US 2011 0014247A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0014247 A1 Kidron (43) Pub. Date: Jan. 20, 2011

(54) METHODS AND COMPOSITIONS FOR ORAL Related U.S. Application Data ADMINISTRATION OF PROTEINS (60) Provisional application No. 61/064,779, filed on Mar. 26, 2008. (75) Inventor: Miriam Kidron, Jerusalem (IL) Publication Classification (51) Int. Cl. Correspondence Address: A638/16 (2006.01) WOLF GREENFIELD & SACKS, PC. A6IR 9/00 (2006.01) 6OO ATLANTIC AVENUE A638/19 (2006.01) BOSTON, MA 02210-2206 (US) A638/2 (2006.01) A638/43 (2006.01) A638/28 (2006.01) (73) Assignee: ORAMED LTD., Jerusalem (IL) A638/27 (2006.01) A6IP3/10 (2006.01) (21) Appl. No.: 12/934,754 (52) U.S. Cl...... 424/400; 424/85.1; 424/85.5; 424/85.7; 424/94.1: 514/1.1 : 514/6.5: 514/6.9; 514/11.3 (22) PCT Fled: Feb. 26, 2009 (57) ABSTRACT This invention provides compositions that include a protein (86) PCT NO.: PCT/IL09/00223 and at least two protease inhibitors, method for treating dia betes mellitus, and methods for administering same, and S371 (c)(1), methods for oral administration of a protein with an enzy (2), (4) Date: Sep. 27, 2010 matic activity, including orally administering same. Patent Application Publication Jan. 20, 2011 Sheet 1 of 3 US 2011/0014247 A1

Figure 1A

8 mg insulin/150 mg EDTA 125 mg SBTI

10 105 100 95 90 85 80

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Figure 1B

8 mg insulin/150 mg EDTA/150000 KIU Aprotinin

S is S S S eS & KS & S Time (min.)

Figure 1C

8 mg insulin/150 mg EDTA/150000 KIU Aprotinin/125 mg SBTI

Time (min.) Patent Application Publication Jan. 20, 2011 Sheet 2 of 3 US 2011/0014247 A1 Figure 1D

8 mg insulin/150 mg EDTA/125 mg SBTI

14 is 12 g O E i 4 - 2 - O

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Figure E

8 mg insulin/150 mg EDTA/150000 KUAprotinin

SS O E 5 2

Time (min.)

Figure 1F

8 mg insulin/150 mg EDTA/150000 KIU Aprotinin/125 mg SBTI

2 O 1 O O

Time (min.) Patent Application Publication Jan. 20, 2011 Sheet 3 of 3 US 2011/0014247 A1 Figure 1G

8 mg insulin/150 mg EDTA/125 mg SBTI

2.

Time (min.)

Figure 1H

8 mg insulin/150 mg EDTA/150000 KIU Aprotinin

2

O

Q s Time (min.)

Figure 1I

8 mg insulin/150 mg EDTA/150000 KIU Aprotinin/125 mg SBTI

SS 2 1.5 3 i 0.5 cs 0

Time (min.) US 2011/0014247 A1 Jan. 20, 2011

METHODS AND COMPOSITIONS FOR ORAL thereof and a first protease inhibitor and a second protease ADMINISTRATION OF PROTEINS inhibitor, thereby treating diabetes mellitus.

FIELD OF INVENTION BRIEF DESCRIPTION OF THE DRAWINGS 0010 FIG.1. is a set of bar graphs showing the changes in 0001. This invention provides oral compositions compris blood glucose levels after treatment with the formulations of ing a protein and at least two protease inhibitors and a method the invention. for administering same. 0011. In FIG. 1 A-C blood glucose levels were signifi cantly reduced in human subjects treated with formulation (3) BACKGROUND OF THE INVENTION (8 mg insulin, 150 mg EDTA, 150000 KIU Aprotinin, 125 mg SBTI in 1 ml fish oil in a soft-gel capsule (SwissCup). 0002. Due to improved biotechnology, the accessibility of 0012 FIG. 1D-F shows that total blood insulin was sig biologically active peptides to the pharmaceutical industry nificantly higher especially between 220-300 minutes in has increased considerably. However, a limiting factor in the human subjects treated with formulation (3). development of peptide drugs is the relative ineffectiveness (0013 FIG. 1G-I, shows that blood C-peptide levels were when given perorally. Almost all peptide drugs are parenter significantly reduced in human Subjects treated with formu ally administered, although parenterally administered pep tide drugs are often connected with low patient compliance. lation (3) 0003 Insulin is a medicament used to treat patients suf DETAILED DESCRIPTION OF THE INVENTION fering from diabetes, and is the only treatment for insulin dependent diabetes mellitus. Diabetes Mellitus is character 0014. This invention provides compositions and methods ized by a pathological condition of absolute or relative insulin comprising a protein and at least two protease inhibitors. In deficiency, leading to hyperglycemia, and is one of the main another, embodiment, the present invention provides compo threats to human health in the 21st century. The global figure sitions and methods comprising a protein and a first protease of people with diabetes is set to rise to 220 million in 2010, inhibitor and a second protease inhibitors. In another, and 300 million in 2025. Type I diabetes is caused primarily embodiment, the present invention provides compositions by the failure of the pancreas to produce insulin. Type II and methods comprising a protein having a molecular weight diabetes, involves a lack of responsiveness of the body to the of up to 100,000 Daltons and a first protease inhibitor and a action of insulin. second protease inhibitors. 0004. Approximately 20%-30% of all diabetics use daily 0015. In another, embodiment, the protein of the present insulin injections to maintain their glucose levels. An esti invention has a molecular weight of 1,000-5,000 Daltons. In mated 10% of all diabetics are totally dependent on insulin another, embodiment, the protein of the present invention has injections. a molecular weight of 5,000-10,000 Daltons. In another, 0005. Currently, the only route of insulin administration is embodiment, the protein of the present invention has a injection. Daily injection of insulin is causes considerable molecular weight of 10,000-20,000 Daltons. In another, suffering for patients. Side effects such as lipodystrophy at embodiment, the protein of the present invention has a the site of the injection, lipatrophy, lipohypertrophy, and molecular weight of 20,000-30,000 Daltons. In another, occasional hypoglycemia are known to occur. In addition, embodiment, the protein of the present invention has a Subcutaneous administration of insulin does not typically molecular weight of 40,000-50,000 Daltons. In another, provide the fine continuous regulation of metabolism that embodiment, the protein of the present invention has a occurs normally with insulin secreted from the pancreas molecular weight of 50,000-60,000 Daltons. In another, directly into the liver via the portal vein. embodiment, the protein of the present invention has a molecular weight of 60,000-70,000 Daltons. In another, 0006. The present invention addresses the need for an embodiment, the protein of the present invention has a alternate solution for administration of insulin. molecular weight of 70,000-80,000 Daltons. In another, embodiment, the protein of the present invention has a SUMMARY OF THE INVENTION molecular weight of 80,000-90,000 Daltons. In another, embodiment, the protein of the present invention has a 0007. This invention provides, in one embodiment, a com molecular weight of 90,000-100,000 Daltons. In another, position comprising a protein or a combination of proteins embodiment, the protein of the present invention has a having a molecular weight of up to 100,000 Daltons and a first molecular weight of 100,000-150,000 Daltons. protease inhibitor and a second protease inhibitor. 0016. In another embodiment, the protein has a molecular 0008. In another embodiment, the present invention pro weight (MW) of 1-50 kilodalton (kDa). In another embodi vides a method for oral administration of a protein having a ment, the MW is 1-45 kDa. In another embodiment, the MW molecular weight up to 100,000 Daltons to a subject, whereby is 1-40 kDa. In another embodiment, the MW is 1-35kDa. In a substantial fraction of the protein retains its activity after another embodiment, the MW is 1-30 kDa. In another absorption, through an intestinal mucosalbarrier of a subject, embodiment, the MW is 1-25 kDa. In another embodiment, comprising administering orally to a Subject a pharmaceutical the MW is 1-20 kDa. In another embodiment, the MW is composition comprising the protein and a first protease 10-50 kDa. In another embodiment, the MW is 15-50kDa. In inhibitor and a second protease inhibitor. another embodiment, the MW is 20-50 kDa. In another 0009. In another embodiment, the present invention pro embodiment, the MW is 25-50 kDa. In another embodiment, vides a method for treating diabetes mellitus in a subject, the MW is 30-50 kDa. In another embodiment, the MW is comprising administering orally to a Subject a pharmaceutical 35-50kDa. In another embodiment, the MW is 1-100kDa. In composition comprising insulin, Exenatide, or a combination another embodiment, the MW is 1-90 kDa. In another US 2011/0014247 A1 Jan. 20, 2011

embodiment, the MW is 1-80 kDa. In another embodiment, ment, the amount is 0.1-2 u?kg. In another embodiment, the the MW is 1-70kDa. In another embodiment, the MW is 1-60 amount is 0.2-2 u?kg. In another embodiment, the amount is kDa. In another embodiment, the MW is 10-100 kDa. In 0.3-2 u/kg. In another embodiment, the amount is 0.5-2 u/kg. another embodiment, the MW is 15-100 kDa. In another In another embodiment, the amount is 0.7-2 u/kg. In another embodiment, the MW is 20-100kDa. In another embodiment, embodiment, the amount is 1-2 u?kg. In another embodiment, the MW is 25-100 kDa. In another embodiment, the MW is the amount is 1.2-2 u?kg. In another embodiment, the amount 30-100 kDa. In another embodiment, the MW is 10-80 kDa. is 1-1.2 u/kg. In another embodiment, the amount is 1-1.5 In another embodiment, the MW is 15-80 kDa. In another u/kg. In another embodiment, the amount is 1-2.5 u?kg. In embodiment, the MW is 20-80 kDa. In another embodiment, another embodiment, the amount is 1-3 u?kg. In another the MW is 25-80 kDa. In another embodiment, the MW is embodiment, the amount is 2-3 u?kg. In another embodiment, 30-80 kDa. Each possibility represents a separate embodi the amount is 1-5u/kg. In another embodiment, the amount is ment of the present invention. 2-5 u/kg. In another embodiment, the amount is 3-5 u/kg. 0017. In another embodiment, the MW is less than 20 kDa. 0022. In another embodiment, the amount of insulin is 0.1 In another embodiment, the MW is less than 25 kDa. In u/kg. In another embodiment, the amount is 0.2 u/kg. In another embodiment, the MW is less than 30 kDa. In another another embodiment, the amount is 0.3 u?kg. In another embodiment, the MW is less than 35kDa. In another embodi embodiment, the amount is 0.4 u/kg. In another embodiment, ment, the MW is less than 40 kDa. In another embodiment, the amount is 0.5u?kg. In another embodiment, the amount is the MW is less than 45kDa. In another embodiment, the MW 0.6 u/kg. In another embodiment, the amount is 0.8 u/kg. In is less than 50 kDa. In another embodiment, the MW is less another embodiment, the amount is 1 u?kg. In another than 55 kDa. In another embodiment, the MW is less than 60 embodiment, the amount is 1.2 u/kg. In another embodiment, kDa. In another embodiment, the MW is less than 65 kDa. In the amount is 1.4 u/kg. In another embodiment, the amount is another embodiment, the MW is less than 70 kDa. In another 1.6 u/kg. In another embodiment, the amount is 1.8 u?kg. In embodiment, the MW is less than 75 kDa. In another embodi another embodiment, the amount is 2 u?kg. In another ment, the MW is less than 80 kDa. In another embodiment, embodiment, the amount is 2.2 u/kg. In another embodiment, the MW is less than 85 kDa. In another embodiment, the MW the amount is 2.5u?kg. In another embodiment, the amount is is less than 90 kDa. In another embodiment, the MW is less 3 u?kg. than 95 kDa. In another embodiment, the MW is less than 100 0023. In another embodiment, the amount of insulin is kDa. 1-10 u. In another embodiment, the amount is 2-10 u. In 0018. In another, embodiment, the protein of the present another embodiment, the amount is 3-10 u. In another invention is insulin. In one embodiment, the insulin of meth embodiment, the amount is 5-10 u. In another embodiment, ods and compositions of the present invention is human insu the amount is 1-20 u. In another embodiment, the amount is lin. In another embodiment, the insulin is recombinant insu 2-20 u. In another embodiment, the amount is 3-20 u. In lin. In another embodiment, the insulin is recombinant human another embodiment, the amount is 5-20 u. In another insulin. In another embodiment, the insulin is bovine insulin. embodiment, the amount is 7-20 u. In another embodiment, In another embodiment, the insulin is porcine insulin. In the amount is 10-20 u. In another embodiment, the amount is another embodiment, the insulin is whale insulin. In another 12-20 u. In another embodiment, the amount is 10-12 u. In embodiment, the insulin is a metal complex of insulin (e.g. a another embodiment, the amount is 10-15 u. In another Zinc complex of insulin, protamine Zinc insulin, or globin embodiment, the amount is 10-25u. In another embodiment, Zinc). the amount is 10-30 u. In another embodiment, the amount is 0019. In another embodiment, the insulin is regular insu 20-30 u. In another embodiment, the amount is 10-50 u. In lin. In another embodiment, the insulin is fast-acting insulin. another embodiment, the amount is 20-50 u. In another In another embodiment, the insulin is lente insulin. In another embodiment, the amount is 30-50 u. In another embodiment, embodiment, the insulin is semilente insulin. In another the amount is 20-100 u. In another embodiment, the amount embodiment, the insulin is Ultralente insulin. In another is 30-100u. In another embodiment, the amount is 100-150 u. embodiment, the insulin is NPH insulin. In another embodi In another embodiment, the amount is 100-250 u. In another ment, the insulin is glargine insulin. In another embodiment, embodiment, the amount is 100-300 u. In another embodi the insulin is lispro insulin. In another embodiment, the insu ment, the amount is 200-300 u. In another embodiment, the lin is aspart insulin. In another embodiment, the insulin is a amount is 100-500 u. In another embodiment, the amount is combination of two or more of any of the above types of 200-500 u. In another embodiment, the amount is 300-500 u. insulin. In another embodiment, the insulin is any other type In another embodiment, the amount is 200-1000 u. In another of insulin known in the art. Each possibility represents a embodiment, the amount is 300-1000 u. separate embodiment of the present invention. 0024. In another embodiment, the amount of insulin is 1 u. 0020. In one embodiment, the amount of insulinutilized in In another embodiment, the amount is 2 u. In another embodi methods and compositions of the present invention is 0.5-3 ment, the amount is 3 u. In another embodiment, the amount units (u)/kg in humans. In one embodiment, the units used to is 4 u. In another embodiment, the amount is 5 u. In another measure insulin in methods and compositions of the present embodiment, the amount is 6 u. In another embodiment, the invention are USP Insulin Units. In one embodiment, the amount is 8 u. In another embodiment, the amount is 10 u. In units used to measure insulin are milligrams. In another another embodiment, the amount is 12 u. In another embodi embodiment, one international Unit (IU) of Insulin is equiva ment, the amount is 14 u. In another embodiment, the amount lent to 45.5 mg insulin. is 16 u. In another embodiment, the amount is 18 u. In another 0021. In another embodiment, the amount of insulin is embodiment, the amount is 20 u. In another embodiment, the 0.1-1 u?kg. In another embodiment, the amount is 0.2-1 u/kg. amount is 22 u. In another embodiment, the amount is 25u. In In another embodiment, the amount is 0.3-1 u/kg. In another another embodiment, the amount is 30 u. In another embodi embodiment, the amount is 0.5-1 u/kg. In another embodi ment, the amount is 50 u. In another embodiment, the amount US 2011/0014247 A1 Jan. 20, 2011

is 80 u. In another embodiment, the amount is 100 u. In another embodiment, the amount of Exenatide in a formula another embodiment, the amount is 120 u. In another embodi tion as described herein is 800 mcg to 900 mcg. In another ment, the amount is 140 u. In another embodiment, the embodiment, the amount of Exenatide in a formulation as amount is 160 u. In another embodiment, the amount is 180 u. described herein is 900 mcg to 1 mg. In another embodiment, the amount is 200 u. In another 0028. In another embodiment, the Exenatide formulation embodiment, the amount is 300 u. In another embodiment, as described herein is taken once a day. In another embodi the amount is 500 u. ment, the Exenatide formulation as described herein is taken 0025. In another embodiment, the protein is Exenatide. In twice a day. In another embodiment, the Exenatide formula another embodiment, the oral formulations of the present tion as described herein is taken three times a day. In another invention protect Exenatide breakdown in the stomach. In embodiment, the Exenatide formulation as described herein another embodiment, Exenatide formulation of the invention is taken four times a day. In another embodiment, the controls blood Sugar levels. In another embodiment, Exenatide formulation as described herein is taken five times Exenatide formulation of the invention helps control blood a day. In another embodiment, one of skill in the art deter Sugar levels. In another embodiment, EXenatide formulation mines the dosage of a Exenatide formulation as described of the invention induces pancreatic production of insulin. In herein. In another embodiment, one of skill in the art deter another embodiment, Exenatide formulation of the invention mines the daily dose of a Exenatide formulation as described is used to treat type 2 (non-insulin dependent) diabetes. In herein. In another embodiment, one of skill in the art deter another embodiment, Exenatide formulation of the invention mines the daily dosing regimen of a Exenatide formulation as is used in conjunction with other diabetes medicines. described herein. 0026. In another embodiment, the amount of Exenatide in 0029. In another embodiment, the Exenatide formulation a formulation as described herein is 10 mcg to 1 mg. In as described herein is taken at least 15 minutes before a meal. another embodiment, the amount of Exenatide in a formula In another embodiment, the Exenatide formulation as tion as described herein is 10 mcg to 25 mcg. In another described herein is taken at least 30 minutes before a meal. In embodiment, the amount of Exenatide in a formulation as another embodiment, the Exenatide formulation as described described herein is 25 mcg to 50 mcg. In another embodi herein is taken at least 45 minutes before a meal. In another ment, the amount of Exenatide in a formulation as described embodiment, the Exenatide formulation as described herein herein is 50 meg to 60 mcg. In another embodiment, the is taken at least 60 minutes before a meal. In another embodi amount of Exenatide in a formulation as described herein is ment, the Exenatide formulation as described herein is taken 60 mcg to 70 mcg. In another embodiment, the amount of at least 75 minutes before a meal. In another embodiment, the Exenatide in a formulation as described herein is 70mcg to 80 Exenatide formulation as described herein is taken at least 90 mcg. In another embodiment, the amount of Exenatide in a minutes before a meal. In another embodiment, the Exenatide formulation as described herein is 80 mcg to 90 mcg. In formulation as described herein is taken at least 100 minutes another embodiment, the amount of Exenatide in a formula before a meal. In another embodiment, the Exenatide formu tion as described herein is 90 mcg to 100 mcg. In another lation as described herein is taken at least 120 minutes before embodiment, the amount of Exenatide in a formulation as a meal. In another embodiment, the Exenatide formulation as described herein is 100 mcg to 110 mcg. In another embodi described herein is taken at least 150 minutes before a meal. ment, the amount of Exenatide in a formulation as described In another embodiment, the Exenatide formulation as herein is 110 mcg to 125 mcg. In another embodiment, the described herein is taken at least 180 minutes before a meal. amount of Exenatide in a formulation as described herein is 0030. In another embodiment, the Exenatide formulation 125 mcg to 150 mcg. In another embodiment, the amount of as described herein reduces the side effects associated with an Exenatide in a formulation as described herein is 150 mcg to injectable dosage form comprising Exenatide. In another 175 mcg. In another embodiment, the amount of Exenatide in embodiment, the Exenatide formulation as described herein a formulation as described herein is 175 mcg to 200 mcg. In reduces nausea as a side effect which is associated with an another embodiment, the amount of Exenatide in a formula injectable dosage form comprising Exenatide. In another tion as described herein is 200 mcg to 220 mcg. In another embodiment, the Exenatide formulation as described herein embodiment, the amount of Exenatide in a formulation as does not induce nausea as a side effect which is associated described herein is 220 mcg to 240 mcg. In another embodi with an injectable dosage form comprising Exenatide. ment, the amount of Exenatide in a formulation as described 0031. As provided herein, protease inhibitors protect the herein is 240 meg to 260 mcg. In another embodiment, the protein of the present invention from cleavage. In another amount of Exenatide in a formulation as described herein is embodiment, the present invention provides that protease 260 meg to 300 mcg. inhibitors protect insulin of the present invention from cleav 0027. In another embodiment, the amount of Exenatide in age. In another, embodiment, the present invention provides a formulation as described herein is 300 mcg to 350 mcg. In that protease inhibitors facilitate the protein absorption in the another embodiment, the amount of Exenatide in a formula intestine of a Subject. In another, embodiment, the present tion as described herein is 350 mcg to 400 mcg. In another invention provides that protease inhibitors facilitate the embodiment, the amount of Exenatide in a formulation as absorption of insulin in the intestine of a Subject. described herein is 400 mcg to 450 mcg. In another embodi 0032. In another embodiment, the present invention pro ment, the amount of Exenatide in a formulation as described vides the use of more than two protease inhibitors in a single herein is 450 mcg to 500 mcg. In another embodiment, the composition or a method. In another, embodiment, the amount of Exenatide in a formulation as described herein is present invention provides that the first and the second pro 550 meg to 600 mcg. In another embodiment, the amount of tease inhibitors are serpins. In another, embodiment, the Exenatide in a formulation as described herein is 600 mcg to present invention provides that serpins are trypsin inhibitors. 700 mcg. In another embodiment, the amount of Exenatide in In another, embodiment, the present invention provides that a formulation as described herein is 700 meg to 800 mcg. In the first and the second protease inhibitors are serpins such as US 2011/0014247 A1 Jan. 20, 2011 but not limited to: Alpha 1-antitrypsin, Antitrypsin-related Aspartic protease inhibitor 7, Aspartic protease inhibitor 8. protein, Alpha 1-antichymotrypsin, Kallistatin, Protein C Aspartic protease inhibitor 9, Pepsin inhibitor Dit33, Asparty1 inhibitor, Cortisol binding globulin, Thyroxine-binding protease inhibitor, or Protease A inhibitor 3. globulin, Angiotensinogen, Centerin, Protein Z-related pro 0037. In another embodiment, the present invention pro tease inhibitor, Vaspin, Monocyte neutrophil elastase inhibi vides that the first protease inhibitor is a serpine and the tor, Plasminogen activator inhibitor-2, Squamous cell carci second protease inhibitor is a Metalloprotease inhibitor. In noma antigen-1 (SCCA-1), Squamous cell carcinoma another, embodiment, the present invention provides that antigen-2 (SCCA-2), Maspin, PI-6, Megsin, PI-8, PI-9, Metalloprotease inhibitors of the invention comprise: Angio Bomapin, Yukopin, Hurpin/Headpin, Antithrombin, Heparin tensin-1-converting enzyme inhibitory peptide, Antihemor cofactor II, Plasminogen activator inhibitor 1, Glia derived ragic factor BJ46a, Beta-casein, Proteinase inhibitor CeKI, nexin/Protease nexin I, Pigment epithelium derived factor, Venom metalloproteinase inhibitor DM43, Carboxypepti Alpha2-antiplasmin, Complement 1-inhibitor, 47 kDa Heat dase A inhibitor, Smpl, IMPI, Alkaline proteinase, inh, shock protein (HSP47), Neuroserpin, or Pancpin. Latexin, Carboxypeptidase inhibitor, Antihemorragic factor 0033. In another embodiment, the present invention pro HSF, Testican-3, SPOCK3, TIMP1, Metalloproteinase vides that the first and the second protease inhibitors are inhibitor 1, Metalloproteinase inhibitor 2, TIMP2, Metallo trypsin inhibitors such as but not limited to: Lima bean trypsin proteinase inhibitor 3, TIMP3, Metalloproteinase inhibitor 4. inhibitor, Aprotinin, soy bean trypsin inhibitor (SBTI), or TIMP4, Putative metalloproteinase inhibitor tag-225, Tissue Ovomucoid. In another, embodiment, the present invention inhibitor of metalloprotease, WAP. kazal, immunoglobulin, provides that the first and the second protease inhibitors are or kunitz and NTR domain-containing protein 1. Lima bean trypsin inhibitor and Aprotinin. In another, 0038. In another embodiment, the present invention pro embodiment, the present invention provides that the first and vides that the first protease inhibitor is a Cysteine protease the second protease inhibitors are Lima bean trypsin inhibitor inhibitor and the second protease inhibitor is a Metallopro and soybean trypsin inhibitor (SBTI). In another, embodi tease inhibitor. In another embodiment, the present invention ment, the present invention provides that the first and the provides that the first protease inhibitor is a Cysteine protease second protease inhibitors are Lima bean trypsin inhibitor, inhibitor and the second protease inhibitor is a Trypsin inhibi and OVomucoid. In another, embodiment, the present inven tor. In another embodiment, the present invention provides tion provides that the first and the second protease inhibitors that the first protease inhibitor is a Cysteine protease inhibitor are Aprotinin and soy bean trypsin inhibitor (SBTI). In and the second protease inhibitor is a Threonine protease another, embodiment, the present invention provides that the inhibitor. In another embodiment, the present invention pro first and the second protease inhibitors are Aprotinin and vides that the first protease inhibitor is a Cysteine protease Ovomucoid. In another, embodiment, the present invention inhibitor and the second protease inhibitor is an Aspartic provides that the first and the second protease inhibitors are protease inhibitor. In another embodiment, the present inven soybean trypsin inhibitor (SBTI) and Ovomucoid. In another, tion provides that the first protease inhibitor is a Cysteine embodiment, the present invention provides that the first pro protease inhibitor and the second protease inhibitor is a Met tease inhibitor is kunitz. In another, embodiment, the present alloprotease inhibitor. In another embodiment, the present invention provides that the second protease inhibitor is invention provides that the first protease inhibitor is a Trypsin kunitz. In another, embodiment, the present invention pro inhibitor and the second protease inhibitor is a Metallopro vides that the first protease inhibitor is Bowman-Birk pro tease inhibitor. In another embodiment, the present invention tease inhibitor (BBI). In another, embodiment, the present provides that the first protease inhibitor is a Trypsin inhibitor invention provides that the second protease inhibitor is Bow and the second protease inhibitor is a Threonine protease man-Birk protease inhibitor (BBI). inhibitor. In another embodiment, the present invention pro 0034. In another embodiment, the present invention pro vides that the first protease inhibitor is a Trypsin inhibitor and vides that the first protease inhibitor is a serpine and the the second protease inhibitor is an Aspartic protease inhibitor. second protease inhibitor is a Cysteine protease inhibitor. In In another embodiment, the present invention provides that another, embodiment, the present invention provides that the first protease inhibitor is a Trypsin inhibitor and the sec Cysteine protease inhibitors of the invention comprise: cys ond protease inhibitor is a Metalloprotease inhibitor. In tatin, type 1 cystatins (or Stefins), CyStatins of type 2, human another embodiment, the present invention provides that the cystatins C, D, S, SN, and SA, cystatin E/M, cystatin F, type first protease inhibitor is an Aspartic protease inhibitor and 3 cystatins, or kininogens. the second protease inhibitoris a Metalloprotease inhibitor. In 0035. In another embodiment, the present invention pro another embodiment, the present invention provides that the vides that the first protease inhibitor is a serpine and the first protease inhibitor is an Aspartic protease inhibitor and second protease inhibitor is a Threonine protease inhibitor. In the second protease inhibitor is a Threonine protease inhibi another, embodiment, the present invention provides that tor. In another embodiment, the present invention provides Threonine protease inhibitors of the invention comprise: that the first protease inhibitor is an Aspartic protease inhibi Bortezomib, MLN-519, ER-807446, TMC-95A. tor and the second protease inhibitor is a Metalloprotease 0036. In another embodiment, the present invention pro inhibitor. vides that the first protease inhibitor is a serpine and the 0039. In another embodiment, the present invention pro second protease inhibitor is an Aspartic protease inhibitor. In vides that the first protease inhibitor is a Cysteine protease another, embodiment, the present invention provides that inhibitor. In another embodiment, the present invention pro Aspartic protease inhibitors of the invention comprise: vides that the first protease inhibitor is a Metalloprotease C-Macroglobulin, Pepstatin A, Aspartic protease inhibitor inhibitor. In another embodiment, the present invention pro 11, Aspartic protease inhibitor 1, Aspartic protease inhibitor vides that the first protease inhibitor is a Trypsin inhibitor. In 2, Aspartic protease inhibitor 3, Aspartic protease inhibitor 4. another embodiment, the present invention provides that the Aspartic protease inhibitor 5, Aspartic protease inhibitor 6, first protease inhibitor is a Threonine protease inhibitor. In US 2011/0014247 A1 Jan. 20, 2011

another embodiment, the present invention provides that the mg/dosage unit. In another embodiment, the amount is 70 first protease inhibitor is an Aspartic protease inhibitor. In mg/dosage unit. In another embodiment, the amount is 100 another embodiment, the present invention provides that the mg/dosage unit. first protease inhibitor is a Metalloprotease inhibitor. In 0043. In another embodiment, the amount of a first or a another embodiment, the present invention provides that the second protease inhibitor is 0.1-1 mg/dosage unit. In another second protease inhibitor is a Trypsin inhibitor. In another embodiment, the amount of protease inhibitor is 0.2-1 embodiment, the present invention provides that the second mg/dosage unit. In another embodiment, the amount is 0.3-1 protease inhibitor is a Metalloprotease inhibitor. In another mg/dosage unit. In another embodiment, the amount is 0.5-1 embodiment, the present invention provides that the second mg/dosage unit. In another embodiment, the amount is 0.1-2 protease inhibitor is a Threonine protease inhibitor. In another mg/dosage unit. In another embodiment, the amount is 0.2-2 embodiment, the present invention provides that the second mg/dosage unit. In another embodiment, the amount is 0.3-2 protease inhibitor is an Aspartic protease inhibitor. mg/dosage unit. In another embodiment, the amount is 0.5-2 mg/dosage unit. In another embodiment, the amount is 1-2 0040. In some embodiments, protease inhibitors comprise mg/dosage unit. In another embodiment, the amount is 1-10 Suicide inhibitor, transition State inhibitor, or chelating mg/dosage unit. In another embodiment, the amount is 2-10 agents. In some embodiments, the first and second protease mg/dosage unit. In another embodiment, the amount is 3-10 inhibitors of the present invention comprise any combination mg/dosage unit. In another embodiment, the amount is 5-10 of two different protease inhibitors such as but not limited to: mg/dosage unit. In another embodiment, the amount is 1-20 AEBSF-HCl, (epsilon)-aminocaproic acid, (alpha) 1-anti mg/dosage unit. In another embodiment, the amount is 2-20 chymotypsin, antipain, antithrombin III, (alpha) 1-antitrypsin mg/dosage unit. In another embodiment, the amount is 3-20 (alpha 1-proteinase inhibitor), APMSF-HC1 (4-amidi mg/dosage unit. In another embodiment, the amount is 5-20 nophenyl-methane Sulfonyl-fluoride), sprotinin, benzami mg/dosage unit. In another embodiment, the amount is 10-20 dine-HCl, chymostatin, DFP (diisopropylfluoro-phosphate), mg/dosage unit. In another embodiment, the amount is leupeptin, PEFABLOC(R) SC (4-(2-Aminoethyl)-benzene 10-100 mg/dosage unit. In another embodiment, the amount sulfonyl fluoride hydrochloride), PMSF (phenylmethyl sul is 20-100 mg/dosage unit. In another embodiment, the fonyl fluoride), TLCK (1-Chloro-3-tosylamido-7-amino-2- amount is 30-100 mg/dosage unit. In another embodiment, heptanone HCl), TPCK (1-Chloro-3-tosylamido-4-phenyl-2- the amount is 50-100 mg/dosage unit. In another embodi butanone), Ovomucoid, trypsin inhibitor from Soybean, ment, the amount is 10-200 mg/dosage unit. In another Aprotinin, pentamidine isethionate, pepstatin, guanidium, embodiment, the amount is 20-200 mg/dosage unit. In alpha2-macroglobulin, a chelating agent of zinc, iodoacetate, another embodiment, the amount is 30-200 mg/dosage unit. Zinc. Each possibility represents a separate embodiment of In another embodiment, the amount is 50-200 mg/dosage the present invention. unit. In another embodiment, the amount is 100-200 mg/dos 0041. In another embodiment, a formulation comprising a age unit. combination of protease inhibitors as described comprise 0044. In another embodiment, the amount of a first or a Aprotinin and BBI. In another embodiment, a formulation second protease inhibitor utilized in methods and composi comprising a combination of protease inhibitors as described tions of the present invention is 1000k.i.u. (kallikrein inacti comprise Aprotinin and Kunitz. In another embodiment, a vator units)/pill. In another embodiment, the amount is 10 formulation comprising a combination of protease inhibitors ki.u./dosage unit. In another embodiment, the amount is 12 as described comprise BBI and Kunitz. In another embodi ki.u./dosage unit. In another embodiment, the amount is 15 ment, a formulation comprising a combination of protease ki.u./dosage unit. In another embodiment, the amount is 20 inhibitors as described comprise SBTI and BBI. In another ki.u./dosage unit. In another embodiment, the amount is 30 embodiment, a formulation comprising a combination of pro ki.u./dosage unit. In another embodiment, the amount is 40 tease inhibitors as described comprise kunitz and SBTI. ki.u./dosage unit. In another embodiment, the amount is 50 0042. In another embodiment, the amount of a first or a ki.u./dosage unit. In another embodiment, the amount is 70 second protease inhibitor utilized in methods and composi ki.u./dosage unit. In another embodiment, the amount is 100 tions of the present invention is 0.1 mg/dosage unit. In another ki.u./dosage unit. In another embodiment, the amount is 150 embodiment, the amount of protease inhibitor is 0.2 mg/dos ki.u./dosage unit. In another embodiment, the amount is 200 age unit. In another embodiment, the amount is 0.3 mg/dos ki.u./dosage unit. In another embodiment, the amount is 300 age unit. In another embodiment, the amount is 0.4 mg/dos ki.u./dosage unit. In another embodiment, the amount is 500 age unit. In another embodiment, the amount is 0.6 k.i.u./dosage unit. In another embodiment, the amount is 700 mg/dosage unit. In another embodiment, the amount is 0.8 ki.u./dosage unit. In another embodiment, the amount is mg/dosage unit. In another embodiment, the amount is 1 1500 ki.u./dosage unit. In another embodiment, the amount mg/dosage unit. In another embodiment, the amount is 1.5 is 3000 ki.u./dosage unit. In another embodiment, the mg/dosage unit. In another embodiment, the amount is 2 amount is 4000 ki.u./dosage unit. In another embodiment, mg/dosage unit. In another embodiment, the amount is 2.5 the amount is 5000 k.i.u./dosage unit. Each amount of a first mg/dosage unit. In another embodiment, the amount is 3 or a second protease inhibitor represents a separate embodi mg/dosage unit. In another embodiment, the amount is 5 ment of the present invention. mg/dosage unit. In another embodiment, the amount is 7 0045. In some embodiments, omega-3 fatty acid can be mg/dosage unit. In another embodiment, the amount is 10 found in vegetable sources such as the seeds of chia, perilla, mg/dosage unit. In another embodiment, the amount is 12 flax, walnuts, purslane, lingonberry, Seabuckthorn, and hemp. mg/dosage unit. In another embodiment, the amount is 15 In some embodiments, omega-3 fatty acids can also be found mg/dosage unit. In another embodiment, the amount is 20 in the fruit of the acai palm. In another embodiment, the mg/dosage unit. In another embodiment, the amount is 30 omega-3 fatty acid has been provided in the form of a syn mg/dosage unit. In another embodiment, the amount is 50 thetic omega-3 fatty acid. In one embodiment, the omega-3 US 2011/0014247 A1 Jan. 20, 2011

fatty acid of methods and compositions of the present inven raacetic acid (EDTA) or egtazic acid EGTA. In another tion has been provided to the composition in the form of a fish embodiment, EDTA is sodium-EDTA. In some embodi oil. In another embodiment, the omega-3 fatty acid has been ments, the enhancer is NO donor. In some embodiments, the provided in the form of canola oil. In another embodiment, enhancer is abile acid, glycine-conjugated form of abile acid, the omega-3 fatty acid has been provided in the form of or an alkali metal salt. In one embodiment, absorption flaxseed oil. In another embodiment, the omega-3 fatty acid enhancement is achieved through utilization of a combination has been provided in the form of any other omega-3 fatty of C-galactosidase and B-mannanase. In some embodiments, acid-rich source known in the art. In another embodiment, the the enhancer is a fatty acid such as sodium caprate. In one omega-3 fatty acid has been provided in the form of a syn embodiment, the enhancer is sodium glycocholate. In one thetic omega-3 fatty acid. Each form of omega-3 fatty acids embodiment, the enhancer is sodium salicylate. In one represents a separate embodiment of the present invention. embodiment, the enhancer is n-dodecyl-B-D-maltopyrano 0046. In another embodiment, the omega-3 fatty acid of side. In some embodiments, Surfactants serve as absorption methods and compositions of the present invention is an enhancer. In one embodiment, the enhancer is chitisan Such as omega-3 polyunsaturated fatty acid. In another embodiment, N.N.N-trimethyl chitosan chloride (TMC). the omega-3 fatty acid is DHA, an omega-3, polyunsaturated, 0049. In one embodiment, NO donors of the present inven 22-carbon fatty acid also referred to as 4, 7, 10, 13, 16, tion comprise 3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohy 19-docosahexaenoic acid. In another embodiment, the drazino)-1-propanamine, N-ethyl-2-(1-ethyl-hydroxy-2-ni omega-3 fatty acid is D-linolenic acid (9, 12, 15-octadecatri troSohydrazino)-ethanamine, O S-Nitroso-N- enoic acid). In another embodiment, the omega-3 fatty acid is acetylpenicillamine Stearidonic acid (6, 9, 12, 15-octadecatetraenoic acid). In 0050. In another embodiment, the bile acid is cholic acid. another embodiment, the omega-3 fatty acid is eicosatrienoic In another embodiment, the bile acid is chenodeoxycholic acid (ETA: 11, 14, 17-eicosatrienoic acid). In another acid. In another embodiment, the bile acid is taurocholic acid. embodiment, the omega-3 fatty acid is eicSoatetraenoic acid In another embodiment, the bile acid is taurochenodeoxy (8, 11, 14, 17-eicosatetraenoic acid). In one embodiment, the cholic acid. In another embodiment, the bile acid is glyco omega-3 fatty acid is eicosapentaenoic acid (EPA; 5, 8, 11, cholic acid. In another embodiment, the bile acid is glyco 14, 17-eicosapentaenoic acid). In another embodiment, the chenocholic acid. In another embodiment, the bile acid is 3 omega-3 fatty acid is eicosahexaenoic acid (also referred to as beta-monohydroxychloric acid. In another embodiment, the “EPA'; 5, 7, 9, 11, 14, 17-eicosahexaenoic acid). In another bile acid is lithocholic acid. In another embodiment, the bile embodiment, the omega-3 fatty acid is docosapentaenoic acid acid is 5 beta-cholanic acid. In another embodiment, the bile (DPA; 7, 10, 13, 16, 19-docosapenatenoic acid). In another acid is 3,12-diol-7-one-5 beta-cholanic acid. In another embodiment, the omega-3 fatty acid is tetracosahexaenoic embodiment, the bile acid is 3 alpha-hydroxy-12-ketocholic acid (6, 9, 12, 15, 18, 21-tetracosahexaenoic acid). In another acid. In another embodiment, the bile acid is 3 beta-hydroxy embodiment, the omega-3 fatty acid is any other omega-3 12-ketocholic acid. In another embodiment, the bile acid is 12 fatty acid known in the art. Each omega-3 fatty acid repre alpha-3 beta-dihydrocholic acid. In another embodiment, the sents a separate embodiment of the present invention. bile acid is ursodesoxycholic acid. 0047. In another embodiment, compositions of the present 0051. In one embodiment, the enhancer is a nonionic sur invention further comprise a Substance that enhances absorp factant. In one embodiment, the enhancer is a nonionic poly tion of a protein of the invention through an intestinal mucosal oxyethylene ether Surface active agent (e.g. one having an barrier. In another embodiment, compositions of the present HLB value of 6 to 19, wherein the average number of poly invention further comprise a Substance that enhances absorp oxyethylene units is 4 to 30). In another embodiment, the tion of insulin through an intestinal mucosal barrier. In enhancer is an anionic Surface active agents. In another another embodiment, compositions of the present invention embodiment, the enhancer is a cationic Surface active agent. further comprise a Substance that enhances absorption of In another embodiment, the enhancer is an ampholytic Sur Exenatide through an intestinal mucosal barrier. In another face active agent. In one embodiment, Zwitteruionic Surfac embodiment, compositions of the present invention further tants such as acylcarnitines serve as absorption enhancers. comprise a Substance that reduces the degradation of 0052. In another embodiment, the amount of enhancer Exenatide in the digestive system. In another embodiment, utilized in methods and compositions of the present invention compositions of the present invention further comprise a Sub is 0.1 mg/dosage unit. In another embodiment, the amount of stance that reduces the degradation of Exenatide in the stom enhancer is 0.2 mg/dosage unit. In another embodiment, the ach. In another embodiment, compositions of the present amount is 0.3 mg/dosage unit. In another embodiment, the invention further comprise a Substance that reduces the deg amount is 0.4 mg/dosage unit. In another embodiment, the radation of Exenatide in the intestine. Such a substance is amount is 0.6 mg/dosage unit. In another embodiment, the referred to herein as an "enhancer. As provided herein, amount is 0.8 mg/dosage unit. In another embodiment, the enhancers, when used together with omega-3 fatty acids or amount is 1 mg/dosage unit. In another embodiment, the protease inhibitors, enhance the ability of a protein to be amount is 1.5 mg/dosage unit. In another embodiment, the absorbed in the intestine. As provided herein, enhancers, amount is 2 mg/dosage unit. In another embodiment, the when used together with omega-3 fatty acids and/or protease amount is 2.5 mg/dosage unit. In another embodiment, the inhibitors, enhance the ability of insulin to be absorbed in the amount is 3 mg/dosage unit. In another embodiment, the intestine. As provided herein, enhancers, when used together amount is 5 mg/dosage unit. In another embodiment, the with omega-3 fatty acids and/or protease inhibitors, enhance amount is 7 mg/dosage unit. In another embodiment, the the ability of Exenatide to be absorbed in the intestine. amount is 10 mg/dosage unit. In another embodiment, the 0.048. In one embodiment, the enhancer is dide amount is 12 mg/dosage unit. In another embodiment, the canoylphosphatidylcholine (DDPC). In one embodiment, the amount is 15 mg/dosage unit. In another embodiment, the enhancer is a chelating agent such as ethylenediaminetet amount is 20 mg/dosage unit. In another embodiment, the US 2011/0014247 A1 Jan. 20, 2011

amount is 30 mg/dosage unit. In another embodiment, the 0056. In another embodiment, EudragitR), an acrylic poly amount is 50 mg/dosage unit. In another embodiment, the mer, is used as the enteric coating. The use of acrylic poly amount is 70 mg/dosage unit. In another embodiment, the mers for the coating of pharmaceutical preparations is well amount is 100 mg/dosage unit. known in the art. Eudragit Acrylic Polymers have been shown 0053. In another embodiment, the amount of enhancer is to be safe, and are neither absorbed nor metabolized by the 0.1-1 mg/dosage unit. In another embodiment, the amount of body, but rather are eliminated. enhancer is 0.2-1 mg/dosage unit. In another embodiment, the 0057. In another embodiment, the coating is a gelatin coat amount is 0.3-1 mg/dosage unit. In another embodiment, the ing. In another embodiment, microencapsulation is used to amount is 0.5-1 mg/dosage unit. In another embodiment, the protect the insulin against decomposition in the stomach. In amount is 0.1-2 mg/dosage unit. In another embodiment, the another embodiment, the coating is a gelatin coating. In amount is 0.2-2 mg/dosage unit. In another embodiment, the another embodiment, microencapsulation is used to protect amount is 0.3-2 mg/dosage unit. In another embodiment, the Exenatide against decomposition in the stomach. Methods for amount is 0.5-2 mg/dosage unit. In another embodiment, the applying a gelatin coating and for microencapsulation are amount is 1-2 mg/dosage unit. In another embodiment, the well known in the art. Each method represents a separate amount is 1-10 mg/dosage unit. In another embodiment, the embodiment of the present invention. amount is 2-10 mg/dosage unit. In another embodiment, the 0058. In another embodiment, the coating is a film-coat amount is 3-10 mg/dosage unit. In another embodiment, the ing. In another embodiment, the coating is ethylcellulose. In amount is 5-10 mg/dosage unit. In another embodiment, the another embodiment, the coating is a water-based dispersion amount is 1-20 mg/dosage unit. In another embodiment, the of ethylcellulose, e.g. hydroxypropylmethylcelullose amount is 2-20 mg/dosage unit. In another embodiment, the (HPMC) E15. In another embodiment, the coating is a gastro amount is 3-20 mg/dosage unit. In another embodiment, the resistant coatings, e.g. a polymer containing carboxylic acid amount is 5-20 mg/dosage unit. In another embodiment, the groups as a functional moiety. In another embodiment, the amount is 10-20 mg/dosage unit. In another embodiment, the coating is a monolithic matrix. In another embodiment, the amount is 10-100 mg/dosage unit. In another embodiment, coating is a cellulose ether (e.g. hypromellose (HPMC). Each the amount is 20-100 mg/dosage unit. In another embodi type of coating represents a separate embodiment of the ment, the amount is 30-100 mg/dosage unit. In another present invention. embodiment, the amount is 50-100 mg/dosage unit. In 0059. In one embodiment, the protein is a recombinant another embodiment, the amount is 10-200 mg/dosage unit. protein. In one embodiment, the protein is an insulin. In In another embodiment, the amount is 20-200 mg/dosage another embodiment, the protein is a glucagon. In another unit. In another embodiment, the amount is 30-200 mg/dos embodiment, the protein is an interferon gamma. In another age unit. In another embodiment, the amount is 50-200 embodiment, the protein is an interferon alpha. In another mg/dosage unit. In another embodiment, the amount is 100 embodiment, the protein is a growth hormone. In another 200 mg/dosage unit. Each type and amount of enhancer rep embodiment, the protein is an erythropoietin. In another resents a separate embodiment of the present invention. embodiment, the protein is Exenatide. In another embodi 0054. In another embodiment, compositions of the present ment, the protein is granulocyte colony Stimulating factor invention further comprise a coating that inhibits digestion of (G-CSF). In another embodiment, the protein is any other the composition in the stomach of a Subject. In one embodi protein known in the art. ment, coating inhibits digestion of the composition in the 0060. In another embodiment, the protein is a growth hor stomach of a Subject. In one embodiment, the coated dosage mone. In one embodiment, the growth hormone is somatotro forms of the present invention release drug when pH move pin. In another embodiment, the growth hormone is Insulin towards alkaline range. In one embodiment, coating is a Growth Factor-I (IGF-I). In another embodiment, the growth monolayer, wherein in other embodiments coating applied in hormone is any other growth hormone known in the art. multilayers. In one embodiment, coating is a bioadhesive 0061 The molecular weights of some of the proteins men polymer that selectively binds the intestinal mucosa and thus tioned above are as follows: insulin—6 kilodalton (kDa); enables drug release in the attachment site. In one embodi glucagon—3.5 kDa, interferon, 28 kDa, growth hormone— ment, the enteric coating is an enteric film coating. In some 21.5-47 kDa; human serum albumin 69 kDa; erythropoi embodiment, coating comprises biodegradable polysaccha etin 34 kDa; G-CSF 30-34 kDa. Thus, in one embodi ride, chitosan, aquateric aqueous, aquacoat ECD, azo poly ment, the molecular weight of these proteins is appropriate mer, cellulose acetate phthalate, cellulose acetate trimelliate, for administration by methods of the present invention. hydroxypropylmethyl cellulose phthalate, gelatin, poly vinyl 0062. In another embodiment, methods and compositions acetate phthalate, hydrogel, pulsincap, or a combination of the present invention are used to administer a human serum thereof. In one embodiment, pH sensitive coating will be used albumin. Human serum albumin is not, in one embodiment, according to the desired release site and/or profile as knownto considered to be a pharmaceutically-active component; how one skilled in the art. ever, it can be used in the context of the present invention as 0055. In one embodiment, the coating is an enteric coat a therapeutically-beneficial carrier for an active component. ing. Methods for enteric coating are well known in the art, and Each type of protein represents a separate embodiment of the are described, for example, in Siepmann F. Siepmann Jet al. present invention. Blends of aqueous polymer dispersions used for pellet coat 0063. In one embodiment, the protein is an enzyme. In ing: importance of the particle size. J Control Release 2005; Some embodiments, the protein is a receptor ligand, trans 105(3): 226-39; and Huyghebaert N. Vermeire A, Remon.JP. porter, or a storage protein. In one embodiment, the protein is In vitro evaluation of coating polymers for enteric coating and a structural protein. humanileal targeting. IntJ Pharm 2005; 298 (1): 26-37. Each 0064. In some embodiments, the enzyme is an oxi method represents a separate embodiment of the present doreductase, transferase, hydrolase, lyase, isomerase, or invention. ligase. In some embodiments, oxidoreductases act on the US 2011/0014247 A1 Jan. 20, 2011

aldehyde or oxo group of donors, on the CH-CH group of Exenatide and 3 protease inhibitors. In one embodiment, the donors, on the CH-NH(2) group of donors, on the CH NH present invention provides a composition comprising an insu group of donors, on NADH or NADPH, on the CH-OH lin protein and 4 protease inhibitors. In one embodiment, the group of donors, on nitrogenous compounds as donors, on a present invention provides a composition comprising a Sulfur group of donors, on a heme group of donors, on diphe Exenatide and 4 protease inhibitors. In one embodiment, the nols and related Substances as donors, on a peroxide as accep present invention provides a composition comprising an insu tor, on hydrogen as donor, on single donors with incorpora lin protein and 5 protease inhibitors. In one embodiment, the tion of molecular oxygen, on paired donors, on Superoxide as present invention provides a composition comprising a acceptor, oxidizing metal ions, on CH or CH(2) groups, on Exenatide and 5 protease inhibitors. In one embodiment, the iron-sulfur proteins as donors, on reduced flavodoxin as present invention provides a composition comprising an insu donor, on phosphorus or arsenic in donors, or on X-Handy-H lin protein and at least 5 protease inhibitors. In one embodi to form an X-y bond. ment, the present invention provides a composition compris 0065. In some embodiments, transferases are acyltrans ing an insulin protein and at least 7 protease inhibitors. In one ferases or glycosyltransferases. In some embodiments, trans embodiment, the present invention provides a composition ferases transferaldehyde or ketone residues. In some embodi comprising an insulin protein and at least 10 protease inhibi ments, transferases transfer alkyl or aryl groups, other than tOrS. methyl groups. In some embodiments, transferases transfer 0075. In another embodiment, the present invention pro nitrogenous, phosphorous, Sulfur or selenium containing vides a composition comprising an active protein of the groups. invention, at least two protease inhibitors, and an omega-3 0066. In some embodiments, hydrolases are glycosylases fatty acid. In another embodiment, the present invention pro or act on ether bonds, on peptide bonds, on carbon-nitrogen vides a composition comprising an active protein of the bonds, other than peptide bonds, on acid anhydrides, on car invention, at least two protease inhibitors, EDTA or a salt bon-carbon bonds, on halide bonds, on phosphorus-nitrogen thereof (such as Na-EDTA), and an omega-3 fatty acid. bonds, on Sulfur-nitrogen bonds, on carbon-phosphorus 0076. In another embodiment, the present invention pro bonds, on sulfur-sulfur bonds, or on carbon-sulfur bonds. vides that the use of two protease inhibitors in a single oral 0067. In some embodiments, lyases are carbon-carbon composition dramatically, unexpectedly, increase the bio lyases, carbon-oxygen lyases, carbon-nitrogen lyases, car availability of a protein of the invention. In another embodi bon-sulfur lyases, carbon-halide lyases, phosphorus-oxygen ment, the present invention provides that the use of two pro lyases, or other lyases. tease inhibitors in a single oral composition dramatically, 0068. In some embodiments, isomerases are racemases or unexpectedly, increase the bioavailability of insulin. In epimerases, cis-trans-isomerases, intramolecular oxi another embodiment, the present invention provides that the doreductases, intramolecular transferases, intramolecular use of two protease inhibitors in a single oral composition lyases, or other isomerases. dramatically, unexpectedly, increase the bioavailability of 0069. In some embodiments, ligases form carbon-sulfur Exenatide. In another embodiment, the present invention pro bonds, carbon-nitrogen bonds, carbon-carbon bonds, phos vides that the use of two serpins in a single oral composition phoric ester bonds, or nitrogen-metal bonds. dramatically, unexpectedly, increase the bioavailability of a 0070. In some embodiments, transporter proteins are protein of the invention. In another embodiment, the present annexins, ATP-binding cassette transporters, hemoglobin, invention provides that the use of two serpins in a single oral ATPases, calcium channels, potassium channels, sodium composition dramatically, unexpectedly, increase the bio channels, or solute carriers. availability of insulin. In another embodiment, the present 0071. In some embodiments, storage proteins comprise invention provides that the use of two serpins in a single oral albumins, lactoglobulins, casein ovomucin, ferritin, phos composition dramatically, unexpectedly, increase the bio vitin, lactoferrin, or Vitellogenin. In one embodiment, albu availability of Exenatide. In another embodiment, the present mins comprise avidin, ovalbumin, serum albumin, parvalbu invention provides that the use of two trypsin inhibitors in a min, c-reactive protein prealbumin, conalbumin, ricin, single oral composition dramatically, unexpectedly, increase lactalbumin, methemalbumin, or transthyretin. the bioavailability of a protein of the invention. In another 0072. In some embodiments, structural proteins comprise embodiment, the present invention provides that the use of amyloid, collagen elastin, or fibrillin. two trypsin inhibitors in a single oral composition dramati 0073. In some embodiments, the protein is a viral protein, cally, unexpectedly, increase the bioavailability of insulin. In bacterial protein, invertebrate protein, or vertebrate protein. another embodiment, the present invention provides that the In some embodiments, the protein is a recombinant protein. use of two trypsin inhibitors in a single oral composition In one embodiment, the protein is a recombinant protein. In dramatically, unexpectedly, increase the bioavailability of one embodiment, the recombinant protein is a recombinant Exenatide. In another embodiment, the present invention pro human protein. vides that the use of SBTI and Aprotinin in a single oral 0074. In one embodiment, the present invention provides a composition dramatically, unexpectedly, increase the bio composition comprising an insulin protein and at least two availability of a protein of the invention. In another embodi protease inhibitors. In one embodiment, the present invention ment, the present invention provides that the use of SBTI and provides a composition comprising a Exenatide and at least Aprotinin in a single oral composition dramatically, unex two protease inhibitors. In one embodiment, the present pectedly, increase the bioavailability of insulin. In another invention provides a composition comprising an insulin pro embodiment, the present invention provides that the use of tein and two protease inhibitors. In one embodiment, the SBTI and Aprotinin in a single oral composition dramatically, present invention provides a composition comprising a unexpectedly, increase the bioavailability of Exenatide. Exenatide and two protease inhibitors. In one embodiment, 0077. In another embodiment, the present invention pro the present invention provides a composition comprising a vides that the use of a serpin and a Cysteine protease inhibitor US 2011/0014247 A1 Jan. 20, 2011

in a single oral composition dramatically, unexpectedly, 0079. In another embodiment, the present invention pro increase the bioavailability of a protein of the invention. In vides that the use of two protease inhibitors in a single oral another embodiment, the present invention provides that the composition dramatically, unexpectedly, increase the bio use of a serpin and a Cysteine protease inhibitor in a single availability of insulin in a human subject by at least 10%. In oral composition dramatically, unexpectedly, increase the another embodiment, the present invention provides that the bioavailability of insulin. In another embodiment, the present use of two protease inhibitors in a single oral composition invention provides that the use of a serpin and a Cysteine dramatically, unexpectedly, increase the bioavailability of protease inhibitor in a single oral composition dramatically, Exenatide in a human subject by at least 10%. In another unexpectedly, increase the bioavailability of Exenatide. In embodiment, the present invention provides that the use of another embodiment, the present invention provides that the two protease inhibitors in a single oral composition dramati use of a serpin and a Threonine protease inhibitor in a single cally, unexpectedly, increase the bioavailability of insulin in a oral composition dramatically, unexpectedly, increase the human subject by at least 20%. In another embodiment, the bioavailability of a protein of the invention. In another present invention provides that the use of two protease inhibi embodiment, the present invention provides that the use of a tors in a single oral composition dramatically, unexpectedly, serpin and a Threonine protease inhibitor in a single oral increase the bioavailability of Exenatide in a human subject composition dramatically, unexpectedly, increase the bio by at least 20%. In another embodiment, the present invention availability of insulin. In another embodiment, the present provides that the use of two protease inhibitors in a single oral invention provides that the use of a serpin and a Threonine composition dramatically, unexpectedly, increase the bio protease inhibitor in a single oral composition dramatically, availability of insulin in a human subject by at least 30%. In unexpectedly, increase the bioavailability of Exenatide. In another embodiment, the present invention provides that the use of two protease inhibitors in a single oral composition another embodiment, the present invention provides that the dramatically, unexpectedly, increase the bioavailability of use of a serpin and a Metalloprotease inhibitor in a single oral Exenatide in a human subject by at least 30%. In another composition dramatically, unexpectedly, increase the bio embodiment, the present invention provides that the use of availability of a protein of the invention. In another embodi two protease inhibitors in a single oral composition dramati ment, the present invention provides that the use of a serpin cally, unexpectedly, increase the bioavailability of insulin in a and a Metalloprotease inhibitor in a single oral composition human subject by at least 40%. In another embodiment, the dramatically, unexpectedly, increase the bioavailability of present invention provides that the use of two protease inhibi insulin. In another embodiment, the present invention pro tors in a single oral composition dramatically, unexpectedly, vides that the use of a serpin and a Metalloprotease inhibitor increase the bioavailability of Exenatide in a human subject in a single oral composition dramatically, unexpectedly, by at least 40%. In another embodiment, the present invention increase the bioavailability of Exenatide. In another embodi provides that the use of two protease inhibitors in a single oral ment, the present invention provides that the use of a serpin composition dramatically, unexpectedly, increase the bio and an Aspartic protease inhibitorina single oral composition availability of insulin in a human subject by at least 50%. In dramatically, unexpectedly, increase the bioavailability of a another embodiment, the present invention provides that the protein of the invention. In another embodiment, the present use of two protease inhibitors in a single oral composition invention provides that the use of a serpin and an Aspartic dramatically, unexpectedly, increase the bioavailability of protease inhibitor in a single oral composition dramatically, Exenatide in a human subject by at least 50%. In another unexpectedly, increase the bioavailability of insulin. In embodiment, the present invention provides that the use of another embodiment, the present invention provides that the two protease inhibitors in a single oral composition dramati use of a serpin and an Aspartic protease inhibitor in a single cally, unexpectedly, increase the bioavailability of insulin in a oral composition dramatically, unexpectedly, increase the human subject by at least 60%. In another embodiment, the bioavailability of Exenatide. present invention provides that the use of two protease inhibi 0078. In another embodiment, the present invention pro tors in a single oral composition dramatically, unexpectedly, vides that the use of two protease inhibitors in a single oral increase the bioavailability of Exenatide in a human subject composition dramatically, unexpectedly, increase the bio by at least 60%. In another embodiment, the present invention availability of a protein of the invention. In another embodi provides that the use of two protease inhibitors in a single oral ment, the present invention provides that the use of two pro composition dramatically, unexpectedly, increase the bio tease inhibitors in a single oral composition dramatically, availability of insulin in a human subject by at least 70%. In unexpectedly, increase the bioavailability of insulin. In another embodiment, the present invention provides that the another embodiment, the present invention provides that the use of two protease inhibitors in a single oral composition use of two protease inhibitors in a single oral composition dramatically, unexpectedly, increase the bioavailability of dramatically, unexpectedly, increase the bioavailability of Exenatide in a human subject by at least 70%. In another Exenatide. In another embodiment, the present invention pro embodiment, the present invention provides that the use of vides that the use of two protease inhibitors in a single oral two protease inhibitors in a single oral composition dramati composition dramatically, unexpectedly, increase the bio cally, unexpectedly, increase the bioavailability of insulin in availability of insulin in a human subject. In another embodi a human subject by at least 80%. In another embodiment, the ment, the present invention provides that the use of two pro present invention provides that the use of two protease inhibi tease inhibitors in a single oral composition dramatically, tors in a single oral composition dramatically, unexpectedly, unexpectedly, increase the bioavailability of Exenatide in a increase the bioavailability of Exenatide in a human subject human Subject. In another embodiment, the present invention by at least 80%. In another embodiment, the present invention provides that the use of two protease inhibitors in a single oral provides that the use of two protease inhibitors in a single oral composition dramatically, unexpectedly, increase the bio composition dramatically, unexpectedly, increase the bio availability of a protein of the invention in a human subject. availability of insulin in a human subject by at least 90%. In US 2011/0014247 A1 Jan. 20, 2011

another embodiment, the present invention provides that the in a single oral composition dramatically, unexpectedly, use of two protease inhibitors in a single oral composition increase the bioavailability of Exenatide in a human subject dramatically, unexpectedly, increase the bioavailability of by at least 40%. In another embodiment, the present invention Exenatide in a human subject by at least 90%. In another provides that the use of Aprotinin and SBTI in a single oral embodiment, the present invention provides that the use of composition dramatically, unexpectedly, increase the bio two protease inhibitors in a single oral composition dramati availability of Exenatide in a human subject by at least 50%. cally, unexpectedly, increase the bioavailability of insulin in a In another embodiment, the present invention provides that human subject by at least 100%. In another embodiment, the the use of Aprotinin and SBTI in a single oral composition present invention provides that the use of two protease inhibi dramatically, unexpectedly, increase the bioavailability of tors in a single oral composition dramatically, unexpectedly, Exenatide in a human subject by at least 60%. In another increase the bioavailability of Exenatide in a human subject embodiment, the present invention provides that the use of by at least 100%. Aprotinin and SBTI in a single oral composition dramatically, 0080. In another embodiment, the present invention pro unexpectedly, increase the bioavailability of Exenatide in a vides that the use of Aprotinin and SBTI in a single oral composition dramatically, unexpectedly, increase the bio human subject by at least 70%. In another embodiment, the availability of insulin in a human subject by at least 10%. In present invention provides that the use of Aprotinin and SBTI another embodiment, the present invention provides that the in a single oral composition dramatically, unexpectedly, use of Aprotinin and SBTI in a single oral composition dra increase the bioavailability of Exenatide in a human subject matically, unexpectedly, increase the bioavailability of insu by at least 80%. In another embodiment, the present invention lin in a human subject by at least 20%. In another embodi provides that the use of Aprotinin and SBTI in a single oral ment, the present invention provides that the use of Aprotinin composition dramatically, unexpectedly, increase the bio and SBTI in a single oral composition dramatically, unex availability of Exenatide in a human subject by at least 90%. pectedly, increase the bioavailability of insulin in a human In another embodiment, the present invention provides that subject by at least 30%. In another embodiment, the present the use of Aprotinin and SBTI in a single oral composition invention provides that the use of Aprotinin and SBTI in a dramatically, unexpectedly, increase the bioavailability of single oral composition dramatically, unexpectedly, increase Exenatide in a human subject by at least 100%. the bioavailability of insulin in a human subject by at least I0083. In another embodiment, the present invention pro 40%. In another embodiment, the present invention provides vides that the use of two protease inhibitors in a single oral that the use of Aprotinin and SBTI in a single oral composi composition dramatically, unexpectedly, increase the bio tion dramatically, unexpectedly, increase the bioavailability availability of a protein of the invention in a human subject by of insulin in a human subject by at least 50%. In another at least 10%. In another embodiment, the present invention embodiment, the present invention provides that the use of provides that the use of two protease inhibitors in a single oral Aprotinin and SBTI in a single oral composition dramatically, composition dramatically, unexpectedly, increase the bio unexpectedly, increase the bioavailability of insulin in a availability of a protein of the invention in a human subject by human subject by at least 60%. In another embodiment, the at least 20%. In another embodiment, the present invention present invention provides that the use of Aprotinin and SBTI provides that the use of two protease inhibitors in a single oral in a single oral composition dramatically, unexpectedly, composition dramatically, unexpectedly, increase the bio increase the bioavailability of insulin in a human subject by at availability of a protein of the invention in a human subject by least 70%. In another embodiment, the present invention pro at least 30%. In another embodiment, the present invention vides that the use of Aprotinin and SBTI in a single oral provides that the use of two protease inhibitors in a single oral composition dramatically, unexpectedly, increase the bio composition dramatically, unexpectedly, increase the bio availability of insulin in a human subject by at least 80%. in availability of a protein of the invention in a human subject by another embodiment, the present invention provides that the at least 40%. In another embodiment, the present invention use of Aprotinin and SBTI in a single oral composition dra provides that the use of two protease inhibitors in a single oral matically, unexpectedly, increase the bioavailability of insu composition dramatically, unexpectedly, increase the bio lin in a human subject by at least 90%. In another embodi availability of a protein of the invention in a human subject by ment, the present invention provides that the use of Aprotinin at least 50%. In another embodiment, the present invention and SBTI in a single oral composition dramatically, unex provides that the use of two protease inhibitors in a single oral pectedly, increase the bioavailability of insulin in a human composition dramatically, unexpectedly, increase the bio subject by at least 100%. availability of a protein of the invention in a human subject by 0081. In another embodiment, the present invention pro at least 60%. In another embodiment, the present invention vides that the use of Aprotinin and SBTI in a single oral provides that the use of two protease inhibitors in a single oral composition dramatically, unexpectedly, increase the bio composition dramatically, unexpectedly, increase the bio availability of availability of a protein of the invention in a human subject by 0082) Exenatide in a human subject by at least 10%. In at least 70%. In another embodiment, the present invention another embodiment, the present invention provides that the provides that the use of two protease inhibitors in a single oral use of Aprotinin and SBTI in a single oral composition dra composition dramatically, unexpectedly, increase the bio matically, unexpectedly, increase the bioavailability of availability of a protein of the invention in a human subject by Exenatide in a human subject by at least 20%. In another at least 80%. In another embodiment, the present invention embodiment, the present invention provides that the use of provides that the use of two protease inhibitors in a single oral Aprotinin and SBTI in a single oral composition dramatically, composition dramatically, unexpectedly, increase the bio unexpectedly, increase the bioavailability of Exenatide in a availability of a protein of the invention in a human subject by human subject by at least 30%. In another embodiment, the at least 90%. In another embodiment, the present invention present invention provides that the use of Aprotinin and SBTI provides that the use of two protease inhibitors in a single oral US 2011/0014247 A1 Jan. 20, 2011

composition dramatically, unexpectedly, increase the bio of Exenatide to a subject, whereby a substantial fraction of availability of a protein of the invention in a human subject by Exenatide retains its activity after absorption through an at least 100%. intestinal mucosal barrier of the Subject, comprising admin 0084. In another embodiment, this invention further pro istering orally to the Subject a pharmaceutical composition vides the use of sustained release dosage forms (e.g. Sustained comprising Exenatide and at least two protease inhibitors, release microencapsulation) that enable the treatment fre thereby orally administering a protein with an enzymatic quency to be reduced to once or twice a day. In another activity to a subject. embodiment, the insulin dosage is increased correspondingly I0088. In another embodiment, the present invention pro with decreasing frequency of administration. In another vides a method for oral administration of a protein with an embodiment, the Exenatide dosage is increased correspond enzymatic activity to a Subject, whereby a Substantial fraction ingly with decreasing frequency of administration. Each type of the protein retains the enzymatic activity after absorption of coating, dosage form, etc, that inhibits digestion of the through an intestinal mucosal barrier of the Subject, compris composition in the stomach represents a separate embodi ing administering orally to the Subject a pharmaceutical com ment of the present invention. position comprising the protein at least two protease inhibi 0085 Methods of measuring insulin levels are well known tors and an omega-3 fatty acid, thereby orally administering a in the art. In one embodiment, levels of recombinant insulin protein with an enzymatic activity to a Subject. In another are measuring using a human insulin radio-immunoassay embodiment, the present invention provides a method for oral (RIA) kit, e.g. the kit manufactured by. Linco Research Inc. administration of a protein with an enzymatic activity to a (St. Charles, Mo.). In another embodiment, levels of C pep subject, whereby a substantial fraction of the protein retains tide are measured as well, to determine the relative contribu the enzymatic activity after absorption through an intestinal tions of endogenous and exogenous insulin to observed rises mucosal barrier of the Subject, comprising administering in insulin levels. In another embodiment, insulin ELISA kits orally to the Subject a pharmaceutical composition compris are used. In another embodiment, insulin levels are measured ing the protein, at least two protease inhibitors and an by any other method known in the art. In another embodi omega-3 fatty acid, thereby orally administering a protein ment, Exenatide levels are measured by a method known in with an enzymatic activity to a human Subject. In another the art. Each possibility represents a separate embodiment of embodiment, the present invention provides a method for oral the present invention. administration of insulin to a subject, whereby a substantial I0086. In another embodiment, a multiparticulate dosage fraction of insulin retains its activity after absorption through forms is used to inhibit digestion of the composition in the an intestinal mucosal barrier of the subject, comprising stomach. In another embodiment, a multiparticulate dosage administering orally to the Subject a pharmaceutical compo forms is used to inhibit digestion of the composition in the sition comprising insulin, at least two protease inhibitors and stomach. In another embodiment, the present invention com an omega-3 fatty acid, thereby orally administering a protein prises administering separate dosage forms wherein each pro with an enzymatic activity to a Subject. In another embodi tease inhibitor is in a separate dosage form and the protein is ment, the present invention provides a method for oral admin in an additional dosage form. In another embodiment, the istration of Exenatide to a subject, whereby a substantial present invention comprises administering separate dosage fraction of Exenatide retains its activity after absorption forms wherein the two protease inhibitors are in a single through an intestinal mucosal barrier of the Subject, compris dosage form and the protein is in an additional dosage form. ing administering orally to the Subject a pharmaceutical com 0087. In another embodiment, the present invention pro position comprising Exenatide, at least two protease inhibi vides a method for oral administration of a protein with an tors and an omega-3 fatty acid, thereby orally administering a enzymatic activity to a subject, whereby a Substantial fraction protein with an enzymatic activity to a subject. of the protein retains the enzymatic activity after absorption I0089. In another embodiment, the present invention pro through an intestinal mucosal barrier of the Subject, compris vides a method for oral administration of a protein with an ing administering orally to the Subject a pharmaceutical com enzymatic activity to a Subject, whereby a Substantial fraction position comprising the protein and at least two protease of the protein retains the enzymatic activity after absorption inhibitors, thereby orally administering a protein with an through an intestinal mucosal barrier of the Subject, compris enzymatic activity to a subject. In another embodiment, the ing administering orally to the Subject a pharmaceutical com present invention provides a method for oral administration position comprising the protein at least two protease inhibi of a protein with an enzymatic activity to a subject, whereby tors, an omega-3 fatty acid, and Na-EDTA, thereby orally a Substantial fraction of the protein retains the enzymatic administering a protein with an enzymatic activity to a Sub activity after absorption through an intestinal mucosal barrier ject. In another embodiment, the present invention provides a of the Subject, comprising administering orally to the Subject method for oral administration of a protein with an enzymatic a pharmaceutical composition comprising the protein and at activity to a subject, whereby a substantial fraction of the least two protease inhibitors, thereby orally administering a protein retains the enzymatic activity after absorption protein with an enzymatic activity to a human Subject. In through an intestinal mucosal bather of the Subject, compris another embodiment, the present invention provides a method ing administering orally to the Subject a pharmaceutical com for oral administration of insulin to a subject, whereby a position comprising the protein, at least two protease inhibi substantial fraction of insulin retains its activity after absorp tors, an omega-3 fatty acid, and Na-EDTA, thereby orally tion through an intestinal mucosal barrier of the Subject, administering a protein with an enzymatic activity to a human comprising administering orally to the Subject a pharmaceu Subject. In another embodiment, the present invention pro tical composition comprising insulin and at least two protease vides a method for oral administration of insulinto a Subject, inhibitors, thereby orally administering a protein with an whereby a substantial fraction of insulin retains its activity enzymatic activity to a subject. In another embodiment, the after absorption through an intestinal mucosal bather of the present invention provides a method for oral administration Subject, comprising administering orally to the Subject a phar US 2011/0014247 A1 Jan. 20, 2011 maceutical composition comprising insulin, at least two pro omega-3 fatty acid, and at least two protease inhibitors, tease inhibitors, an omega-3 fatty acid, and Na-EDTA, thereby treating diabetes mellitus. thereby orally administering a protein with an enzymatic 0093. In another embodiment, the present invention pro activity to a Subject. In another embodiment, the present vides a method for treating diabetes mellitus in a human invention provides a method for oral administration of Subject, comprising administering orally to the Subject a phar Exenatide to a subject, whereby a substantial fraction of maceutical composition comprising an insulin, Na-EDTA, Exenatide retains its activity after absorption through an omega-3 fatty acid, and at least two protease inhibitors, intestinal mucosal bather of the Subject, comprising admin thereby treating diabetes mellitus. In another embodiment, istering orally to the Subject a pharmaceutical composition the present invention provides a method for treating diabetes comprising Exenatide, at least two protease inhibitors, an mellitus in a human Subject, comprising administering orally omega-3 fatty acid, and Na-EDTA, thereby orally adminis to the Subject a pharmaceutical composition comprising an tering a protein with an enzymatic activity to a Subject. insulin, Na-EDTA, omega-3 fatty acid, and at least two pro 0090. In another embodiment, the present invention pro tease inhibitors, thereby treating diabetes mellitus. vides a method for oral administration of a protein with an 0094. In another embodiment, the present invention pro enzymatic activity to a subject, whereby a Substantial fraction vides a method for treating diabetes mellitus in a human of the protein retains the enzymatic activity after absorption Subject, comprising administering orally to the Subject a phar through an intestinal mucosal bather of the Subject, compris maceutical composition comprising a Exenatide and at least ing administering orally to the Subject a pharmaceutical com two protease inhibitors, thereby treating diabetes mellitus. In position comprising the protein at least two protease inhibi another embodiment, the present invention provides a method tors and Na-EDTA, thereby orally administering a protein for treating diabetes mellitus in a human Subject, comprising with an enzymatic activity to a Subject. In another embodi administering orally to the Subject a pharmaceutical compo ment, the present invention provides a method for oral admin sition comprisinga Exenatide and at least two protease inhibi istration of a protein with an enzymatic activity to a subject, tors, thereby treating diabetes mellitus. whereby a substantial fraction of the protein retains the enzy 0095. In another embodiment, the present invention pro matic activity after absorption through an intestinal mucosal vides a method for treating diabetes mellitus in a human barrier of the Subject, comprising administering orally to the Subject, comprising administering orally to the Subject a phar Subject a pharmaceutical composition comprising the pro maceutical composition comprising a Exenatide, omega-3 tein, at least two protease inhibitors and Na-EDTA, thereby fatty acid, and at least two protease inhibitors, thereby treat orally administering a protein with an enzymatic activity to a ing diabetes mellitus. In another embodiment, the present human Subject. In another embodiment, the present invention invention provides a method for treating diabetes mellitus in provides a method for oral administration of insulin to a a human Subject, comprising administering orally to the Sub subject, whereby a substantial fraction of insulin retains its ject a pharmaceutical composition comprising a Exenatide, activity after absorption through an intestinal mucosal barrier omega-3 fatty acid, and at least two protease inhibitors, of the Subject, comprising administering orally to the Subject thereby treating diabetes mellitus. a pharmaceutical composition comprising insulin, at least 0096. In another embodiment, the present invention pro two protease inhibitors and Na-EDTA, thereby orally admin vides a method for treating diabetes mellitus in a human istering a protein with an enzymatic activity to a subject. In Subject, comprising administering orally to the Subject a phar another embodiment, the present invention provides a method maceutical composition comprising a Exenatide, Na-EDTA, for oral administration of Exenatide to a subject, whereby a omega-3 fatty acid, and at least two protease inhibitors, substantial fraction of Exenatide retains its activity after thereby treating diabetes mellitus. In another embodiment, absorption through an intestinal mucosal barrier of the Sub the present invention provides a method for treating diabetes ject, comprising administering orally to the Subject a phar mellitus in a human Subject, comprising administering orally maceutical composition comprising Exenatide, at least two to the Subject a pharmaceutical composition comprising a protease inhibitors and Na-EDTA, thereby orally administer Exenatide, Na-EDTA, omega-3 fatty acid, and at least two ing a protein with an enzymatic activity to a subject. protease inhibitors, thereby treating diabetes mellitus. 0091. In another embodiment, the present invention pro 0097. In one embodiment, the diabetes mellitus is Type I vides a method for treating diabetes mellitus in a human diabetes. In another embodiment, the diabetes mellitus is Subject, comprising administering orally to the Subject a phar Type II diabetes. In another embodiment, the diabetes melli maceutical composition comprising an insulin and at least tus is insulin-dependent diabetes. In another embodiment, the two protease inhibitors, thereby treating diabetes mellitus. In diabetes mellitus is non-insulin-dependent diabetes. In another embodiment, the present invention provides a method another embodiment, the diabetes mellitus is any other type for treating diabetes mellitus in a human Subject, comprising of diabetes known in the art. Each possibility represents a administering orally to the Subject a pharmaceutical compo separate embodiment of the present invention. sition comprising an insulin and at least two protease inhibi 0098. In one embodiment, three treatments a day of the tors, thereby treating diabetes mellitus. insulin composition are administered. In another embodi 0092. In another embodiment, the present invention pro ment, two treatments a day are administered. In another vides a method for treating diabetes mellitus in a human embodiment, four treatments a day are administered. In Subject, comprising administering orally to the Subject a phar another embodiment, one treatment a day is administered. In maceutical composition comprising an insulin, omega-3 fatty another embodiment, more than four treatments a day are acid, and at least two protease inhibitors, thereby treating administered. Each possibility represents a separate embodi diabetes mellitus. In another embodiment, the present inven ment of the present invention. tion provides a method for treating diabetes mellitus in a 0099. Any of the methods of the present invention may human Subject, comprising administering orally to the Sub utilize, in various embodiments, any of the compositions of ject a pharmaceutical composition comprising an insulin, the present invention. US 2011/0014247 A1 Jan. 20, 2011

0100. In another embodiment, the present invention pro tissues. Similarly, insulin administered according to the vides a composition for oral administration of insulin, com present invention reaches the intestine and is absorbed in the prising an insulin protein and at least two protease inhibitors, body through the intestine and through the portal system to whereby a substantial fraction of the insulin retains the enzy the liver. This absorption route thus resembles the physiologi matic activity after absorption through an intestinal mucosal cal secretion of insulin by the pancreas, enabling, in this barrier of a human subject. In another embodiment, the embodiment, delicate control of the blood glucose level and present invention provides a composition for oral administra the metabolic activities of the liver and the peripheral organs tion of Exenatide, comprising an insulin protein and at least controlled by insulin. By contrast, when insulin is adminis two protease inhibitors, whereby a substantial fraction of the tered to insulin-deficient diabetic patients via the peripheral Exenatide retains the enzymatic activity after absorption venous system, the concentration of insulin in the portal vein through an intestinal mucosal bather of a human Subject. In is similar to that in the peripheral circulation, resulting in one embodiment, the present invention provides a composi hypoinsulinemia in the portal vein and the liver and hyperin tion for oral administration of a protein, comprising a protein Sulinemia in the peripheral venous system. This leads, in one and at least two protease inhibitors, whereby a substantial embodiment, to an abnormal pattern of glucose disposal. fraction of the protein retains the enzymatic activity after 0105. In another embodiment, different constituents of absorption through an intestinal mucosal barrier of the Sub compositions of the present composition are absorbed at dif ject. ferent rates from the intestinal lumen into the blood stream. 0101. In one embodiment, the present invention provides The absorption of the bile acid, in one embodiment, is sig the use of a protein and at least two protease inhibitors in the nificantly faster than the absorption of insulin. manufacture of a medicament for oral administration of a 0106 For this reason, in another embodiment, a drug regi protein with an enzymatic activity to a subject, whereby a men involving ingestion of a pair of pills at spaced intervals, Substantial fraction of the protein retains the enzymatic activ e.g., a second pill containing a higher concentration of ity after absorption through an intestinal mucosal barrier of enhancer is taken at a defined interval (e.g. 30 minutes) after the Subject. In one embodiment, the present invention pro the first pill. In another embodiment, certain of the constitu vides the use of a protein, at least two protease inhibitors, and ents are microencapsulated to enhance the absorption of the an omega-3 fatty acid in the manufacture of a medicament for insulin into the system. In another embodiment, certain of the oral administration of a protein with an enzymatic activity to constituents are microencapsulated to enhance the absorption a subject, whereby a substantial fraction of the protein retains of the Exenatide into the system. the enzymatic activity after absorption through an intestinal 0107. In one embodiment, a treatment protocol of the mucosal barrier of the subject. In one embodiment, the present invention is therapeutic. In another embodiment, the present invention provides the use of a protein, at least two protocol is prophylactic. Each possibility represents a sepa protease inhibitors, Na-EDTA, and an omega-3 fatty acid in rate embodiment of the present invention. the manufacture of a medicament for oral administration of a 0108. In another embodiment, solid carriers/diluents for protein with an enzymatic activity to a subject, whereby a use in methods and compositions of the present invention Substantial fraction of the protein retains the enzymatic activ include, but are not limited to, a gum, a starch (e.g. corn ity after absorption through an intestinal mucosal barrier of starch, pregeletanized starch), a Sugar (e.g., lactose, mannitol, the subject. Sucrose, dextrose), a cellulosic material (e.g. microcrystalline 0102. In one embodiment, the present invention provides cellulose), an acrylate (e.g. polymethylacrylate), calcium car the use of an insulin protein and at least two protease inhibi bonate, magnesium oxide, talc, or mixtures thereof. tors in the manufacture of a medicament for treating diabetes 0109. In another embodiment, the compositions further mellitus in a Subject. In one embodiment, the present inven comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, tion provides the use of an insulin protein, at least two pro ethyl cellulose, guar gum, hydroxypropyl cellulose, hydrox tease inhibitors, and an omega-3 fatty acid in the manufacture ypropyl methyl cellulose, povidone), disintegrating agents of a medicament for treating diabetes mellitus in a Subject. In (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, one embodiment, the present invention provides the use of an croScarmelose sodium, crospovidone, guar gum, Sodium insulin protein, at least two protease inhibitors, Na-EDTA, starch glycolate), buffers (e.g., Tris-HCI., acetate, phosphate) and an omega-3 fatty acid in the manufacture of a medica of various pH and ionic strength, additives such as albuminor ment for treating diabetes mellitus in a Subject. gelatin to prevent absorption to Surfaces, detergents (e.g., 0103) In one embodiment, the present invention provides Tween 20, Tween 80, Pluronic F68, bile acid salts), protease the use of a Exenatide protein and at least two protease inhibi inhibitors, Surfactants (e.g. sodium lauryl Sulfate), perme tors in the manufacture of a medicament for treating diabetes ation enhancers, Solubilizing agents (e.g., glycerol, polyeth mellitus in a Subject. In one embodiment, the present inven ylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium tion provides the use of a Exenatide protein, at least two metabisulfite, butylated hydroxyanisole), stabilizers (e.g. protease inhibitors, and an omega-3 fatty acid in the manu hydroxypropyl cellulose, hyroxypropylmethyl cellulose), facture of a medicament for treating diabetes mellitus in a Viscosity increasing agents (e.g. carbomer, colloidal silicon Subject. In one embodiment, the present invention provides dioxide, ethyl cellulose, guar gum), Sweeteners (e.g. aspar the use of a Exenatide protein, at least two protease inhibitors, tame, citric acid), preservatives (e.g., Thimerosal, benzyl Na-EDTA, and an omega-3 fatty acid in the manufacture of a alcohol, parabens), lubricants (e.g. Stearic acid, magnesium medicament for treating diabetes mellitus in a Subject. Stearate, polyethylene glycol, Sodium lauryl Sulfate), flow 0104. In one embodiment, methods and compositions of aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl the present invention have the advantage of more closely phthalate, triethyl citrate), emulsifiers (e.g. carbomer, mimicking physiological insulin secretion by the pancreas. hydroxypropyl cellulose, sodium lauryl Sulfate), polymer When insulin is secreted into the portal vein, the liver is coatings (e.g., poloxamers or poloxamines), coating and film exposed to a greater insulin concentration than peripheral forming agents (e.g. ethyl cellulose, acrylates, polymethacry US 2011/0014247 A1 Jan. 20, 2011

lates) and/or adjuvants. Each of the above excipients repre 0116 Each of the above additives, excipients, formula sents a separate embodiment of the present invention. tions and methods of administration represents a separate 0110. In some embodiments, the dosage forms of the embodiment of the present invention. present invention are formulated to achieve an immediate 0117. In one embodiment, the term “treating” refers to release profile, an extended release profile, or a delayed curing a disease. In another embodiment, “treating refers to release profile. In some embodiments, the release profile of preventing a disease. In another embodiment, “treating the composition is determined by using specific excipients refers to reducing the incidence of a disease. In another that serve for example as binders, disintegrants, fillers, or embodiment, “treating refers to ameliorating symptoms of a coating materials. In one embodiment, the composition will disease. In another embodiment, “treating refers to inducing beformulated to achieve a particular release profile as known remission. In another embodiment, “treating refers to slow to one skilled in the art. ing the progression of a disease. 0111. In one embodiment, the composition is formulated Experimental Details Section as an oral dosage form. In one embodiment, the composition is a solid oral dosage form comprising tablets, chewable Example 1 tablets, or capsules. In one embodiment the capsules are soft Capsules Comprising a Protein and a Combination gelatin capsules. In another embodiment, capsules as of Protease Inhibitors described herein are hard-shelled capsules. In another embodiment, capsules as described herein are soft-shelled 0118 Materials and Experimental Methods capsules. In another embodiment, capsules as described herein are made from gelatine. In another embodiment, cap Formulation Sules as described herein are made from plant-based gelling 0119) 4 days prior to dosing, formulation were prepared Substances like carrageenans and modified forms of starch containing: (1) 8 mg insulin, 150 mg EDTA, 125 mg SBTI in and cellulose. 1 ml fish oil in a soft-gel capsule (SwissCup), (2). 8 mg 0112. In other embodiments, controlled- or sustained-re insulin, 150 mg EDTA, 150000 KIU Aprotinin in 1 ml fish oil lease coatings utilized in methods and compositions of the in a soft-gel capsule (SwissCup), (3) 8 mg insulin, 150 mg present invention include formulation in lipophilic depots EDTA, 150000 KIU Aprotinin, 125 mg SBTI in 1 ml fish oil (e.g. fatty acids, waxes, oils). in a soft-gel capsule (SwissCup). The formulations were 0113. The compositions also include, in another embodi stored in the refrigerator (4°C.) until dosing. ment, incorporation of the active material into or onto par 0120 Results ticulate preparations of polymeric compounds such as poly I0121. In the next experiment, the formulations as lactic acid, polglycolic acid, hydrogels, etc, or onto describes in the “Formulation' section were orally consumed liposomes, microemulsions, micelles, unilamellar or multila by healthy human subjects. As shown in FIG.1, blood glucose mellar vesicles, erythrocyte ghosts, or spheroplasts.) Such levels were significantly reduced and more stable in human compositions will influence the physical state, solubility, sta subjects treated with formulation (3) (8 mg insulin, 150 mg bility, rate of in vivo release, and rate of in vivo clearance. In EDTA, 150000 KIU Aprotinin, 125 mg SBTI in 1 ml fish oil another embodiment, particulate compositions of the active in a soft-gel capsule (SwissCup). These results also Suggest ingredients are coated with polymers (e.g. poloxamers or that the two protease inhibitors in formulation (3) had a syn poloxamines) ergistic effect in lowering blood glucose levels. 0114. In another embodiment, the compositions contain I0122. It should be emphasized that no differences in the ing the insulin and omega-3 fatty acid are delivered in a reduction of blood glucose levels were observed in a previous vesicle, e.g. a liposome (see Langer, Science 249:1527-1533 experiment in dogs treated with the same formulations com (1990); Treat et al., in Liposomes in the Therapy of Infectious prising a single protease inhibitor (formulations 1 or 2, SBTI Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, or Aprotinin) or a formulation that comprises both SBTI and New York, pp. 353–365 (1989); Lopez-Berestein, ibid., pp. Aprotinin (formulation 3). Thus, the results regarding the 317-327; see generally ibid). In another embodiment, the reduction in blood glucose levels with insulin and a combi compositions containing the Exenatide and omega-3 fatty nation of protease inhibitors was unexpected. acid are delivered in a vesicle, e.g. a liposome (see Langer, (0123. Furthermore, as shown in FIG. 1D-F, total blood Science 249:1527-1533 (1990); Treat et al., in Liposomes in insulin was significantly higher especially between 220-300 the Therapy of Infectious Disease and Cancer, Lopez-Berest minutes in human Subjects treated with formulation (3) (8 mg ein and Fidler (eds.), Liss, New York, pp. 353–365 (1989); insulin, 150 mg EDTA, 150000 KIU Aprotinin, 125 mg SBTI Lopez-Berestein, ibid., pp. 317-327; see generally ibid). in 1 ml fish oil in a soft-gel capsule (SwissCup). These results 0115 The preparation of pharmaceutical compositions also suggest that the two protease inhibitors informulation (3) that contain an active component, for example by mixing, had a synergistic effect in stabling insulin levels. granulating, or tablet-forming processes, is well understood 0.124. As shown in FIG. 1G-I, blood C-peptide levels were in the art. The active therapeutic ingredient is often mixed significantly reduced in human Subjects treated with formu with excipients that are pharmaceutically acceptable and lation (3) (8 mg insulin, 150 mg EDTA, 150000 KIU Apro compatible with the active ingredient. For oral administra tinin, 125 mg SBTI in 1 ml fish oil in a soft-gel capsule tion, the active ingredients of compositions of the present (SwissCup). These results also suggest that the two protease invention are mixed with additives customary for this pur inhibitors in formulation (3) had a synergistic effect in low pose. Such as vehicles, stabilizers, or inert diluents, and con ering blood C-peptide levels. verted by customary methods into suitable forms for admin 0.125. In a similar experiment in healthy humans, formu istration, such as tablets, coated tablets, hard or soft gelatin lations comprising: (1) 150 mg EDTA, 24 mg Aprotinin, 75 capsules, aqueous, alcoholic or oily Solutions. mg BBI, and 10 mg insulin; (2) 150 mg EDTA, 24 mg. Apro US 2011/0014247 A1 Jan. 20, 2011

tinin, 75 mg Kunitz, and 12 mg insulin; and (3) 150 mg of determine the optimal amounts. The most effective enhancer/ EDTA, 24 mg Aprotinin, 75 mg SBTI, and 8 mg of insulin amount is used in Subsequent experiments. were found to be effective in lowering blood glucose levels and maintaining sufficient blood insulin levels for over 3 Example 5 hours after a meal. Optimization of Type and Amount of Insulin 0126. It should be emphasized that no differences in the I0131 Various types and amounts of insulin e.g. those reduction of C-peptide levels were observed in a previous listed above in the specification) are compared for their ability experiment in dogs treated with the same formulations com to regulate blood Sugar in methods and compositions of the prising a single protease inhibitor (formulations 1 or 2, SBTI present invention. Insulin tablets or capsules are formulated or Aprotinin) or a formulation that comprises both SBTI and as described in the above Examples, except that the type and Aprotinin (formulation 3). Thus, the results regarding the amount of insulin is varied. The most effective typefamount reduction in blood C-peptide levels with insulin and a com of insulin is used in clinical trials. bination of protease inhibitors was unexpected. 1-62. (canceled) 0127. The above described formulations are also active 63. An oral pharmaceutical composition comprising a with 0.5 ml fish oil both in dogs and humans. Dogs do not recombinant protein having a molecular weight of up to 100, react to Aprotinin. Therefore in dogs the reduction in blood 000 Daltons, a first protease inhibitor, and a second protease glucose with SBTI alone was equivalent to the reduction in inhibitor, wherein each of said first protease inhibitor and said blood glucose with both Aprotinin and SBTI. Moreover, a second protease inhibitor inhibits trypsin. formulation comprising insulin and Aprotinin but not SBTI 64. The oral pharmaceutical composition of claim 63, did not reduce blood glucose in dogs. wherein said recombinant protein is an enzyme. 65. The oral pharmaceutical composition of claim 63, wherein said recombinant protein is insulin. Example 2 66. The oral pharmaceutical composition of claim 63, wherein said recombinant protein is Exenatide. Optimization of Source of Omega-3 Fatty Acids 67. The oral pharmaceutical composition of claim 63, wherein said first protease inhibitor and said second protease 0128 Various omega-3 fatty acids or sources of omega-3 inhibitor are serpins. fatty acids (e.g. those listed above in the specification) are 68. The oral pharmaceutical composition of claim 63, compared for their ability to preserve insulin following oral wherein at least one of said first protease inhibitor and said administration in methods and compositions of the present second protease inhibitor is selected from group consisting of invention. Insulin tablets or capsules are formulated as Lima bean trypsin inhibitor, Aprotinin, soy bean trypsin described in the above Examples, except that the insulin is inhibitor (SBTI), and Ovomucoid. dissolved in the alternate source instead of in fish oil. The 69. The oral pharmaceutical composition of claim 68, most effective source of omega-3 fatty acids is used in Sub wherein said first protease inhibitor is SBTI and said second sequent Examples. protease inhibitor is Aprotinin. 70. The oral pharmaceutical composition of claim 70, fur Example 3 ther comprising an omega-3 fatty acid. 71. The oral pharmaceutical composition of claim 70. Optimization of Protease Inhibitors wherein said omega-3 fatty acid is derived from fish oil. 72. The oral pharmaceutical composition of claim 63, fur 0129. Various protease inhibitors (either non-toxic or hav ther comprising a substance that enhances absorption of said ing an acceptable toxicity profile; e.g. those listed above in the recombinant protein through an intestinal mucosal barrier. specification) are compared for their ability to preserve insu 73. The oral pharmaceutical composition of claim 72, lin following oral administration in methods and composi wherein said substance is EDTA or a salt thereof. tions of the present invention. Insulin and/or Exenatide tablets 74. The oral pharmaceutical composition of claim 72, or capsules are formulated as described in the above wherein said substance is a bile acid or alkali metal salt Examples, except that the alternate protease inhibitors are thereof. substituted for SBTI and/or Aprotinin. Amounts of the pro 75. The oral pharmaceutical composition of claim 63, fur tease inhibitors are also varied, to determine the optimal ther comprising a coating that inhibits digestion of said oral amounts. The most effective protease inhibitor/amount is pharmaceutical composition in a stomach of a subject. used in Subsequent Examples. 76. The oral pharmaceutical composition of claim 75, wherein said coating is an enteric coating or gelatin coating. Example 4 77. A method for oral administration of a protein having a molecular weight up to 100,000 Daltons to a subject, whereby Optimization of Enhancer a Substantial fraction of said protein retains its activity after absorption through an intestinal mucosal barrier of said Sub 0130 Various enhancers (e.g. those listed above in the ject, comprising administering orally to said Subject a phar specification) are compared for their ability to facilitate maceutical composition comprising said protein, a first pro absorption of insulin following oral administration in meth tease inhibitor and a second protease inhibitor, wherein each ods and compositions of the present invention. Insulin tablets of said first protease inhibitor and said second protease inhibi or capsules are formulated as described in the above tor inhibits trypsin. Examples, except that the alternate enhancers are substituted 78. The method of claim 77, wherein said protein is a for EDTA. Amounts of the enhancers are also varied, to recombinant protein. US 2011/0014247 A1 Jan. 20, 2011

79. The method of claim 77, wherein said protein is an 95. A method for treating diabetes mellitus in a subject, enzyme. comprising administering orally to said Subject a pharmaceu 80. The method of claim 77, wherein said protein is insulin. tical composition comprising insulin, Exenatide, or a combi 81. The method of claim 77, wherein said protein is nation thereof, a first protease inhibitor, and a second protease Exenatide. 82. The method of claim 77, wherein said protein is inhibitor, wherein each of said first protease inhibitor and said selected from the group consisting of a glucagon, an inter second protease inhibitor inhibits trypsin, thereby treating feron gamma, an interferon alpha, a growth hormone, an diabetes mellitus. erythropoietin, and granulocyte colony stimulating factor 96. The method of claim 95, wherein said subject is human. (G-CSF). 97. The method of claim 95, wherein said pharmaceutical 83. The method of claim 77, wherein said protein is composition further comprises an omega-3 fatty acid. selected from the group consisting of a receptor ligand, a transport protein, and a storage protein. 98. The method of claim 97, wherein said omega-3 fatty 84. The method of claim 77, wherein said protein has a acid is derived from fish oil. molecular weight of 1-50 kilodaltons. 99. The method of claim 95, wherein said first protease 85. The method of claim 77, wherein said composition inhibitor and said second protease inhibitor are serpins. further comprises an omega-3 fatty acid. 86. The method of claim 85, wherein said omega-3 fatty 100. The method of claim 95, wherein at least one of said acid is derived from fish oil. first protease inhibitor and said second protease is selected 87. The method of claim 77, wherein said first protease from the group consisting of Lima bean trypsin inhibitor, inhibitor and said second protease inhibitor are serpins. Aprotinin, soybean trypsin inhibitor (SBTI), and Ovomu 88. The method of claim 77, wherein at least one of said coid. first protease inhibitor and said second protease is selected 101. The method of claim 100, wherein said first protease from the group consisting of Lima bean trypsin inhibitor, inhibitor is SBTI and said second protease inhibitor is Apro Aprotinin, soybean trypsin inhibitor (SBTI), and Ovomu tinin. coid. 89. The method of claim 88, wherein said first protease 102. The method of claim 95, wherein said pharmaceutical inhibitor is SBTI and said second protease inhibitor is Apro composition further comprises a Substance that enhances tinin. absorption of said insulin, Exenatide, or combination thereof 90. The method of claim 77, wherein said pharmaceutical through an intestinal mucosal barrier. composition further comprises a Substance that enhances 103. The method of claim 102, wherein said substance is absorption of said recombinant protein through an intestinal EDTA or a salt thereof. mucosal barrier. 104. The method of claim 102, wherein said substance is a 91. The method of claim 90, wherein said substance is bile acid or alkali metal salt thereof. EDTA or a salt thereof. 92. The method of claim 90, wherein said substance is a 105. The method of claim 95, wherein said pharmaceutical bile acid or alkali metal salt thereof. composition further comprises a coating that inhibits diges 93. The method of claim 77, wherein said pharmaceutical tion of said pharmaceutical composition in a stomach of a composition further comprises a coating that inhibits diges Subject. tion of said pharmaceutical composition in a stomach of a 106. The method of claim 105, wherein said coating is an Subject. enteric coating or gelatin coating. 94. The method of claim 93, wherein said coating is an enteric coating or gelatin coating. c c c c c