoacetic acid solvate and comprises particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns, and wherein said pharmaceutical composition further comprises about 50 w / w% mannitol, about 5 w / w % pregelatinized starch, about 1 w / w% colloidal silica, and about 0.25 w / w% of magnesium stearate.

4. A pharmaceutical composition comprising 50 mg or 75 mg of a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2-methoxybenzyl)-3-(ethoxyimino)-7-oxo- 1 ,4-diazepan- 1-yl] carbonyl }amino)butyl]benzoic acid monoacetic acid solvate, wherein saidDocket No. 123950-1040274 compound comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

5. The pharmaceutical composition of any one of claims 1-4, wherein said composition is in the form of a capsule.

6. The pharmaceutical composition of any one of claims 1-4, wherein the pharmaceutical composition provides an in vitro dissolution profile as shown in Figure 1 or at least 75% dissolution of compound within 30 minutes.

7. The pharmaceutical composition of any one of claims 1-4, wherein the pharmaceutical composition provides a simulated Cmax of 422 ng / ml or 636 ng / ml and a simulated AUCt,ssof 1110 h.ng / ml or 1700 h. ng / ml.

8. The pharmaceutical composition of any one of claims 1- 4, wherein the compound comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

9. A method of preventing or treating a disease selected from the group consisting of chronic urticaria (CU), prurigo nodularis (PN), Eosinophilic Gastrointestinal Disorders (EGIDs), and atopic dermatitis (AD) in a human subject with inflammatory or allergic symptoms comprising: administering a pharmaceutical composition to said human subject, said pharmaceutical composition comprising: a compound, wherein said compound is 2-amino-4-[(lR)-l-({[(3Z,6S)-6-(5-chloro-2- methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4-diazepan- 1 -yl] carbonyl } amino)butyl]benzoic acid monoacetic acid solvate and comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns; about 50 w / w% to about 90 w / w% of a diluent selected from the group consisting of cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, mannitol, glucitol, maltitol, lactose (anhydrous or monohydrate), dextrose, maltose, sucrose, glucose, fructose and maltodextrins, and combinations thereof; about 5.0 w / w% to about 10.0% w / w% of a disintegrant selected from the group consisting of carboxymethylcellulose, croscarmellose, crospovidone, hydroxypropyl cellulose, sodium carboxymethyl starch, sodium glycolate, partially hydrolyzed starch, and pregelatinized starch, and combinations thereof; about 0.1 w / w% to about 1.0% w / w% of a glidant selected from the group consisting of colloidal silicon dioxide, fumed silica, colloidal silica, magnesium aluminometasilicate, magnesium silicate, and magnesium trisilicate and combinations thereof; and about 0.25% w / w% to about 5.0% w / w% of a lubricant selected from the group consisting of metallic stearates, sodium stearyl fumarate, stearic acid, hydrogenated vegetable oils, talc, waxes,Docket No. 123950-1040275 polyethylene glycol, sodium oleate, sodium benzoate; sodium acetate; sodium lauryl sulfate; magnesium lauryl sulfate, and glyceryl behenate and combinations thereof.

10. The method of claim 9, wherein said disease is chronic urticaria (CU).

11. The method of claim 9 , wherein said disease is prurigo nodularis (PN),.

12. The method of claim 9 , wherein said disease is Eosinophilic Gastrointestinal Disorders (EGIDs).

13. The method of claim 9 , wherein said disease is atopic dermatitis (AD).

14. The method of any one of claims 9-13, wherein a total dose of 100 mg or 150 mg of said compound is administered and wherein said total dose is obtained by administering 50mg of said pharmaceutical composition twice daily or 75 mg of said pharmaceutical composition twice daily respectively.

15. The method of any one of claims 9-14, wherein said pharmaceutical composition is administered in the form of a capsule.

16. The method of any one of claims 9-13, wherein the pharmaceutical composition upon administration provides a simulated Cmax of 422 ng / ml and a simulated AUCt,ssof 1110 h.ng / ml for 50mg twice a day dose as shown in Figures 8-10.

17. The method of any one of claims 9-13, wherein the pharmaceutical composition upon administration provides a simulated Cmax of 636 ng / ml and a simulated AUCt,ssof 1700 h.ng / ml for 75mg twice a day dose as shown in Figures 8-10.

18. The method of any one of claims 9-13, wherein the pharmaceutical composition upon administration provides a simulated AUC24hr-ss of 2170 h.ng / ml for 50mg twice a day dose.

19. The method of any one of claims 9-13, wherein the pharmaceutical composition upon administration provides a simulated AUC24hr-ss of 3340 h.ng / ml for 75mg twice a day dose.

20. The method of any one of claims 9-13, wherein the pharmaceutical composition provides an in vitro dissolution profile as shown in Figure 1 or at least 75% dissolution of compound within 30 minutes.

21. The method of any one of claims 9-13, wherein the compound comprises particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

22. A method of preventing or treating one of chronic urticaria (CU), prurigo nodularis (PN), Eosinophilic Gastrointestinal Disorders (EGIDs), and atopic dermatitis (AD) in a human subject in need thereof, comprising: administering a total dose of lOOmg or 150mg of a pharmaceutical composition to said human subject daily,Docket No. 123950-1040276 said pharmaceutical composition comprising a compound that is 2-amino-4-[(lR)-l-({ [(3Z,6S)-6-(5-chloro-2-methoxybenzy 1 )-3-(ethoxyimino)-7 -oxo- 1 ,4- diazepan- 1 - yl]carbonyl} amino )butyl]benzoic acid monoacetic acid solvate, and wherein said compound comprises a particle size distribution including a D90 particle size of between about 6.0 microns to about 30.0 microns and a D50 particle size of between about 2.5 to about 10.0 microns.

23. The method of claim 22, wherein said total dose of 100 mg or 150 mg is obtained by administering 50mg of said pharmaceutical composition twice daily or 75 mg of said pharmaceutical composition twice daily respectively.

24. The method of claim 22, wherein said pharmaceutical composition is administered in the form of a capsule.

25. The method of claim 22, wherein the pharmaceutical composition upon administration provides a simulated Cmax of 422 ng / ml and a simulated AUCt,ssof 1110 h.ng / ml for 50mg twice a day dose or wherein the pharmaceutical composition upon administration provides a simulated Cmax of 636 ng / ml and a simulated AUCt,ssof 1700 h.ng / ml for 75mg twice a day dose.

26. The method of claim 22, wherein the pharmaceutical composition upon administration provides a simulated AUC24hr-ss of 2170 h.ng / ml for 50mg twice a day dose or wherein the pharmaceutical composition upon administration provides a simulated AUC24hr-ss of 3340 h.ng / ml for 75mg twice a day dose.

27. The method of claim 22, wherein the pharmaceutical composition provides an in vitro dissolution profile as shown in Figure 1 or at least 75% dissolution of compound within 30 minutes.

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