IL-22 Oral, Rectal, or Other Gastrointestinal-Related Compositions and Methods of Using Same

Abstract

The present invention provides compositions comprising a protein and an absorption enhancer, methods of treating inflammatory disorders such as irritable bowel disease (IBD) comprising administering the same, and methods of oral, rectal, or other gastrointestinal administration of an enzymatically active protein, comprising administering the protein orally, rectally, or other gastrointestinal administration.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 895,179, filed September 3, 2019, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to oral, rectal, or other gut-related compositions comprising a protein, an absorption enhancer, and optionally a protease inhibitor, and methods of administering the same. In particular, the present invention relates to oral, rectal, or other gut-related compositions containing IL-22 or IL-22-Fc, and their use in the treatment of inflammatory disorders such as irritable bowel disease (IBD). [Background technology]

[0003] Interleukin-22 (IL-22) is a class II cytokine upregulated in T cells. One function of IL-22 is to enhance innate immunity in peripheral tissues by inducing the expression of antimicrobial peptides (Wolk et al., Immunity, 21:241-54, 2004; Boniface et al., J. Immunol., 174:3695-3702, 2005). Other studies have shown that IL-22 mRNA expression is induced in vivo in response to LPS administration and that IL-22 regulates parameters indicative of the acute phase response (Dumoutier L. et al., Genes Immunol., 1(8):488-494, (2000); Pittman et al., Genes and Immunity, 2:172, 2001). Collectively, these findings indicate that IL-22 plays a role in inflammation (Kotenko SV, Cytokine & Growth Factor Reviews, 13(3):223-40, 2002). Several T cell disorders are associated with increased levels of IL-22 (Wolk et al., Immunity, 21:241-54, 2004; Ikeuchi H. et al., Arthritis Rheum., 52:1037-1046, 2005; Andoh, A. et al., Gastroenterology, 129:969-984, 2005). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Wolk et al.,Immunity,21:241-54,2004 [Non-patent document 2] Boniface et al., J. Immunol., 174:3695-3702, 2005 [Non-patent document 3] Dumoutier L. et al., Genes Immunol.,1(8):488-494,(2000) [Non-patent document 4] Pittman et al.,Genes and Immunity,2:172,2001 [Non-Patent Document 5] Kotenko SV,Cytokine & Growth Factor Reviews,13(3):223-40,2002 [Non-patent document 6] Ikeuchi H. et al.,Arthritis Rheum.,52:1037-1046,2005 [Non-Patent Document 7] Andoh, A. et al., Gastroenterology, 129:969-984, 2005 Summary of the Invention [Problem to be solved by the invention]

[0005] Currently, there are limitations to the administration of IL-22 for the treatment of inflammatory diseases. The present invention addresses the need for alternative methods of administering IL-22. [Means for solving the problem]

[0006] The present invention is based, in part, on oral, rectal, or other gastrointestinal-related pharmaceutical compositions comprising an IL-22 or IL-22-Fc protein and an absorption enhancer selected from the group consisting of N(8-[2-hydroxybenzoyl]amino)caprylic acid (NAC), N-(10-[2-hydroxybenzoyl]amino)decanoic acid (NAD), or a salt of the NAC or NAD. In some embodiments, the IL-22 has at least 80% identity to SEQ ID NO: 1, 2, 3, or 4. In some embodiments, the absorption enhancer is N-(8-[2-hydroxybenzoyl]amino)caprylic acid. In some embodiments, the salt of the NAC or NAD is selected from the group consisting of a monosodium salt, a disodium salt, and combinations thereof. In some embodiments, the composition further comprises a protease inhibitor selected from the group consisting of a serpin, a suicide inhibitor, a transition state inhibitor, a protein protease inhibitor, a chelating agent, a cysteine ​​protease inhibitor, a threonine protease inhibitor, an aspartic acid protease inhibitor, and a metalloprotease inhibitor. In some embodiments, the composition further comprises EDTA or a salt thereof, and / or a coating that inhibits digestion of the composition in the subject's stomach. Enteric coated It further includes a coating or gelatin coating.

[0007] In some embodiments, the invention is an Fc-fusion protein of any of the above compositions.

[0008] In some embodiments, the invention is a method for treating an inflammatory disorder in a patient in need thereof, comprising administering to the patient any one of the oral, rectal, or other gastrointestinal-related pharmaceutical compositions described above, or any one of the Fc-fusion proteins described above. In some embodiments, the invention comprises an inflammatory disorder selected from inflammatory bowel disorder, Crohn's disease, ulcerative colitis, type 2 diabetes, type 2 diabetes with morbid obesity, wounds (including diabetic wounds and diabetic ulcers), burns, ulcers (including pressure ulcers and venous ulcers), graft versus host disease (GVHD), microbial infections, acute kidney injury, acute pancreatitis, cardiovascular conditions, metabolic syndrome, acute endotoxemia, sepsis, atherosclerosis, cardiovascular disease, endotoxemia (acute and mild), acute coronary heart disease, hypertension, dyslipidemia, obesity, hyperglycemia, lipid metabolism disorders, hepatitis, acute hepatitis, renal failure, acute renal failure, acute kidney injury, renal transplant failure, post-renal transplant dysfunction, contrast-induced nephropathy, pancreatitis, acute pancreatitis, hepatic fibrosis, and pulmonary fibrosis. [Brief explanation of the drawings]

[0009] [Figure 1] Serum levels of (A) IL-22 / SNAC mixture or (B) serum amyloid A are shown. [Figure 2] (A) Serum levels of IL-22 / SNAC mixture or (B) expression of RegIIIβ in the colon of individual mice are shown. [Figure 3] The levels of IL-22 retained in the colon versus systemically absorbed IL-22 from Fc-bound IL-22 (FIG. 3A) and IL-22 alone (FIG. 3B) are shown. [Figure 4] The differences in systemic versus colonic IL-22 levels following intrarectal (FIG. 4A) and intraperitoneal (FIG. 4B) administration are shown. [Figure 5] Figure 5 shows the levels of systemic serum amyloid acid (SAA) or gastrointestinal RegIIIβ after intraperitoneal and intrarectal administration of mouse Fc-IL-22. Figure 5A shows systemic SAA levels, and Figure 5B shows gastrointestinal RegIIIβ levels. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention is directed to compositions and methods for oral, rectal, or other gut-related administration of IL-22. Throughout this application, IL-22 comprises an Fc fusion protein, IL-22-Fc. In some embodiments, IL-22 can be in an oral, rectal, or other gut-related pharmaceutical composition with an absorption enhancer. In some embodiments, IL-22 can be in an oral, rectal, or other gut-related pharmaceutical composition with a protease inhibitor and an absorption enhancer. In some embodiments, the absorption enhancer increases absorption of IL-22 across the intestinal mucosal layer. In some embodiments, the present invention is a method of treating a patient with an inflammatory disorder by administering any of the oral, rectal, or other gut-related pharmaceutical compositions described above and in further detail below. In some embodiments, the inflammatory disorder can be, for example, irritable bowel disease (IBD).

[0011] In another embodiment, the compositions of the invention comprise IL-22, an absorption enhancer, and a carrier. In another embodiment, the compositions of the invention comprise IL-22, a protease inhibitor, and a carrier. In another embodiment, the compositions of the invention are used to deliver IL-22 through various biological, chemical, and physical barriers that can reduce IL-22 degradation. In another embodiment, the compositions comprise IL-22 and SNAC. In another embodiment, the compositions comprise IL-22, SNAC, a protease inhibitor, and EDTA or Na-EDTA. In another embodiment, the compositions comprise IL-22, SNAC, a protease inhibitor, and an omega-3 fatty acid. In another embodiment, the compositions comprise IL-22, SNAC, a protease inhibitor, EDTA or Na-EDTA, and an omega-3 fatty acid. In another embodiment, the compositions are oral, rectal, or other gastrointestinal-related pharmaceutical compositions. In another embodiment, the compositions described herein are oral, rectal, or other gastrointestinal-related encapsulated pharmaceutical compositions. In another embodiment, the compositions described herein are in an oral, rectal, or other gastrointestinal liquid dosage form. In another embodiment, the compositions described herein are in an oral, rectal, or other gastrointestinal dry dosage form (such as a tablet).

[0012] Interleukin-22 (IL-22) is a member of the IL-10 family of cytokines produced by Th22 cells, NK cells, lymphoid tissue inducer (LTi) cells, dendritic cells, and Th17 cells. IL-22 binds to the IL-22R1 / IL-10R2 receptor complex, which is expressed on innate cells such as epithelial cells, hepatocytes, and keratinocytes, as well as on barrier epithelial tissues of several organs, including the dermis, pancreas, intestine, and respiratory system. IL-22 is known to regulate host defense and tissue regeneration, and treatment with IL-22 was effective in treating dextran sulfate sodium (DSS)-induced colitis in mice.

[0013] In certain embodiments, an IL-22 polypeptide comprises the amino acid sequence of human IL-22 SEQ ID NO: 1 (precursor protein) or SEQ ID NO: 2. In some embodiments, an IL-22 polypeptide comprises an amino acid sequence having 80% identity to SEQ ID NO: 1 or 2. In some embodiments, an IL-22 polypeptide comprises an amino acid sequence having 90% identity to SEQ ID NO: 1 or 2. In some embodiments, an IL-22 polypeptide comprises an amino acid sequence having 95% identity to SEQ ID NO: 1 or 2. In some embodiments, an IL-22 polypeptide comprises an amino acid sequence having 99% identity to SEQ ID NO: 1 or 2. The human IL-22 amino acid sequences of SEQ ID NOs: 1 and 2 are as follows: Human IL-22 precursor sequence (Q9GZX6): [ka] Human IL-22 sequence (Q9GZX6): [ka]

[0014] In certain embodiments, the IL-22 polypeptide comprises the amino acid sequence of murine IL-22 SEQ ID NO: 3 (precursor) or SEQ ID NO: 4. In some embodiments, the IL-22 polypeptide comprises an amino acid sequence having 80% identity to SEQ ID NO: 3 or 4. In some embodiments, the IL-22 polypeptide comprises an amino acid sequence having 90% identity to SEQ ID NO: 3 or 4. In some embodiments, the IL-22 polypeptide comprises an amino acid sequence having 95% identity to SEQ ID NO: 3 or 4. In some embodiments, the IL-22 polypeptide comprises an amino acid sequence having 99% identity to SEQ ID NO: 3 or 4. The murine IL-22 amino acid sequences of SEQ ID NOs: 3 and 4 are as follows: Mouse IL-22 precursor sequence: [ka] Mouse IL-22 sequence: [ka]

[0015] IL-22 plays an important role in mucosal immunity by mediating early host defense against the attachment and clearance of bacterial pathogens. See Zheng et al., 2008, Nat. Med. 14:282-89. IL-22 promotes the production of antimicrobial peptides and proinflammatory cytokines from epithelial cells and stimulates the proliferation and migration of colonic epithelial cells in the gastrointestinal tract. See Kumar et al., 2013, J. Cancer, 4:57-65. IL-22 knockout mice exhibit impaired gastrointestinal epithelial regeneration, higher bacterial loads, and increased mortality after bacterial infection. Kumar et al., supra. Similarly, influenza virus infection in IL-22 knockout mice led to severe weight loss and impaired regeneration of tracheal and bronchial epithelial cells. Thus, IL-22 plays a pro-inflammatory role in the suppression of microbial infection and an anti-inflammatory protective role in epithelial regeneration during inflammatory responses. Many of the biological functions of IL-22, which promote pathological inflammation and tissue repair, remain unclear. The seemingly contradictory reports regarding the effects of IL-22 on epithelial cells have yet to be fully elucidated. Kumar et al., op. cit.

[0016] Increased expression of IL-22 has been detected in patients with inflammatory bowel disorders (IBD). See, e.g., Wolk et al., 2007, J. Immunology, 178:5973; Andoh et al., 2005, Gastroenterology, 129:969. IBD, such as Crohn's disease (CD) and ulcerative colitis (UC), is thought to result from dysregulated immune responses to commensal microbiota present in the gastrointestinal tract. Cox et al., 2012, Mucosal Immunol. 5:99-109. Both UC and CD are complex diseases that occur in genetically susceptible individuals exposed to yet-to-be-defined environmental stimuli. CD and UC are mediated by both common and distinct mechanisms and exhibit distinct clinical characteristics. See Sugimoto et al., 2008, J. Clinical Investigation, 1 18:534-544. Mucosal healing is a current targeted approach for treating IBD and can be induced by IL-22, which induces activation of STAT3, serum amyloid A, and RegIIIβ.

[0017] In UC, inflammation first develops in the mucosa of the colon and rectum, leading to debilitating conditions including diarrhea, rectal bleeding, and weight loss. UC is thought to be largely caused by an inappropriate inflammatory response by the host to invading intestinal microorganisms that penetrate a damaged epithelial barrier (Xavier and Podolsky, 2007, Nature 448:427-434). Crohn's disease is characterized by infiltration of activated immune cells into the intestine and distortion of intestinal architecture. See Wolk et al., supra.

[0018] In another aspect, the present invention provides methods of treating any one or combination of the following diseases using an IL-22 polypeptide or IL-22Fc fusion protein of the present invention: type 2 diabetes, type 2 diabetes with morbid obesity, wounds (including diabetic wounds and diabetic ulcers), burns, ulcers (including pressure ulcers and venous ulcers), graft versus host disease (GVHD), microbial infections, acute kidney injury, acute pancreatitis, cardiovascular conditions, metabolic syndrome, acute endotoxemia, sepsis, atherosclerosis, cardiovascular disease, endotoxemia (acute and mild), acute coronary heart disease, hypertension, dyslipidemia, obesity, hyperglycemia, lipid metabolism disorders, hepatitis, acute hepatitis, renal failure, acute renal failure, acute kidney injury, renal transplant failure, post-renal transplant dysfunction, contrast-induced nephropathy, pancreatitis, acute pancreatitis, hepatic fibrosis, and pulmonary fibrosis. In certain embodiments, acute pancreatitis can be mild to moderate or severe disease. In certain embodiments, acute pancreatitis includes post-ERCP (endoscopic retrograde cholangiopancreatography) disease. In some further embodiments, a patient being treated for the above diseases is in need of a change in HDL / LDL lipid profile, and an IL-22 polypeptide or an IL-22Fc fusion protein can alter that profile in the patient to increase HDL and decrease LDL. In a related aspect, the invention provides use of an IL-22 polypeptide or an IL-22Fc fusion protein in the preparation of a medicament for the treatment of any one or a combination of the above diseases.

[0019] In one aspect, the invention features an IL-22Fc fusion protein comprising an IL-22 polypeptide linked to an Fc region. In some embodiments, the IL-22 polypeptide is linked to the Fc region by a linker. In some embodiments, the IL-22 polypeptide is glycosylated, and the IL-22Fc fusion protein has a sialic acid content ranging from 8 to 12 moles of sialic acid per mole of IL-22Fc fusion protein. In certain aspects, 8 to 12 moles of sialic acid per mole of IL-22Fc fusion protein means that 8 to 12 sialic acid moieties are contained in 1 mole of IL-22 fusion protein. In some embodiments, the IL-22Fc fusion protein has a sialic acid content ranging from 8 to 9 moles of sialic acid per mole of IL-22Fc fusion protein.

[0020] The term "Fc polypeptide" or "Fc region," as used herein, includes native and mutein forms of polypeptides derived from the Fc region of an antibody. It also includes truncated forms of such polypeptides containing the hinge region that promotes dimerization. In certain embodiments, the Fc region comprises the CH2 and CH3 domains of an antibody. Fusion proteins (and oligomers formed therefrom) comprising an Fc portion offer the advantages of extended serum half-life as well as easy purification by affinity chromatography on Protein A or Protein G columns. Preferred Fc regions are derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. As used herein, specific residues within the Fc are identified by position. All Fc positions are based on the EU numbering scheme.

[0021] One function of the Fc portion of an antibody is to signal to the immune system when the antibody binds to its target. This is considered an "effector function." This signaling can lead to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated through binding of Fc to Fc receptors on the surface of cells of the immune system. CDC is mediated through binding of Fc to proteins of the complement system, such as C1q.

[0022] IgG subclasses vary in their ability to mediate effector functions. For example, IgG1 is far superior to IgG2 and IgG4 in mediating ADCC and CDC. Therefore, in embodiments where effector function is undesirable, IgG2 Fc is preferred. However, molecules containing IgG2 Fc are known to be more difficult to manufacture and have less attractive biophysical properties, including a shorter half-life, compared to molecules containing IgG1 Fc.

[0023] By introducing one or more mutations into the Fc, the effector function of the antibody can be enhanced or reduced. Embodiments of the present invention include IL-22 mutein Fc fusion proteins having an Fc engineered to enhance effector function (U.S. Pat. No. 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both of which are incorporated herein by reference in their entireties). Exemplary IgG1 with enhanced effector function include: The Fc molecule contains the following substituents: S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; D280H / K290S / S298V; F243L / R292P / Y300L; F243L / R292P / Y300L / P396L; F243L / R292P / Y300L / V305I / P396L; G236A / S239D / I332E; K326A / E333A; K326W / E333S; K290E / S298G / T299A; K290N / S298G / T299A; K290E / S298G / T299A / K326E; or K290N / S298G / T299A / K326E.

[0024] Another method for enhancing the effector function of IgG Fc-containing proteins is by reducing Fc fucosylation. Removal of core fucose from biantennary complex-type oligosaccharides attached to Fc significantly enhanced ADCC effector function without altering antigen binding or CDC effector function. Several methods are known for reducing or eliminating fucosylation of Fc-containing molecules, such as antibodies. These methods include recombinant expression in certain mammalian cell lines, including FUT8 knockout cell lines, mutant CHO line Lec13, rat hybridoma cell line YB2 / 0, cell lines containing small interfering RNA specific for the FUT8 gene, and cell lines coexpressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Alternatively, Fc-containing molecules can be expressed in non-mammalian cells, such as plant cells, yeast, or prokaryotic cells, such as E. coli.

[0025] In some embodiments of the invention, an IL-22-Fc fusion protein comprises an Fc engineered to have reduced effector function. Exemplary Fc molecules with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1).

[0026] Human IgG1 has a glycosylation site at N297 (EU numbering system), and glycosylation is known to contribute to the effector function of IgG1 antibodies. An exemplary IgG1 sequence is shown in SEQ ID NO: 5. To generate aglycosylated antibodies, groups have mutated N297. Mutations have focused on substituting N297 with amino acids similar in physiochemical properties to asparagine, such as glutamine (N297Q), or with alanine (N297A), which mimics asparagine without the polar group.

[0027] As used herein, "aglycosylated antibody" or "aglycosylated fc" refers to the state of glycosylation of Fc at residue 297. An antibody or other molecule may contain glycosylation at one or more other positions and still be considered an aglycosylated antibody or an aglycosylated Fc fusion protein.

[0028] In an attempt to generate an IgG1 Fc lacking effector function, it was found that mutating amino acid N297 of human IgG1 to glycine, i.e., N297G, resulted in significantly superior purification efficiency and biophysical properties compared to other amino acid substitutions at that residue. Thus, in some embodiments, an IL-22-Fc fusion protein comprises a human IgG1 Fc with an N297G substitution.

[0029] Fc, including human IgG1 Fc with an N297G mutation, can also contain further insertions, deletions, and substitutions. In certain embodiments, the human IgG1 Fc contains the N297G substitution and is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 5. In a particularly preferred embodiment, the C-terminal lysine residue is substituted or deleted. The amino acid sequence of human IgG1 containing the N297G substitution and the C-terminal lysine deletion is set forth in SEQ ID NO: 6. [ka] [ka]

[0030] Molecules containing aglycosylated IgG1 Fc have been found to be less stable than molecules containing glycosylated IgG1 Fc. The Fc region can be further engineered to increase the stability of aglycosylated molecules. In some embodiments, one or more amino acids are substituted with cysteine ​​to form disulfide bonds in the dimeric state. Residues V259, A287, R292, V302, L306, V323, or I332 of the amino acid sequence set forth in SEQ ID NO: 3 can be substituted with cysteine. In preferred embodiments, specific pairs of residues are substituted to preferentially form disulfide bonds with each other, thereby limiting or preventing scrambling of the disulfide bonds. Preferred pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C.

[0031] Additional mutations that can be made to IgG1 Fc include those that promote heterodimer formation among Fc-containing polypeptides. In some embodiments, the Fc region is engineered to create "knobs" and "holes" that promote heterodimer formation of two different Fc-containing polypeptide chains when co-expressed in cells. See U.S. Patent No. 7,695,963. In other embodiments, the Fc region is altered to use electrostatic steering to promote heterodimer formation but prevent homodimer formation of two different Fc-containing polypeptide chains when co-expressed in cells. WO 09 / 089,004, the entire contents of which are incorporated herein by reference. Preferred heterodimeric Fc chains include those in which one Fc chain contains D399K and E356K substitutions and the other Fc chain contains K409D and K392D substitutions. In another embodiment, one Fc chain comprises the D399K, E356K, and E357K substitutions, and the other Fc chain comprises the K409D, K392D, and K370D substitutions.

[0032] In yet another aspect, the present invention also provides use of an IL-22 Fc fusion protein described herein in the preparation of a medicament for treating IBD, including UC and CD, in a subject in need thereof. In a related aspect, the present invention provides use of an IL-22 Fc fusion protein described herein in the preparation of a medicament for inhibiting microbial infection in the intestine or maintaining goblet cells in the intestine during microbial infection in a subject in need thereof. In yet another aspect, the present invention provides use of an IL-22 Fc fusion protein described herein in the preparation of a medicament for enhancing epithelial cell integrity, epithelial cell proliferation, epithelial cell differentiation, epithelial cell migration, or epithelial wound healing in the intestine in a subject in need thereof. In another related aspect, the present invention provides use of an IL-22 polypeptide or an IL-22 Fc fusion protein in the preparation of a medicament for accelerating, promoting or improving wound healing, including but not limited to, the treatment of cardiovascular conditions, metabolic syndrome, atherosclerosis, acute kidney injury, acute pancreatitis, healing of chronic wounds, diabetic wounds, infected wounds, pressure ulcers or diabetic foot ulcers, in a subject in need thereof.

[0033] As used herein, IL-22-based compositions (which in certain embodiments comprise an IL-22 Fc fusion protein and an IL-22 polypeptide or agonist) will be formulated, dosed, and administered consistent with sound medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the dosing schedule, and other factors known to a physician. In one embodiment, the composition can be used to extend the survival of a human subject susceptible to or diagnosed with a disease or disease state. Survival is defined as the time from the first administration of the drug to death.

[0034] Pharmaceutical formulations are prepared in the form of lyophilized formulations or aqueous solutions by mixing the active ingredient having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) and Remington's Pharmaceutical Sciences 20th edition, ed. A.F. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.) using standard methods known in the art. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate buffer, citrate buffer, and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 Polypeptides (less than 10 ...Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0035] Optionally, but preferably, the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride, preferably at approximately physiological concentrations. Optionally, the formulations of the present invention can contain a pharmaceutically acceptable preservative. In some embodiments, the concentration of the preservative is 0.1-2.0%, typically in the v / v range. Suitable preservatives include those known in the pharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben, benzalkonium chloride, and propylparaben are preferred preservatives. Optionally, the formulations of the present invention can contain a pharmaceutically acceptable surfactant at a concentration of 0.005-0.02%.

[0036] The present invention provides dosage amounts for IL-22-based therapy. For example, depending on the type and severity of the disease, initial candidate dosages for administration to a subject range from about 1 μg / kg to 15 mg / kg (e.g., 0.1-20 mg / kg) of polypeptide, either in one or more separate administrations or by continuous infusion. Typical daily dosages can range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, treatment is sustained until a desired suppression of disease symptoms is observed, depending on the condition. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0037] The appropriate dosage of a polypeptide of the present invention (whether used alone or in combination with one or more other additional therapeutic agents) for preventing or treating a disease will depend on the type of disease being treated, the type of polypeptide, the severity and cause of the disease, whether the polypeptide is being administered for prophylactic or therapeutic purposes, previous treatments, the subject's medical history and response to the polypeptide, and the judgment of the attending physician. The polypeptide is administered to the subject at one time or over a series of treatments, as appropriate. Depending on the type and severity of the disease, for example, about 1 μg / kg to 20 mg / kg (e.g., 0.1 mg / kg to 15 mg / kg) of polypeptide can be administered to a subject as an initial candidate dose, either in one or more separate administrations or by continuous infusion. A typical daily dosage can range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, treatment will typically be sustained until a desired suppression of disease symptoms is observed, depending on the condition. One exemplary dosage of the polypeptide would be in the range of about 0.05 mg / kg to about 20 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 10 mg / kg, 12 mg / kg, 15 mg / kg, or 20 mg / kg (or any combination thereof) may be administered to a subject. In certain embodiments, about 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg, 6.0 mg / kg, 7.0 mg / kg, 8.0 mg / kg, 9.0 mg / kg, 10 mg / kg, 12 mg / kg, 15 mg / kg, or 20 mg / kg (or any combination thereof) may be administered to a subject. Such doses may be administered intermittently, for example, weekly, every two weeks, or every three weeks (e.g., so that the subject receives from about 2 to about 20 doses, or for example, about 6 doses, of the polypeptide). An initial higher loading dose may be administered, followed by one or more lower doses. An exemplary dosing regimen includes administering an initial loading dose of about 4 mg / kg, followed by weekly maintenance doses of about 2 mg / kg of the antibody. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventional techniques and assays.

[0038] In one aspect, an IL-22 Fc fusion protein is provided for use as a medicament. In a further aspect, an IL-22 Fc fusion protein is provided for use in the treatment of IBD, including UC and CD. In certain embodiments, an IL-22 Fc fusion protein is provided for use in a method of treatment. In certain embodiments, the invention provides an IL-22 Fc fusion protein for use in a method of treating an individual with UC or CD, comprising administering to the individual an effective amount of an IL-22 Fc fusion protein. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further embodiment, the invention provides an IL-22 Fc fusion protein for use in enhancing epithelial proliferation, differentiation, and / or migration. In certain embodiments, the epithelial tissue is intestinal epithelial tissue. In certain embodiments, the invention provides an IL-22 Fc fusion protein for use in a method of enhancing epithelial proliferation, differentiation, and / or migration in an individual, comprising administering to the individual an effective amount of an IL-22 Fc fusion protein to enhance epithelial proliferation, differentiation, and / or migration. In yet another embodiment, the present invention provides an IL-22 Fc fusion protein for use in the treatment of diabetes, particularly type 2 diabetes, diabetic wound healing, metabolic syndrome, and atherosclerosis. In a specific embodiment, the present invention provides an IL-22 Fc fusion protein for use in a method for treating diabetes, particularly type 2 diabetes, diabetic wound healing, metabolic syndrome, and atherosclerosis in an individual, comprising administering to the individual an effective amount of an IL-22 Fc fusion protein.See U.S. Provisional Patent Application No. 61 / 800,795, entitled "Using an IL-22 polypeptide for wound healing," and U.S. Provisional Patent Application No. 61 / 801,144, entitled "Methods of treating cardiovascular conditions and metabolic syndrome using an IL-22 polypeptide," both filed March 15, 2013. The disclosures of both provisional patent applications are incorporated herein by reference in their entireties. An "individual" or "subject" or "patient" according to any of the above embodiments is preferably a human.

[0039] In a further aspect, the present invention provides use of an IL-22 polypeptide or an IL-22 Fc fusion protein in the manufacture or preparation of a medicament. In one embodiment, the medicament is intended for the treatment of IBD and wound healing. In a further embodiment, the medicament is intended for use in a method for the treatment of IBD and wound healing, comprising administering an effective amount of the medicament to an individual with IBD. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further embodiment, the medicament is intended for suppression of an inflammatory response in gastrointestinal epithelial cells. In a further embodiment, the medicament is intended for use in a method for enhancing epithelial proliferation, differentiation, and / or migration in an individual, comprising administering to the individual an effective amount of the agent to enhance epithelial proliferation, differentiation, and / or migration. The "individual" in any of the above embodiments can be a human.

[0040] In a further aspect, the present invention provides methods of treating IBD, including UC and CD. In one embodiment, the method comprises administering to an individual with IBD an effective amount of an IL-22 polypeptide or an IL-22Fc fusion protein. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent described below. The "individual" in any of the above embodiments can be a human.

[0041] As provided herein, a protease inhibitor protects a protein of the present invention from cleavage. In another embodiment, the present invention provides that a protease inhibitor protects an insulin of the present invention from cleavage. In another embodiment, the present invention provides that a protease inhibitor promotes protein absorption in the intestine of a subject. In another embodiment, the present invention provides that a protease inhibitor promotes insulin absorption in the intestine of a subject.

[0042] In another embodiment, the serpin is selected from the group consisting of alpha 1-antitrypsin, antitrypsin-related protein, alpha 1-antichymotrypsin, kallistatin, protein C inhibitor, cortisol-binding globulin, thyroxine-binding globulin, angiotensinogen, centerin, protein Z-related protease inhibitor, vaspin, monocyte neutrophil elastase inhibitor, plasminogen activator inhibitor 2, squamous cell carcinoma antigen-1 (SCCA-1), squamous cell carcinoma antigen-2 (SCCA-2), and the like. ), maspin, PI-6, megsin, PI-8, PI-9, bomapin, yukopin, hurpin / headpin, antithrombin, heparin cofactor II, plasminogen activator inhibitor 1, glial-derived nexin / protease nexin I, pigment epithelium-derived factor, alpha 2-antiplasmin, complement 1-inhibitor, 47 kDa heat shock protein (HSP47), neuroserpin, or pancpin.

[0043] In another embodiment, the present invention provides that the protease inhibitor is a trypsin inhibitor, such as, but not limited to, lima bean trypsin inhibitor, aprotinin, soybean trypsin inhibitor (SBTI), or ovomucoid. In another embodiment, the present invention provides that the protease inhibitor is a cysteine ​​protease inhibitor. In another embodiment, the present invention provides that the cysteine ​​protease inhibitor of the present invention comprises cystatin, type 1 cystatin (or stefin), type 2 cystatin, human cystatin C, D, S, SN, and SA, cystatin E / M, cystatin F, type 3 cystatin, or kininogen. In another embodiment, the present invention provides that the protease inhibitor is a threonine protease inhibitor. In another embodiment, the present invention provides that the threonine protease inhibitor of the present invention comprises bortezomib, MLN-519, ER-807446, or TMC-95A. In another embodiment, the present invention provides that the protease inhibitor is an aspartic protease inhibitor. In another embodiment, the present invention provides that the aspartic protease inhibitor of the present invention comprises α2-macroglobulin, pepstatin A, aspartic protease inhibitor 11, aspartic protease inhibitor 1, aspartic protease inhibitor 2, aspartic protease inhibitor 3, aspartic protease inhibitor 4, aspartic protease inhibitor 5, aspartic protease inhibitor 6, aspartic protease inhibitor 7, aspartic protease inhibitor 8, aspartic protease inhibitor 9, pepsin inhibitor Dit33, aspartyl protease inhibitor, or protease A inhibitor 3. In another embodiment, the present invention provides that the protease inhibitor is a metalloprotease inhibitor. In another embodiment, the present invention provides that the metalloprotease inhibitor of the present invention comprises an angiotensin-1-converting enzyme inhibitor peptide.

[0044] Antihemorrhagic factor BJ46a, beta-casein, proteinase inhibitor CeKI, venom metalloproteinase inhibitor DM43, carboxypeptidase A inhibitor, smpl, IMPI, alkaline proteinase, inh, latexin, carboxypeptidase inhibitor, antihemorrhagic factor HSF, testican-3, SPOCK3, TIMP1, metalloproteinase inhibitor 1, metalloproteinase inhibitor 2, TIMP2, metalloproteinase inhibitor 3, TIMP3, metalloproteinase inhibitor 4, TIMP4, putative metalloproteinase inhibitor tag-225, tissue inhibitor of metalloproteinase, WAP, Kazal, immunoglobulin, or kunitz and NTR domain-containing protein 1.

[0045] In some embodiments, the protease inhibitor is a suicide inhibitor, a transition state inhibitor, or a chelator. In some embodiments, the protease inhibitor of the present invention is AEBSFHCl, (epsilon)-aminocaproic acid, (alpha)1-antichymotrypsin, antipain, antithrombin III, (alpha)1-antitrypsin ([alpha]1-proteinase inhibitor), APMSF-HCl (4-amidinophenyl-methanesulfonyl fluoride), sprotinin, benzamidine-HCl, chymostatin, DFP (diisopropylfluorophosphate), leupeptin, PEFABLOC® SC (4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride), PMSF (phenylmethylsulfonyl fluoride), TLCK (1-chloro-3-tosylamido-7-amino-2-heptanone HCl), TPCK (1-chloro-3-tosylamido-4-phenyl-2-butanone), pentamidine isethionate, pepstatin, guanidinium, alpha-2 macroglobulin, a zinc chelator, or iodoacetic acid, zinc. Each possibility corresponds to a separate embodiment of the present invention.

[0046] In another embodiment, the amount of protease inhibitor utilized in methods and compositions of the present invention is 0.1 mg / dosage unit. In another embodiment, the amount of protease inhibitor is 0.2 mg / dosage unit. In another embodiment, the amount is 0.3 mg / dosage unit. In another embodiment, the amount is 0.4 mg / dosage unit. In another embodiment, the amount is 0.6 mg / dosage unit. In another embodiment, the amount is 0.8 mg / dosage unit. In another embodiment, the amount is 1 mg / dosage unit. In another embodiment, the amount is 1.5 mg / dosage unit. In another embodiment, the amount is 2 mg / dosage unit. In another embodiment, the amount is 2.5 mg / dosage unit. In another embodiment, the amount is 3 mg / dosage unit. In another embodiment, the amount is 5 mg / dosage unit. In another embodiment, the amount is 7 mg / dosage unit. In another embodiment, the amount is 10 mg / dosage unit. In another embodiment, the amount is 12 mg / dosage unit. In another embodiment, the amount is 15 mg / dosage unit. In another embodiment, the amount is 20 mg / dosage unit. In another embodiment, the amount is 30 mg / dosage unit. In another embodiment, the amount is 50 mg / dosage unit. In another embodiment, the amount is 70 mg / dosage unit. In another embodiment, the amount is 100 mg / dosage unit.

[0047] In another embodiment, the compositions of the present invention comprise a substance that enhances the absorption of a protein of the present invention across the intestinal mucosal barrier. In another embodiment, the compositions of the present invention further comprise a substance that enhances the absorption of IL-22 across the intestinal mucosal barrier. In another embodiment, the compositions of the present invention further comprise a substance that reduces the degradation of IL-22 in the digestive system. In another embodiment, the compositions of the present invention further comprise a substance that reduces the degradation of IL-22 in the stomach. In another embodiment, the compositions of the present invention further comprise a substance that reduces the degradation of IL-22 in the intestine. Such substances are referred to herein as "enhancers." As provided herein, an enhancer, when used together with an omega-3 fatty acid or a protease inhibitor, enhances the ability of a protein, such as IL-22, to be absorbed in the intestine. As provided herein, an enhancer, when used together with an omega-3 fatty acid and a protease inhibitor, enhances the ability of insulin to be absorbed in the intestine. As provided herein, an enhancer, when used together with an omega-3 fatty acid and a protease inhibitor, enhances the ability of IL-22 to be absorbed in the intestine.

[0048] In one embodiment, the enhancer is didecanoylphosphatidylcholine (DDPC). In one embodiment, the enhancer is a chelating agent, such as ethylenediaminetetraacetic acid (EDTA) or egtazate EGTA. In another embodiment, the EDTA is sodium EDTA. In some embodiments, the enhancer is an NO donor. In some embodiments, the enhancer is a bile acid, a glycine-conjugated bile acid, or an alkali metal salt. In one embodiment, absorption enhancement is achieved by utilizing a combination of α-galactosidase and p-mannanase. In some embodiments, the enhancer is a fatty acid, such as sodium caprate. In one embodiment, the enhancer is sodium glycocholate. In one embodiment, the enhancer is sodium salicylate. In one embodiment, the enhancer is n-dodecyl-pD-maltopyranoside. In some embodiments, a surfactant acts as an absorption enhancer. In one embodiment, the enhancer is chitosan, such as N,N,N-trimethylchitosan chloride (TMC).

[0049] In one embodiment, the NO donor of the present invention comprises 3-(2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine, N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)ethanamine, or S-nitroso-N-acetylpenicillamine.

[0050] In another embodiment, the bile acid is cholic acid. In another embodiment, the bile acid is chenodeoxycholic acid. In another embodiment, the bile acid is taurocholic acid. In another embodiment, the bile acid is taurochenodeoxycholic acid. In another embodiment, the bile acid is glycocholic acid. In another embodiment, the bile acid is glycochenocholic acid. In another embodiment, the bile acid is 3 beta-monohydroxychloric acid. In another embodiment, the bile acid is lithocholic acid. In another embodiment, the bile acid is 5 beta-cholanic acid. In another embodiment, the bile acid is 3,12-diol-7-one-5 beta-cholanic acid. In another embodiment, the bile acid is 3 alpha hydroxy-12-ketocholic acid. In another embodiment, the bile acid is 3 beta-hydroxy-12-ketocholic acid. In another embodiment, the bile acid is 12 alpha-3 beta-dihydrocholic acid. In another embodiment, the bile acid is ursodeoxycholic acid.

[0051] In one embodiment, the enhancer is a non-ionic surfactant. In one embodiment, the enhancer is a non-ionic polyoxyethylene ether surfactant (e.g., one having an HLB value of 6-19 and an average number of polyoxyethylene units of 4-30). In another embodiment, the enhancer is an anionic surfactant. In another embodiment, the enhancer is a cationic surfactant. In another embodiment, the enhancer is an amphoteric surfactant. In one embodiment, a zwitterionic surfactant, such as an acylcarnitine, serves as an absorption enhancer.

[0052] In another embodiment, the absorption enhancer is an oral, rectal, or other gastrointestinal absorption enhancer effective for large molecule drugs. In another embodiment, the absorption enhancer is highly water soluble. In another embodiment, the absorption enhancer is fully absorbed, i.e., greater than 85%, in the gastrointestinal tract. In another embodiment, the absorption enhancer is in a coarse form. In another embodiment, the absorption enhancer is micronized. In another embodiment, the absorption enhancer is amorphous. In another embodiment, the absorption enhancer is N-(5-chlorosalicyloyl)-8-aminocaprylic acid (CNAC). In another embodiment, the absorption enhancer is N-(10-[2-hydroxybenzoyl]amino)decanoic acid (SNAD). In another embodiment, the absorption enhancer is N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC). Without being bound by theory, CNAC, SNAD, and / or SNAC may non-covalently complex with IL-22 to increase lipophilicity and / or decrease transepithelial electrical resistance. In another embodiment, the absorption enhancer is CNAC, SNAD, SNAC, their monosodium and / or disodium salts, ethanol solvates of their sodium salts, and monohydrates of their sodium salts, and any combination thereof. In another embodiment, the absorption enhancer is 8-(N-2-hydroxy-4-methoxybenzoyl)-aminocaprylic acid (4-MOAC) and its pharmaceutically acceptable salts, and / or amorphous and polymorphic forms of 4-MOAC. In another embodiment, the absorption enhancer is N-(8-[2-hydroxy-5-chlorobenzoyl]-amino)octanoic acid (also known as 8-(N-2-hydroxy-5-chlorobenzoyl)aminocaprylic acid)) (5-CNAC) and its pharmaceutically acceptable salts, and / or amorphous and polymorphic forms of 5-CNAC. In another embodiment, the absorption enhancer is 4-[(2-hydroxy-4-chlorobenzoyl)amino]butanoate (also known as 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid) (4-CNAB) and its pharmaceutically acceptable salts, including its monosodium salt, and / or amorphous and polymorphic forms of 4-CNAB.

[0053] In another embodiment, the pharmaceutical compositions of the present invention comprise a delivery-effective amount of one or more absorption enhancers. In another embodiment, the pharmaceutical compositions of the present invention comprise an amount of an active agent sufficient to deliver the desired effect.

[0054] In another embodiment, the pharmaceutical composition of the present invention comprises an absorption enhancer in an amount of 2.5% to 99.4% by weight. In another embodiment, the pharmaceutical composition of the present invention comprises an absorption enhancer in an amount of 2.5% to 10% by weight. In another embodiment, the pharmaceutical composition of the present invention comprises an absorption enhancer in an amount of 8% to 15% by weight. In another embodiment, the pharmaceutical composition of the present invention comprises an absorption enhancer in an amount of 10% to 20% by weight. In another embodiment, the pharmaceutical composition of the present invention comprises an absorption enhancer in an amount of 15% to 30% by weight. In another embodiment, the pharmaceutical composition of the present invention comprises an absorption enhancer in an amount of 20% to 40% by weight. In another embodiment, the pharmaceutical composition of the present invention comprises an absorption enhancer in an amount of 30% to 50% by weight. In another embodiment, the pharmaceutical composition of the present invention comprises an absorption enhancer in an amount of 40% to 60% by weight. In another embodiment, the pharmaceutical composition of the present invention comprises an absorption enhancer in an amount of 50% to 70% by weight. In another embodiment, the pharmaceutical composition of the present invention comprises an absorption enhancer in an amount of 70% to 99.4% by weight. In another embodiment, the amount of absorption enhancer in the compositions of the present invention is a delivery-effective amount, which can be determined by methods known to those of skill in the art for any particular carrier or biologically or chemically active agent.

[0055] In another embodiment, the amount of potentiator utilized in methods and compositions of the present invention is 0.1 mg / dosage unit. In another embodiment, the amount is 0.2 mg / dosage unit. In another embodiment, the amount is 0.3 mg / dosage unit. In another embodiment, the amount is 0.4 mg / dosage unit. In another embodiment, the amount is 0.6 mg / dosage unit. In another embodiment, the amount is 0.8 mg / dosage unit. In another embodiment, the amount is 1 mg / dosage unit. In another embodiment, the amount is 1.5 mg / dosage unit. In another embodiment, the amount is 2 mg / dosage unit. In another embodiment, the amount is 2.5 mg / dosage unit. In another embodiment, the amount is 3 mg / dosage unit. In another embodiment, the amount is 5 mg / dosage unit. In another embodiment, the amount is 7 mg / dosage unit. In another embodiment, the amount is 10 mg / dosage unit. In another embodiment, the amount is 12 mg / dosage unit. In another embodiment, the amount is 15 mg / dosage unit. In another embodiment, the amount is 20 mg / dosage unit. In another embodiment, the amount is 30 mg / dosage unit. In another embodiment, the amount is 50 mg / dosage unit. In another embodiment, the amount is 70 mg / dosage unit. In another embodiment, the amount is 100 mg / dosage unit.

[0056] In another embodiment, the composition of the present invention further comprises a coating that inhibits digestion of the composition in the subject's stomach. In one embodiment, the coating inhibits digestion of the composition in the subject's stomach. In one embodiment, the coated dosage form of the present invention releases the drug when the pH moves into the alkaline range. In one embodiment, the coating is a single layer, while in another embodiment, the coating is applied in multiple layers. In one embodiment, the coating is a bioadhesive polymer that specifically binds to the intestinal mucosa, thereby allowing drug release at the attachment site. In one embodiment, the enteric coating is an enteric film coating. In some embodiments, the coating comprises a biodegradable polysaccharide, chitosan, Aquateric® Aqueous, Aquacoat® ECD, an azopolymer, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, gelatin, polyvinyl acetate phthalate, hydrogel, Pulsincap, or a combination thereof. In one embodiment, a pH-sensitive coating will be used according to the desired release site and / or release profile known to those skilled in the art.

[0057] In one embodiment, the coating is an enteric coating. Methods of enteric coating are well known in the art and are described, for example, in Siepmann F, Siepmann J et al., Blends of aqueous polymer dispersions used for pellet coating: importance of the particle size. J Control Release 2005;105(3):226-39; and Huyghebaert N, Vermeire A, Remon JP. In vitro evaluation of coating polymers for enteric coating and human ileal targeting. Int J Pharm 2005;298(1):26-37. Each method corresponds to a separate embodiment of the present invention.

[0058] In another embodiment, the acrylic polymer Eudragit® is used as an enteric coating. The use of acrylic polymers for coating pharmaceutical formulations is well known in the art. Eudragit acrylic polymers have been shown to be safe and are not absorbed or metabolized by the body, but rather excreted.

[0059] In another embodiment, the coating is a gelatin coating. In another embodiment, microencapsulation is used to protect insulin from degradation in the stomach. In another embodiment, microencapsulation is used to protect exenatide from degradation in the stomach. Methods of applying gelatin coatings and microencapsulation are well known in the art. Each method corresponds to a separate embodiment of the present invention.

[0060] In another embodiment, the coating is a film coating. In another embodiment, the coating is ethylcellulose. In another embodiment, the coating is an aqueous dispersion of ethylcellulose, e.g., hydroxypropylmethylcellulose (HPMC) E15. In another embodiment, the coating is a gastroresistant coating, e.g., a polymer containing carboxylic acid groups as functional moieties. In another embodiment, the coating is a monolithic matrix. In another embodiment, the coating is a cellulose ether (e.g., hypromellose (HPMC)). Each type of coating corresponds to a separate embodiment of the present invention.

[0061] In another embodiment, the present invention provides that the use of at least one protease inhibitor and absorption enhancer in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of the proteins of the present invention. In another embodiment, the present invention provides that the use of at least one protease inhibitor and absorption enhancer in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22.

[0062] In another embodiment, the present invention provides that the use of at least one protease inhibitor and SNAC in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of the proteins of the present invention. In another embodiment, the present invention provides that the use of at least one protease inhibitor and SNAC in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22.

[0063] In another embodiment, the present invention provides that the use of at least one protease inhibitor and a SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of the proteins of the present invention. In another embodiment, the present invention provides that the use of at least one protease inhibitor and a SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22.

[0064] In another embodiment, the present invention provides that the use of a protease inhibitor and SNAC or SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22 in a human subject by at least 10%. In another embodiment, the present invention provides that the use of a protease inhibitor and SNAC or SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22 in a human subject by at least 20%. In another embodiment, the present invention provides that the use of a protease inhibitor and SNAC or SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22 in a human subject by at least 30%. In another embodiment, the present invention provides that the use of a protease inhibitor and SNAC or SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22 in a human subject by at least 40%. In another embodiment, the present invention provides that the use of a protease inhibitor and SNAC or SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22 in a human subject by at least 50%. In another embodiment, the present invention provides that the use of a protease inhibitor and SNAC or SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22 in a human subject by at least 60%. In another embodiment, the present invention provides that the use of a protease inhibitor and SNAC or SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22 in a human subject by at least 70%. In another embodiment, the present invention provides that the use of a protease inhibitor and SNAC or SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22 in a human subject by at least 80%.In another embodiment, the present invention provides that the use of a protease inhibitor and SNAC or SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22 in a human subject by at least 90%. In another embodiment, the present invention provides that the use of a protease inhibitor and SNAC or SNAD in a single oral, rectal, or other gut-related composition dramatically and unexpectedly increases the bioavailability of IL-22 in a human subject by at least 100%.

[0065] In another embodiment, the present invention provides a method for oral, rectal, or other gastrointestinal-related administration of an enzymatically active protein, such as IL-22, to a subject, whereby a significant proportion of the protein retains enzymatic activity after absorption across the subject's intestinal mucosal barrier, the method comprising orally, rectally, or other gastrointestinal-related administering to the subject a pharmaceutical composition comprising the protein, a protease inhibitor, and an absorption enhancer, thereby orally, rectally, or other gastrointestinal-related administering the enzymatically active protein to the subject.

[0066] In another embodiment, the present invention provides a method for oral, rectal, or other gut-related administration of IL-22 to a subject, whereby a substantial proportion of IL-22 retains its activity after absorption across the subject's intestinal mucosal barrier, the method comprising orally, rectally, or other gut-related administration of IL-22 to a subject. In another embodiment, the present invention provides a method for oral, rectal, or other gut-related administration of IL-22 to a subject, whereby a substantial proportion of IL-22 retains its activity after absorption across the subject's intestinal mucosal barrier, the method comprising orally, rectally, or other gut-related administration of IL-22 to a subject.

[0067] In another embodiment, the present invention provides a method for oral, rectal, or other gastrointestinal administration of IL-22 to a subject, whereby a significant proportion of the administered IL-22 retains activity after absorption across the subject's intestinal mucosal barrier, the method comprising orally, rectally, or other gastrointestinal administration of IL-22 to a subject, a pharmaceutical composition comprising IL-22, at least one protease inhibitor, SNAC or SNAD, and an omega-3 fatty acid, thereby orally, rectally, or other gastrointestinal administration of IL-22 to a subject. In another embodiment, the present invention provides a method for oral, rectal, or other gastrointestinal administration of IL-22 to a subject, whereby a significant proportion of the administered IL-22 retains enzymatic activity after absorption across the subject's intestinal mucosal barrier, the method comprising orally, rectally, or other gastrointestinal administration of IL-22 to a human subject, a pharmaceutical composition comprising IL-22, at least one protease inhibitor, and SNAC or SNAD, EDTA (or a salt thereof), and an omega-3 fatty acid, thereby providing a method for orally, rectally, or other gastrointestinal administration of IL-22 to a human subject.

[0068] In another embodiment, the present invention provides a method of treating an inflammatory disorder in a human subject, comprising orally, rectally, or otherwise administering to the subject a pharmaceutical composition comprising IL-22, at least one protease inhibitor, and SNAC or SNAD, thereby treating the inflammatory disorder. In another embodiment, the present invention provides a method of treating an inflammatory disorder in a human subject, comprising orally, rectally, or otherwise administering to the subject a pharmaceutical composition comprising IL-22, at least one protease inhibitor, SNAC or SNAD, and an omega-3 fatty acid, thereby treating the inflammatory disorder. In another embodiment, the present invention provides a method of treating an inflammatory disorder in a human subject, comprising orally, rectally, or otherwise administering to the subject a pharmaceutical composition comprising IL-22, at least one protease inhibitor, and SNAC or SNAD, EDTA (or a salt thereof), and an omega-3 fatty acid, thereby treating the inflammatory disorder. In some embodiments, the inflammatory disorder is irritable bowel disease (IBD). In some embodiments, the inflammatory disorder is ulcerative colitis, Crohn's disease, or fistulas associated with ulcerative colitis or Crohn's disease. In some embodiments, the inflammatory disorder is ulcerative colitis. In some embodiments, the inflammatory disorder is Crohn's disease. It has been found that intraguinary administration of IL-22 or IL-22-Fc allows for greater retention of IL-22 or IL-22-Fc in the gut environment rather than systemically, compared to parenteral administration. Furthermore, oral or rectal administration can prevent spikes in systemic serum levels of IL-22.

[0069] In one embodiment, the present invention provides the use of a protein such as IL-22, at least one protease inhibitor, and SNAC or SNAD in the manufacture of a medicament for orally, rectally, or other gastrointestinal administration to a subject, whereby a significant proportion of the protein retains its activity after absorption across the subject's intestinal mucosal barrier. In one embodiment, the present invention provides the use of a protein, at least one protease inhibitor, SNAC or SNAD, and omega-3 fatty acids in the manufacture of a medicament for orally, rectally, or other gastrointestinal administration to a subject, whereby a significant proportion of the protein retains its activity after absorption across the subject's intestinal mucosal barrier. In one embodiment, the present invention provides the use of a protein, at least one protease inhibitor, SNAC or SNAD, Na-EDTA, and omega-3 fatty acids in the manufacture of a medicament for orally, rectally, or other gastrointestinal administration to a subject, whereby a significant proportion of the protein retains its activity after absorption across the subject's intestinal mucosal barrier. In some embodiments, the use of a protein such as IL-22, at least one protease inhibitor, and SNAC or SNAD in the manufacture of a medicament for orally, rectally, or otherwise gastrointestinal-related administration of the protein to a subject, whereby a significant proportion of the protein retains its activity after absorption across the subject's intestinal mucosal barrier, is for use in treating an inflammatory disorder. In some embodiments, the inflammatory disorder is irritable bowel disease (IBD). In some embodiments, the inflammatory disorder is ulcerative colitis, Crohn's disease, or fistulas associated with ulcerative colitis or Crohn's disease. In some embodiments, the inflammatory disorder is ulcerative colitis. In some embodiments, the inflammatory disorder is Crohn's disease.

[0070] In another embodiment, solid carriers / diluents for use in the methods and compositions of the present invention include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulose-derived materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0071] In another embodiment, the composition may contain binders (e.g., gum arabic, corn starch, gelatin, carbomer, ethylcellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone), disintegrants (e.g., corn starch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), buffers of various pH and ionic strength (e.g., Tris-HCl, acetate buffer, phosphate buffer), additives such as albumin or gelatin to prevent absorption to surfaces, surfactants (e.g., Tween 20, Tween 80, Pluronic F68, bile salts), surfactants (e.g., sodium lauryl sulfate), penetration enhancers, solubilizers (e.g., glycerol, polyethyleneglycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose), thickeners (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., aspartame, citric acid), preservatives (e.g., thimerosal, benzyl alcohol Further examples of suitable additives include, but are not limited to, cellulose acetate, cellulose esters, cellulose acetate copolymer ...

[0072] In some embodiments, the dosage forms of the present invention are formulated to achieve immediate release, sustained release, or delayed release characteristics. In some embodiments, the release characteristics of the composition are determined by the use of specific additives that serve, for example, as binders, disintegrants, fillers, or coating materials. In one embodiment, the composition will be formulated to achieve a specific release characteristic known to those skilled in the art.

[0073] In one embodiment, the composition is formulated as an oral, rectal, or other gastrointestinal dosage form. In one embodiment, the composition is a solid oral, rectal, or other gastrointestinal dosage form, including a tablet, chewable tablet, or capsule. In one embodiment, the capsule is a soft gelatin capsule. In another embodiment, the capsules described herein are hard-shell capsules. In another embodiment, the capsules described herein are soft-shell capsules. In another embodiment, the capsules described herein are made from gelatin. In another embodiment, the capsules described herein are made from plant-based gelling substances, such as carrageenan and modified forms of starch and cellulose.

[0074] In other embodiments, the controlled- or sustained-release coating utilized in the methods and compositions of the present invention comprises a lipophilic depot formulation (eg, fatty acids, waxes, oils).

[0075] The preparation of pharmaceutical compositions containing an active component, for example, by mixing, granulating, or tablet-forming processes, is well understood in the art. Therapeutic active ingredients are often mixed with pharmaceutically acceptable additives that are compatible with the active ingredient. For oral, rectal, or other gastrointestinal administration, the active ingredient of the compositions of the present invention is mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into a form suitable for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous solutions, alcoholic solutions, or oily solutions.

[0076] The compositions of the present invention can generally be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0077] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention. To the extent that any definitions or terminology provided in a reference incorporated by reference differ from the terms and discussion provided herein, the terms and definitions herein shall control.

[0078] equivalent The foregoing specification is believed to be sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and set forth the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0079] The following examples, including the experiments conducted and results obtained, are provided for illustrative purposes only and are not to be construed as limiting the invention. [Example]

[0080] Example 1: Crossing the intestinal barrier using SNAC-IL-22 complexes C57Bl / 6 mice (n = 4) were intrarectally administered 100 μl of a mixture of 1 mg / ml IL-22 and 100 mg / ml SNAC preparation. Serum was collected at the indicated times for IL-22 ELISA or serum amyloid A (SAA) ELISA, respectively. After intrarectal administration of the IL-22 / SNAC mixture, serum IL-22 levels increased to over 10e6 pg / ml at 15 min and remained elevated for at least 4 h (Figure 1A). Serum amyloid A (SAA) is a highly conserved acute-phase protein synthesized primarily by the liver. During acute inflammation, serum SAA levels can increase up to 1000-fold. SAA exhibits significant immunological activity, for example, by inducing the synthesis of multiple cytokines and by being chemotactic for neutrophils and mast cells, and is thought to protect the colonic epithelium from acute injury in a murine colitis model. Figure 1B shows that serum SAA levels from the liver increased after rectal administration of the IL-22 / SNAC mixture, reaching peak levels 24 hours after administration. Figure 2A shows the serum IL-22 levels in each mouse tested. Figure 2B shows increased expression of RegIIIβ in the colon after rectal administration of IL-22 / SNAC.

[0081] Example 2: Gastrointestinal versus systemic PK study of IL-22 Mice were intrarectally administered either IL-22 (16.5 kDa) or murine IL-22Fc (85 kDa) to investigate the level of IL-22 retained in the colon versus the level of IL-22 systemically absorbed. For rectal administration, C57Bl / 6 mice were intrarectally administered 100 μl of 1 mg / ml IL-22 plus 100 mg / ml SNAC, or murine IL-22Fc plus 100 mg / ml SNAC using a 20G flexible plastic tube. For measurement of colonic IL-22 levels, whole colons were collected 15 min, 30 min, 1 h, 2 h, 4 h, or 24 h after administration. After removal of feces, the colons were cut longitudinally and washed with 45 ml of wash buffer (PBS + 0.2% Tween 20) for 5 min. After four repeated washes, the colons were placed in safe-lock tubes containing 1 ml of NP40 cell lysis buffer (Invitrogen). Colon samples were lysed using a TissueLyser II (Qiagen). Supernatants were collected for IL-22 measurement by ELISA (mouse / rat IL-22 ELISA kit from R&D Systems). Serum was collected 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 24 hours after administration to measure serum IL-22 levels. IL-22 levels were measured by ELISA (mouse / rat IL-22 ELISA kit from R&D Systems). The results are shown in Figure 3. Higher colonic IL-22 levels were observed with Fc-conjugated IL-22 (Figure 3A), and an initial spike in systemic IL-22 levels was observed with IL-22 (Figure 3B).

[0082] Example 3: Gut versus systemic delivery of IL-22 Mice were administered murine IL-22Fc either intrarectally or intraperitoneally to investigate differences in systemic versus colonic IL-22 levels. C57Bl / 6 mice were administered 78 μg of murine IL-22Fc plus 100 mg / ml SNAC intrarectally using a 20G flexible plastic tube, or 3 μg of murine IL-22Fc intraperitoneally. For measurement of colonic IL-22 levels, whole colons were collected 15 min, 30 min, 1 h, 2 h, 4 h, or 24 h after administration. After removing feces, the colons were cut longitudinally and washed with 45 ml of wash buffer (PBS + 0.2% Tween 20) for 5 min. After four repeated washes, the colons were placed in safe-lock tubes containing 1 ml of NP40 cell lysis buffer (Invitrogen). The colons were lysed using a TissueLyser II (Qiagen). Supernatants were collected for IL-22 measurement by ELISA (mouse / rat IL-22 ELISA kit from R&D Systems). Serum was collected 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 24 hours after administration for measurement of serum IL-22 levels. IL-22 levels were measured by ELISA (mouse / rat IL-22 ELISA kit from R&D Systems). The results are shown in Figure 4. Higher levels of colon signaling were observed with intrarectal administration (Figure 4A). Higher levels of serum signaling were observed with intraperitoneal administration (Figure 4B).

[0083] Example 4: Pharmacodynamic study of gut-delivered IL-22 Intraperitoneal and intrarectal administration of murine IL22Fc was investigated for its ability to induce systemic PD responses, as measured by increased serum amyloid acid levels, or in the gut, as measured by colonic Reg3β expression in the gut. Murine IL22Fc was administered intraperitoneally, and murine IL22Fc plus SNAC was administered intrarectally. SNAC alone served as a control. Serum amyloid acid levels were measured by ELISA (SAA ELISA kit, R&D Systems). Colonic RegIIIβ expression was measured by qPCR. Briefly, mesocolons were harvested and flash-frozen in liquid nitrogen. For RNA extraction, colon tissues were placed in 0.8 ml of RTL lysis buffer (Qiagen) in safelock tubes and lysed in a TissueLyser II. The supernatant was used for RNA purification and qPCR in a Quant Studio 7. The results are shown in Figure 5. Systemic levels of SAA are shown in Figure 5A, demonstrating higher SAA levels with intraperitoneally administered murine IL22Fc. Gastrointestinal levels of RegIIIβ are shown in Figure 5B, demonstrating high levels of RegIIIβ in the gut from both intraperitoneally and intrarectally administered murine IL22Fc. Compared with systemic administration of murine IL22Fc via the intraperitoneal route, the local versus systemic pharmacodynamic effects of intrarectally administered murine IL22Fc plus SNAC are highly selective.

Claims

1. A pharmaceutical composition for oral, rectal, or intraperitoneal administration comprising an IL-22 protein and an absorption enhancer selected from the group consisting of N(8-[2-hydroxybenzoyl]amino)caprylic acid (NAC) and salts of said NAC, in the absence of a protease inhibitor.

2. 2. The pharmaceutical composition of claim 1, wherein the IL-22 has a sequence that is at least 90% identical to SEQ ID NO: 1, 2, 3, or 4.

3. 2. The pharmaceutical composition of claim 1, wherein the IL-22 has a sequence that is at least 95% identical to SEQ ID NO: 1, 2, 3, or 4.

4. 2. The pharmaceutical composition of claim 1, wherein the IL-22 has the sequence of SEQ ID NO: 1, 2, 3, or 4.

5. The pharmaceutical composition according to any one of claims 1 to 4, which is a solid pharmaceutical composition.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the absorption enhancer is a salt of NAC, the salt being selected from the group consisting of a monosodium salt, a disodium salt, and combinations thereof.

7. The pharmaceutical composition according to any one of claims 1 to 6, further comprising EDTA or a salt thereof.

8. 8. The pharmaceutical composition of claim 1, further comprising a coating that inhibits digestion of the composition in the subject's stomach, an enteric coating, or a gelatin coating.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the IL-22 protein is an IL-22 Fc fusion protein comprising an Fc bound to the IL-22 protein.

10. The pharmaceutical composition of claim 9, wherein the Fc is a human IgG1 Fc.

11. 11. The pharmaceutical composition of claim 10, wherein the human IgGl Fc comprises one or more mutations that alter the effector function of the Fc, and wherein the human IgGl comprises a substitution at N297.

12. 12. The pharmaceutical composition of claim 11, wherein the substitution at N297 is N297G.

13. The pharmaceutical composition of any one of claims 9 to 12, wherein a linker connects the Fc and the human IL-22 portion of the protein.

14. A pharmaceutical composition according to any one of claims 1 to 13 for treating an inflammatory disorder in a patient in need thereof.

15. 15. The pharmaceutical composition of claim 14, wherein the inflammatory disorder is selected from the group consisting of inflammatory bowel disorder, Crohn's disease, ulcerative colitis, type 2 diabetes, type 2 diabetes with morbid obesity, wounds (including diabetic wounds and diabetic ulcers), burns, ulcers (including pressure ulcers and venous ulcers), graft versus host disease (GVHD), microbial infections, acute kidney injury, acute pancreatitis, cardiovascular conditions, metabolic syndrome, acute endotoxemia, sepsis, atherosclerosis, cardiovascular disease, endotoxemia (acute and mild), acute coronary heart disease, hypertension, dyslipidemia, obesity, hyperglycemia, lipid metabolism disorders, hepatitis, acute hepatitis, renal failure, acute renal failure, acute kidney injury, renal transplant failure, post-renal transplant dysfunction, contrast-induced nephropathy, pancreatitis, acute pancreatitis, hepatic fibrosis, and pulmonary fibrosis.

16. 16. The pharmaceutical composition of claim 15, wherein the inflammatory disorder is inflammatory bowel disorder, Crohn's disease, or ulcerative colitis.

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