Recombinant protease inhibitor-containing compositions, methods for producing and using the same

Abstract

To provide compositions for oral administration of therapeutic proteins and peptides.SOLUTION: A composition comprises a therapeutic peptide or therapeutic protein of up to 100 kilodalton, a chelator of divalent cations, and an isolated recombinantly expressed Bowman-Birk inhibitor (BBI), and the composition provides improved sustained activity of the therapeutic protein and peptide. A method of use of the composition is also provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] Provided herein are oral compositions for the administration of therapeutic proteins, including recombinant protease inhibitors, that exhibit improved sustained activity. [Background technology]

[0002] Protein / peptide-based drugs are generally susceptible to degradation in the gastrointestinal tract and / or are not efficiently absorbed in a biologically active form from the small intestine into the bloodstream. Orally delivered formulations have been developed for protein-based drugs, such as insulin, exenatide, other hormones, etc.

[0003] Trypsin and chymotrypsin inhibitors derived from soybean (Glycine max) are already available and are considered safe for human consumption. One example of such inhibitors is named SBTI (soybean trypsin inhibitor) and consists of KTI (Kunitz Trypsin Inhibitor), which inhibits trypsin, and BBI (Bowman-Birk inhibitor), which inhibits trypsin and chymotrypsin. Various commercial sources of SBTI are known, and improved methods for isolating the active ingredients are described, for example, in U.S. Patent No. 5,399,633, which is incorporated herein by reference in its entirety.

[0004] There has been significant progress and promise in providing a platform technology for the oral delivery of peptide-based therapeutics, particularly those targeting metabolic diseases, such that the ability to improve upon the current state of the art is difficult to imagine. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] PCT International Publication No. WO / 2013 / 114369 Summary of the Invention

[0006] Surprisingly, it has now been shown that a stable oral composition comprising a peptide / protein based therapeutic, an enhancer / stabilizer, an omega-3 fatty acid, and a protease inhibitor(s), already proven to provide sustained efficacy in oral formulations, can be further improved if the protease inhibitor is derived from a recombinant source.

[0007] SBTI, chemically extracted to obtain separate preparations of purified KTI and BBI, have been used to prepare oral protein therapeutic drug-containing formulations having known quantities of BBI and KTI, respectively, with known purity and activity when included in the oral formulation, and effective in treating known diseases. For example, in US Patent No. 5,999,633, which is incorporated herein by reference in its entirety, an improved method for the purification of SBTI was developed in which each product was prepared at its own specifications with high levels of activity, levels of high molecular weight (MW) contaminants were minimized, and high purity industrial yield preparations were achieved.

[0008] Surprisingly, it has now been found that when protease inhibitors are prepared / derived from recombinant heterologous production means, even though downstream purification steps are shared between chemically purified and recombinantly expressed proteases, the resulting therapeutic protein / peptide activity is greater and longer sustained when a recombinant source is used for the initial protease production source.

[0009] The invention provides an oral pharmaceutical composition comprising a therapeutic peptide or protein of up to 100 kilodaltons, a chelator of divalent cations, and isolated recombinantly expressed BBI.

[0010] In some embodiments, recombinantly expressed BBI has a nucleotide sequence that shares at least 95% identity with that set forth in SEQ ID NO:1, or in some embodiments, recombinantly expressed BBI has a nucleotide sequence that shares at least 97% identity with that set forth in SEQ ID NO:1, or in some embodiments, recombinantly expressed BBI has a nucleotide sequence that shares at least 99% identity with that set forth in SEQ ID NO:1. In some embodiments, recombinantly expressed BBI has the nucleotide sequence set forth in SEQ ID NO:1.

[0011] In some embodiments, recombinantly expressed BBI is expressed in a yeast expression system, which in some embodiments is expressed in a Pichia pastoris yeast expression system.

[0012] In some embodiments, recombinantly expressed BBI is expressed in a heterologous expression system, wherein the nucleotide sequence encoding the same is optimized for codon usage in the organism being used as such heterologous expression system.

[0013] In some embodiments, the formulation further comprises, in addition to the BBI, a trypsin inhibitor, which in some embodiments is KTI3, which in some embodiments is chemically purified, and in some embodiments is recombinantly expressed.

[0014] In some embodiments, the therapeutic protein is useful in treating a subject having a metabolic disease or disorder. In some embodiments, the therapeutic peptide or protein is selected from the group consisting of insulin, influenza hemagglutinin, influenza neuraminidase, glucagon, interferon gamma, interferon beta, interferon alpha, growth hormone, erythropoietin, GLP-1, GLP-1 analogs, granulocyte colony-stimulating factor (G-CSF), renin, growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon. , coagulation factors, anticoagulation factors, atrial natriuretic factor, surfactant protein A (SP-A), surfactant protein B (SP-B), surfactant protein C (SP-C), surfactant protein D (SP-D), plasminogen activator, bombesin, hematopoietic growth factors (colony stimulating factors, multiple), tumor necrosis factor (TNF) proteins, enkephalinase, RANTES (Regulatory Antibody for Activation of T Cell Expression and Secretion) control), human macrophage inflammatory protein (MIP-1-α), serum albumin, Mullerian inhibitory substance, relaxin, mouse gonadotropin-releasing hormone, deoxyribonuclease, inhibin, activin, vascular endothelial growth factor (VEGF), neurotrophin, neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), nerve growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor, transforming growth factor (TGF), insulin-like growth factor-I and -II (I In the present invention, the IGF-I and IGF-II are selected from the group consisting of des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein 1 (IGFBP-1), IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, keratinocyte growth factor, osteoinductive protein, bone morphogenetic protein (BMP)-2, BMP-7, colony stimulating factor (CSF), interleukin (IL), superoxide dismutase, dissociation accelerating factor, chemokine family members, and complement factors.In some embodiments, the therapeutic peptide or protein is selected from the group consisting of insulin and GLP-1 analogs. In some embodiments, the therapeutic peptide or protein is selected from the group consisting of insulin and GLP-1 analogs, leptin, bone morphogenetic proteins, including BMP-4, and nerve growth factors, including NGF-1.

[0015] In some embodiments, the therapeutic peptide or protein is an immunomodulatory agent, and in some embodiments, the therapeutic peptide or protein is glatiramer acetate, hi some embodiments, the therapeutic peptide or protein is provided to promote autotolerance to allergens or antigens associated with an autoimmune disease.

[0016] In some embodiments, a therapeutic peptide or protein is useful in treating, alleviating, preventing, or reducing the incidence or severity of a metabolic disease or disorder.

[0017] In some embodiments, the invention provides an oral pharmaceutical composition as described herein for use as a medicament for orally administering a therapeutic peptide or protein to a subject.

[0018] In some aspects, the oral pharmaceutical compositions of the invention prevent rapid degradation of a therapeutic peptide or protein in a subject, or in some embodiments, promote sustained activity of an orally administered therapeutic peptide or protein in a subject, or in some embodiments, promote sustained bioavailability of an orally administered therapeutic peptide or protein in a subject. According to this aspect, and in some embodiments, the sustained activity or bioavailability of an orally administered therapeutic peptide or protein exceeds the sustained activity or bioavailability obtained following use of an otherwise identical oral formulation that contains chemically purified BBI instead of the isolated recombinantly expressed BBI.

[0019] In some embodiments, the invention provides for the use of an oral pharmaceutical composition as described herein for orally administering a therapeutic protein to a subject. In some embodiments, the oral pharmaceutical composition prevents rapid degradation of the therapeutic peptide or protein in the subject. In some embodiments, the oral pharmaceutical composition promotes sustained activity of the orally administered therapeutic peptide or protein in the subject. In some embodiments, the oral pharmaceutical composition promotes sustained bioavailability of the orally administered therapeutic peptide or protein in the subject. In some embodiments, the use provides a therapeutically effective amount of the peptide or protein, wherein the BBI, or the up to 100 kilodalton therapeutic peptide or protein, or combinations thereof, is provided in the formulation at a lower concentration than would be therapeutically effective in an otherwise identical oral formulation that contains chemically purified BBI instead of the isolated recombinantly expressed BBI, and wherein the use achieves a comparable therapeutic effect in the subject.

[0020] In some embodiments, the invention provides a method for orally administering a therapeutic peptide or protein to a subject, the method comprising administering to the subject an oral pharmaceutical composition as described herein, thereby administering the therapeutic protein to the subject.

[0021] In some embodiments, the invention provides a method of preventing degradation of an orally administered therapeutic peptide or protein in a subject, the method comprising administering to the subject an oral pharmaceutical composition as described herein.

[0022] In some embodiments, the invention provides a method of promoting sustained activity of an orally administered therapeutic peptide or protein in a subject, the method comprising administering to the subject an oral pharmaceutical composition as described herein.

[0023] In some embodiments, the invention provides a method of promoting sustained bioavailability of an orally administered therapeutic peptide or protein in a subject, the method comprising administering to the subject an oral pharmaceutical composition as described herein.

[0024] In some embodiments, the invention provides a method of providing a therapeutically effective amount of a peptide or protein to a subject in an oral formulation, the method comprising the step of preparing an oral pharmaceutical composition comprising a therapeutic peptide or protein of up to 100 kilodaltons, a divalent cation chelator, and an isolated recombinantly expressed Bowman-Birk Inhibitor (BBI), wherein the BBI, or the therapeutic peptide or protein of up to 100 kilodaltons, or a combination thereof, is provided in the formulation at a lower concentration than is therapeutically effective in an otherwise identical oral formulation that contains chemically purified BBI instead of the isolated recombinantly expressed BBI, and achieves a comparable therapeutic effect in the subject. [Brief description of the drawings]

[0025] The following figures are by way of illustrative example and are not to be construed as limiting the claimed invention. [Figure 1] Chromatogram of purified rBBI. Results of RPHPLC analysis are overlaid with available standard samples. [Diagram 2] Protein assessment by BCA. Lyophilized powder samples were gravimetrically weighed and dissolved in respective buffer (SOP number: QC-006) to a final concentration of 1mg / ml for quantification. Protein yield result by BCA: 94% [Diagram 3] SDS-PAGE analysis of rBBI. Electrophoresis was performed, gels were scanned, and bands were quantified using Syngene's gel documentation system in combination with software Genesys for image capture and Gene tool for densitometric quantification studies. Samples at 1 milligram per milliliter (mg / ml) were loaded onto a 20% Phastgel™. Lane 1: protein marker; lane 2, blank; lane 3, 2 μg rBBI under reducing conditions; lane 4, 5 μg rBBI under reducing conditions; lane 5, 10 μg rBBI under reducing conditions; lane 6, blank; lane 7, 2 μg rBBI under non-reducing conditions; lane 4, 5 μg rBBI under non-reducing conditions; lane 5, 10 μg rBBI under non-reducing conditions. [Figure 4-1] Chymotrypsin Inhibition Assay: Results demonstrated that sample showed 2.3 mg of chymotrypsin was inhibited by 1 mg of inhibitor. [Figure 4-2] Chymotrypsin Inhibition Assay: Results demonstrated that sample showed 2.3 mg of chymotrypsin was inhibited by 1 mg of inhibitor. [Figure 5-1] Trypsin Inhibition Assay. Results demonstrated that sample showed 1.7 mg of trypsin was inhibited by 1 mg of inhibitor. [Figure 5-2] Trypsin Inhibition Assay. Results demonstrated that sample showed 1.7 mg of trypsin was inhibited by 1 mg of inhibitor. [Figure 6] Average blood glucose profile over time after administration of oral insulin formulations. In vivo studies were performed in fasting pigs treated with oral insulin formulations (8 mg) prepared with protease inhibitors (BBI) derived from natural sources or engineered using recombinant expression technology. The formulations were inserted into the duodenum using an endoscope to give a final dose of 4 mg of insulin. Blood samples were taken over a 5-hour monitoring period for determination of blood glucose concentration. In addition to 8 mg insulin and 150 mg EDTA, each formulation contains SBTI as described in US Pat. No. 5,999,433, or recombinant BBI (rBBI) 100 mg, rBBI 100 mg and 25 mg KTI, or rBBI 50 mg and KTI 25 mg, as indicated. Each group receiving the rBBI containing composition experienced a more rapid decline in circulating glucose levels compared to animals receiving BBI chemically purified from the SBTI starting material. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention provides improved oral compositions comprising one or more therapeutic peptides or proteins, including recombinant protease inhibitor BBI, that surprisingly outperform compositions comprising the same chemically purified protease inhibitors, despite the respective downstream purification steps being comparable. Surprisingly, it has now been shown that stable oral compositions comprising peptide / protein-based therapeutics, enhancers / stabilizers, omega-3 fatty acids, and protease inhibitor(s), already proven to provide sustained efficacy in oral formulations, can be further improved when the protease inhibitor is derived from a recombinant source.

[0027] The present invention provides an oral pharmaceutical composition comprising an oil-based liquid formulation, wherein the oil-based liquid formulation comprises a therapeutic peptide or protein of up to 100 kilodaltons, a divalent cation chelator, and isolated recombinantly expressed BBI.

[0028] In some embodiments, recombinantly expressed BBI has a nucleotide sequence that shares at least 95% identity with that set forth in SEQ ID NO:1, or in some embodiments, recombinantly expressed BBI has a nucleotide sequence that shares at least 97% identity with that set forth in SEQ ID NO:1, or in some embodiments, recombinantly expressed BBI has a nucleotide sequence that shares at least 99% identity with that set forth in SEQ ID NO:1. In some embodiments, recombinantly expressed BBI.

[0029] As used herein, the terms "protein" and "peptide" are used interchangeably. Neither term is intended to impose a limitation on the number of amino acids present, except where a limitation is expressly stated.

[0030] In some embodiments, oral formulations as described herein contain recombinantly expressed BBI, and in some embodiments may further contain additional protease inhibitors, chemically purified from conventional commercially available SBTI preparations, such as KTI3 isolated from soy flour, as described.

[0031] As shown herein in Example 4, oral formulations containing a subclinical 4 mg insulin dose provided excellent glycemic control of rapid circulating blood glucose levels, with the effect lasting for at least 3 hours when 25 mg rBBI was included in the formulation, an effect that was 75% longer than the duration of effect measured in control animals. Similarly, when 8 mg insulin was administered, the rBBI-containing formulation outperformed a formulation containing chemically extracted SBTI.

[0032] For the ratio of antitrypsin to antichymotrypsin activity present in rBBI-containing preparations, the activities ranged from 1.6:1 to 1:1. For the ratio of antitrypsin to antichymotrypsin activity present in rBBI-containing preparations, the activities were also demonstrated to range from 1.5:1 to 1:1.

[0033] Unless otherwise indicated, antichymotrypsin activity referred to herein is measured using chymotrypsin having an activity of 40 BTEE units per mg of chymotrypsin and is expressed as mg of chymotrypsin inhibited per mg of protein being tested. BTEE refers to N-benzoyl-L-tyrosine ethyl ester (see instructions for Sigma-Aldrich product no. B6125).

[0034] Unless otherwise indicated, antitrypsin activity referred to herein is measured using trypsin with an activity of 10,000 BAEE per mg of trypsin and is expressed as mg of trypsin inhibited per mg of protein being tested. BAEE refers to Na-benzoyl-L-arginine ethyl ester solution (see instructions for Sigma-Aldrich product number B4500). For example, in a typical assay, one unit corresponds to the amount of inhibitor that reduces trypsin activity by one benzoyl-L-arginine ethyl ester unit (BAEE-U). One BAEE-U is the amount of enzyme that increases the absorbance at 253 nm by 0.001 per minute at pH 7.6 and 25°C. See, e.g., K. Ozawa, M. Laskowski, 1966, J. Biol. Chem. 241:3955; and Y. Birk, 1976, Meth. Enzymol. 45:700.

[0035] In an additional aspect, KTI3 isolated from soy flour is provided, wherein in various embodiments, the KTI3 is at least 85% pure as measured by SDS-PAGE, brilliant blue staining, or imager quantitation.

[0036] In yet another aspect, there is provided KTI3 isolated from soy flour, wherein the protein content of the KTI3 is greater than 95% as determined by BCA analysis.

[0037] In yet another aspect, KTI3 isolated from soy flour is provided, wherein the KTI3 contains less than 0.1% high MW contaminants as assessed, for example, by SDS-PAGE and imager quantification.

[0038] One of skill in the art will understand that each of the above purity requirements, with respect to its protein content, level of contaminants, or potency, will generally be evaluated before the KTI3 is mixed with one or more other components of the pharmaceutical composition.

[0039] In further embodiments, the pharmaceutical compositions described above include a coating that resists degradation in the stomach, hi more specific embodiments, the coating is a pH sensitive capsule, or alternatively, a soft gelatin capsule.

[0040] In another embodiment, the pharmaceutical composition further comprises a therapeutic protein of up to 100 kilodaltons as an active ingredient, hi another embodiment, the active ingredient is a non-protein molecule that is susceptible to degradation or inactivation in the human digestive tract.

[0041] In another embodiment, an oral pharmaceutical composition is provided comprising an oil-based liquid formulation, wherein the oil-based liquid formulation comprises a therapeutic protein of up to 100 kilodaltons (kDa), a divalent cation chelator, and a recombinant BBI as described herein, and optionally an additional protease inhibitor. In another embodiment, the liquid formulation consists essentially of a therapeutic protein of up to 100 kDa, a divalent cation chelator, a recombinant BBI as described herein, and an oil. In another embodiment, the liquid formulation consists essentially of a therapeutic protein of up to 100 kDa, a divalent cation chelator, a recombinant BBI as described herein, an oil, and an emulsifier. In another embodiment, the liquid formulation consists essentially of a therapeutic protein of up to 100 kDa, a divalent cation chelator, a recombinant BBI as described herein, an oil, and two emulsifiers.

[0042] In another aspect, an oral pharmaceutical composition is provided comprising an oil-based liquid formulation, wherein the oil-based liquid formulation comprises a therapeutic protein of up to 100 kDa, a chelator of divalent cations, and a recombinant BBI as described herein, such that said liquid formulation has an antichymotrypsin activity of at least 50 mg of chymotrypsin inhibited per ml of liquid formulation. In other embodiments, the liquid formulation has an antichymotrypsin activity of at least 35, 40, 45, 55, or 60 mg of chymotrypsin inhibited per ml of liquid formulation. In yet other embodiments, the liquid formulation has an antichymotrypsin activity in the range of 35-70, 40-70, 45-70, 50-70, or 40-60 mg of chymotrypsin inhibited per ml of liquid formulation. In other embodiments, the liquid formulation further comprises an antitrypsin activity of at least 25 mg of trypsin inhibited per ml of liquid formulation. In other embodiments, the liquid formulation further comprises an antitrypsin activity such that at least 30, 35, 40, 45, or 50 mg of trypsin is inhibited per ml of liquid formulation. Alternatively, the liquid formulation further comprises an antitrypsin activity such that at least 25-50, 30-50, 35-50, 25-40, or 25-45 mg of trypsin is inhibited per ml of liquid formulation.

[0043] In another aspect, a method is provided for creating a pharmaceutical composition, comprising: (a) providing a preparation of recombinant BBI as described herein, a therapeutic protein of up to 100 kilodaltons, and a divalent cation chelator; and (b) mixing said recombinant BBI, therapeutic protein, and chelator as described herein into an oil-based liquid formulation. Additionally, each of the embodiments described herein for other ingredients and additional ingredients that may be present can be incorporated into this method. In another embodiment, a pharmaceutical composition created by this method is provided.

[0044] "Liquid" as used herein refers to a composition having a viscosity in the range of 1 to 1000 millipascal seconds at 20° C. Fish oil, for example, is a liquid under ambient conditions. The term includes oil-based solutions, suspensions and combinations thereof.

[0045] In one embodiment, BBI refers to Bowman-Birk inhibitor; Uniprot number P01055 [database accessed Jan. 28, 2013]).

[0046] A representative precursor sequence for BBI is as follows:

[0047] [ka]

[0048] In some embodiments, KTI as used herein refers to KTI3. (Uniprot number P01070: database accessed January 3, 2013). A representative precursor sequence of KTI3 is as follows:

[0049] [ka]

[0050] In some embodiments, KTI3 is commercially available as chemically purified, e.g., purified from soybean flour, and can be included in oral compositions as described herein, hi other embodiments, KTI3 can be recombinantly produced as well.

[0051] Additional Protease Inhibitors

[0052] Trypsin inhibitors known in the art are specific for trypsin, while others inhibit trypsin and other proteases such as chymotrypsin. Trypsin inhibitors can be derived from animal or vegetable sources: for example, soybean, corn, lima bean, and other beans, pumpkin, sunflower, pancreas and lungs of cows and other animals, egg whites of chickens and turkeys, soy-based infant formula, and mammalian blood. Trypsin inhibitors can also be of microbial origin: for example, antipain; see H. Umezawa, 1976, Meth. Enzymol. 45,678. Trypsin inhibitors can also be arginine or lysine mimics, or other synthetic compounds: for example, arylguanidines, benzamidines, 3,4-dichloroisocoumarin, diisopropylfluorophosphate, gabexate mesylate, or phenylmethanesulfonyl fluoride. As used herein, an arginine or lysine mimic is a P 1 Compounds capable of binding to the pocket and / or interfering with trypsin active site function.

[0053] In certain embodiments, additional trypsin inhibitors utilized in the described methods and compositions include lima bean trypsin inhibitor and aprotinin, (also known as pancreatic trypsin inhibitor or basic pancreatic trypsin inhibitor [BPTI]; Uniprot No. P00974 [database accessed Jan. 2, 2013]), Kazal inhibitor (pancreatic secretory trypsin inhibitor), ovomucoid, alpha 1-antitrypsin, cortisol-binding globulin, Centerin ([SERPINA9 / GCET1 (germinal center B cell expressed transcript 1)], PI-6 (Sun et al. 1995), PI-8 (Sprecher et al. 1995), bomapin, clade A serpin, serpin), [e.g., Serpina3 (NCBI gene ID: 12), Serpina6 (NCBI gene ID: 866), Serpina12 (NCBI gene ID: 145264); Serpina10 (NCBI gene ID: 51156); Serpina7 (NCBI gene ID: 6906); Serpina9 (NCBI gene ID: 327657); Serpina11 (NCBI gene ID: 256394); Serpina13 (NC and Serpina4 (NCBI Gene ID: 5104)], Yukopin (Serpinb12; Gene ID: 89777), antipain, benzamidine, 3,4-dichloroisocoumarin, diisopropylfluorophosphate, and gabexate mesylate. In other embodiments, one of the above inhibitors is selected.

[0054] The representative precursor sequence of aprotinin is:

[0055] [ka]

[0056] In other embodiments, the oil-based liquid formulations utilized in the described methods and compositions contain recombinant BBI and isolated or recombinant KTI, or both isolated and recombinant aprotinin, or a combination thereof.

[0057] In certain embodiments, the rBBI and optional second protease inhibitor, such as KTI, utilized in the described methods and compositions, if any, are stored using a preservative, hi other embodiments, the rBBI and optional second protease inhibitor, such as KTI, are prepared and stored without the use of a preservative.

[0058] Therapeutic Proteins

[0059] The therapeutic proteins for the compositions and methods described herein are, in some embodiments, isolated prior to inclusion in the described pharmaceutical compositions. "Isolated" in this context excludes providing the therapeutic protein as a homogenized tissue preparation or other form that contains significant amounts of contaminating proteins. A preferred example of an isolated protein or peptide is a recombinant protein or peptide. An even more preferred embodiment is a synthetic protein, in other words, a protein produced in a cell-free device. Those skilled in the art will understand in light of the present disclosure that both wild-type and mutant forms of therapeutic proteins may be utilized.

[0060] Certain proteins and peptides are known to be specific inhibitors of trypsin and / or chymotrypsin, including, but not limited to, those described herein as being trypsin and / or chymotrypsin inhibitors. Such proteins are not intended for use as therapeutic components in the described compositions and are excluded from the definition of "therapeutic protein" as used herein.

[0061] Those skilled in the art will appreciate in light of the present disclosure that a variety of therapeutic proteins may be used in the described methods and compositions. In certain embodiments, the therapeutic protein is up to 100 kilodaltons (kDa) in size, generally between 1 and 100 kDa. In more specific embodiments, the size is up to 90 kDa. In other embodiments, the size is up to 80 kDa. In other embodiments, the size is up to 70 kDa. In other embodiments, the size is up to 60 kDa. In other embodiments, the size is up to 50 kDa. Preferably, the size is between 1 and 90 kDa. In other embodiments, the size is between 1 and 80 kDa. In other embodiments, the size is between 1 and 70 kDa. In other embodiments, the size is between 1 and 60 kDa. In other embodiments, the size is between 1 and 50 kDa.

[0062] Therapeutic proteins suitable for use herein include derivatives that are modified (by covalent attachment of non-amino acid moieties to the protein). For example, but not limited to, proteins include proteins modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, genetically added polypeptides as fusion proteins, or derivatization with known protecting / blocking groups. High MW PEG can be attached to therapeutic proteins through site-specific attachment to the N-terminus or C-terminus of PEG, with or without a multifunctional linker, or through the epsilon amino group present in lysine residues. Additionally, derivatives can include one or more non-classical amino acids.

[0063] In certain, more specific embodiments, the therapeutic protein utilized in the described methods and compositions is insulin, influenza hemagglutinin, influenza neuraminidase, glucagon, interferon gamma, interferon beta, interferon alpha, growth hormone, erythropoietin, GLP-1, GLP-1 analogs, leptin, granulocyte colony-stimulating factor (G-CSF), renin, growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, coagulation factors (e.g., Factor VII, Factor VIII, Factor C, DC factor, tissue factor (TF) and thrombin), anticoagulant factors (e.g. protein C), atrial natriuretic factor, surfactant protein A (SP-A), surfactant protein B (SP-B), surfactant protein C (SP-C), surfactant protein D (SP-D), plasminogen activators (e.g. urokinase or human urinary or human tissue plasminogen activator (t-PA)), bombesin, hematopoietic growth factors (also known as colony-stimulating factors, multiple), tumor necrosis factor (TNF) proteins (e.g. TNFα, TNFβ, TNFβ-2,4-1BBL), enkephalinase, RANTES (regulatory regulation of normal T cell expression and activation), human macrophage inflammatory protein (MIP-1-α), serum albumin, Mullerian inhibitory substance, relaxin, mouse gonadotropin releasing hormone, deoxyribonuclease, inhibin, activin, vascular endothelial growth factor (VEGF), neurotrophic factors (e.g., brain-derived neurotrophic factor [BDNF]), neurotrophic factor-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), nerve growth factor, platelet-derived growth factor (PDGF), fibroblast growth factors (e.g., α-FGF and β-FGF), transforming growth factors (TGFs) (e.g., TGFα and TGFβ, including TGF-1, TGF-2, TGF-3, TGF-4, and TGF-5), NGF-1, including nerve growth factor, insulin-like growth factor-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins (including IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6), keratinocyte growth factor, osteoinductive factors, bone morphogenetic protein (BMP)-2, BMP-7, BMP-4, colony stimulating factors (CSFs) (e.g., M-CSF and GM-CSF), interleukins (ILs) (e.g., IL-1 through IL-13, IL-15, IL-18, and IL-23), superoxide dismutase, dissociation accelerating factors, chemokine family members (e.g., iotaxin and MCP-1), and complement factors (e.g., C3 and C5).

[0064] In yet another aspect, the therapeutic proteins / peptides for incorporation into the oral formulations of the invention function as enzyme replacement therapy (ERT) in patients suffering from inherited metabolic diseases, for example, where a genetic defect results in a structural defect or absence of an endogenous enzyme essential to a metabolic pathway. These metabolic disorders then result in the accumulation of substrates and / or their intermediates, leading to pathological conditions. For example, and in some embodiments, the therapeutic proteins / peptides may include glucocerebrosidase, alpha galactosidase, acid alpha glucosidase, alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylgalactosamine 4-sulfatase, arylsulfatase A, acid sphingomyelinase, alkaline phosphatase, pyridoxal 5-phosphate, porphobilinogen deaminase, and phenylalanine ammonia lyase.

[0065] In other embodiments, therapeutic proteins / peptides serve to alleviate the symptoms of a pathological condition by augmenting existing endogenous pathways or by exerting novel catalytic functions. Such therapeutic enzymes have been successfully utilized in a variety of pathological conditions ranging from oncology to neuromuscular dysfunction, hemostasis, and even cosmetic surgery. For example, and in some embodiments, therapeutic proteins / peptides may include

[0066] Alteplase, Reteplase, Tenecteplase, human tissue plasminogen activator, Urokinase, Streptokinase, Anistreplace Eminases, Drotrecognin-a, Factor VIIa, human coagulation Factor VIIa, L-Asparaginase, Carboxypeptidase Rasburicase, Adenosine deaminase, Botulinum toxin type A, Hyaluronidase, etc.

[0067] In other embodiments, the therapeutic protein / peptide is selected from the group consisting of Factor VIIa, Factor XIII, Factor IX, Factor X, Factor XI, vWF, Protein C, Antithrombin III, Fibrinogen, C1 esterase inhibitor, Alpha-1 proteinase inhibitor (α1-PI), Glucocerebrosidase, Alpha-L-iduronidase, Iduronate sulfatase, N-acetylgalactosamine-4-sulfatase, N-acetylgalactosamine-6-sulfatase, Heparan sulfate sulfatase, Alpha-galactosidase A, Alpha-glucosidase, Acid sphingomyelinase, Alpha-mannosidase, Arylsulphatase A, Lysosomal acid lipase (LAL), Sucrase-isomaltase, Adenosine deaminase, and the like. deaminase), insulin-like growth factor 1 (IGF-1), alkaline phosphatase, porphobilinogen deaminase, or a combination thereof.

[0068] In yet more specific embodiments, the therapeutic protein is insulin. Alternatively, the therapeutic protein may be a GLP-1 inhibitor. In some more specific embodiments, the therapeutic protein is exenatide. In other embodiments, both insulin and exenatide are present in the described compositions. In other embodiments, the liquid formulation consists essentially of insulin, exenatide, a divalent cation chelator, isolated BBI, and oil. In other embodiments, the liquid formulation consists essentially of insulin, exenatide, a divalent cation chelator, isolated BBI, at least one emulsifier, and oil. In yet other embodiments, the liquid formulation consists essentially of insulin, exenatide, a divalent cation chelator, isolated KTI3, aprotinin, and oil. In further embodiments, the liquid formulation consists essentially of insulin, exenatide, a divalent cation chelator, isolated KTI3, aprotinin, at least one emulsifier, and oil.

[0069] Those skilled in the art will appreciate in light of this disclosure that various types of insulin are suitable for the described methods and compositions. Exemplary insulin proteins include, but are not limited to, both wild-type insulin and mutant insulins, including synthetic human insulin, synthetic bovine insulin, synthetic porcine insulin, synthetic whale insulin, and metal complexes of insulin, such as zinc complexes of insulin, protamine zinc insulin, and globin zinc.

[0070] Various classes of insulin may also be utilized, such as, for example, rapid acting insulin, lente insulin, semilente insulin, ultralente insulin, NPH insulin, glargine insulin, lispro insulin, aspart insulin, or a combination of two or more of the above types of insulin.

[0071] In a particularly preferred embodiment, the insulin of the described methods and compositions is wild-type human insulin (Uniprot ID P01308). Of the 110 amino acids, 1-24 are the signal peptide, 25-54 form the insulin B chain, 57-87 form the C peptide, and 90-110 form the insulin A chain. In one preferred embodiment, human insulin is produced as a recombinant protein in bacterial cells. In another preferred embodiment, human insulin is produced synthetically.

[0072] GLP-1 analogs are also referred to in the art as GLP-1 mimetics. One of skill in the art will understand in light of the present disclosure that the described compositions can include at least one of the following GLP-1 analogs: exenatide (Byetta™ CAS No. 141732-76-5), lixisenatide (CAS No. 320367-13-3), liraglutide (CAS No. 204656-20-2), exendin-9 (CAS No. 133514-43-9), AC3174 ([Leu(14)]exendin-4, Amylin Pharmaceuticals, Inc.), taspoglutide (CAS No. 275371-94-3), albiglutide (CAS No. 782500-75-8), semaglutide (CAS No. 910463-68-2), LY2189265 (dulaglutide™; CAS No. 923950-08-7), and CJC-1134-PC (a modified exendin-4 analog that binds recombinant human albumin manufactured by ConjuChem™). All CAS records were accessed on Dec. 19, 2011. Thus, in certain embodiments, the described methods or compositions utilize any of the GLP-1 analogs listed above. In other embodiments, one of the GLP-1 analogs listed above is selected. Those of skill in the art will recognize, in light of the findings presented herein, that other GLP-1 analogs may also be utilized in the methods and compositions described.

[0073] In some embodiments, reference to a "therapeutic peptide" or "therapeutic protein" is intended to include any such molecule, which when provided to a subject, provides a beneficial effect. In some cases, the molecule is therapeutic in that it functions to replace the absence or reduced presence of such a molecule in a subject. In one embodiment, the molecule replaces a missing protein, such as in the case of an endogenous null mutant that is supplemented by oral delivery of a foreign protein. In other embodiments, an endogenous protein is mutated to create a non-functional protein, which is supplemented by oral delivery of a heterologous functional protein. In other embodiments, oral delivery of a heterologous protein is additive to low endogenous levels, resulting in a cumulative and enhanced availability of a given protein. In other embodiments, the molecule stimulates a signaling cascade that provides expression or secretion, or other of a key component for cellular or host function.

[0074] emulsifier

[0075] "Weight / weight" percentages of emulsifiers and surfactants referenced herein utilize the amount of oil base in the formulation, e.g., fish oil, as the denominator; thus, 60 mg of Gelucire in 500 mg of fish oil is considered to be 12% w / w, regardless of the weight of other ingredients. Similarly, 50 mg of Tween-80 mixed with 500 mg of fish oil is considered to be 10% Tween-80.

[0076] In one embodiment, the oil-based liquid formulation utilized in the described methods and pharmaceutical compositions, and in another embodiment, each of the oil-based liquid formulations present, comprises, in addition to the therapeutic protein, the chelating agent, and the BBI, a polyethylene glycol (PEG) ester of a fatty acid, such as, for example, a PEG ester of a monoglyceride, a diglyceride, a triglyceride, or a mixture thereof. In a more specific embodiment, the PEG ester may be provided as a mixture with (a) a free monoacylglycerol, a free diacylglycerol, a free triacylglycerol, or a mixture thereof; and (b) a PEG ester of a fatty acid, such as, for example, a monoglyceride, a diglyceride, a triglyceride, or a mixture thereof. In this regard, each of the terms "monoacylglycerol", "diacylglycerol", and "triacylglycerol" need not refer to a single compound, but rather may include a mixture of compounds, such as, for example, a mixture of monoacylglycerols, diacylglycerols, or triacylglycerols having fatty acids of various lengths. In a preferred embodiment, the monoacylglycerol, diacylglycerol, or triacylglycerol utilized in the described methods and compositions, such as those used in common PEG esters, is derived from an oil source that is generally recognized as safe (GRAS). Examples of GRAS oils are coconut oil, corn oil, peanut oil, soybean oil, and Myvacet 9-45 (a diacetylated monoacylglycerol of C-18 fatty acids). A more specific embodiment of (a) is derived from C 8 -C 18 A more specific embodiment of component (b) is a mixture of monoacylglycerol, diacylglycerol, and triacylglycerol. 8 -C 18 It is a mixture of PEG mono- and diesters of fatty acids.

[0077] In more specific embodiments, the liquid formulation further comprises free PEG in addition to the PEG ester of a fatty acid, and in even more specific embodiments, an additional non-ionic surfactant, such as a polysorbate-based surfactant, is present in addition to the PEG ester and free PEG.

[0078] In still more specific embodiments of the described methods and compositions, the liquid formulation comprises: 8 -C 18 (b) a mixture of monoacylglycerols, diacylglycerols, and triacylglycerols; (b) C 8 -C 18 PEG-32 mono- and diesters of a mixture of fatty acids; and (c) free PEG-32. In even more specific embodiments, the weight / weight ratio of component (a) to the sum of components (b)+(c) is 10:90 to 30:70; more specifically, 15:85 to 25:75; more specifically, 20:80. In some embodiments, components (a)-(c) collectively comprise an 8-16% weight / weight ratio of the oil-based liquid formulation. In more specific embodiments, the amount is 9-15%. In more specific embodiments, the amount is 10-14%. In more specific embodiments, the amount is 11-13%. In more specific embodiments, the amount is 12%.

[0079] In other embodiments, the oil-based liquid formulation utilized in the described methods and pharmaceutical compositions includes a self-emulsifying component in addition to the therapeutic protein, chelating agent, and BBI. Some embodiments of the self-emulsifying component are mixtures of components described in the preceding paragraphs, but these mixtures do not limit the definition of the term "self-emulsifying component" as used herein. As used herein, "self-emulsifying component" refers to a component that naturally forms an emulsion. Generally, such components form emulsions upon contact with an aqueous medium, forming fine dispersions, or microemulsions (SMEDDS). Some embodiments of such components include triacylglycerol mixtures of triacylglycerols and hydrophilic / lipophilic balance (HLB; see Griffin WC: "Calculation of HLB Values ​​of Non-Ionic Surfactants," J Soc Cosmetic Chemists 5:259 (1954)) surfactants. Other embodiments of the self-emulsifying component have a waxy, semi-solid viscosity.

[0080] Preferably, the HLB of the self-emulsifying component utilized in the described methods and compositions is 10 or greater. In other embodiments, it is 11-19. In other embodiments, it is 12-18. In other embodiments, it is 12-17. In other embodiments, it is 12-16, which represents an oil-in-water (O / W) emulsifier. In other embodiments, it is 13-15. In other embodiments, it is 14. Even more specific embodiments of the self-emulsifying component have an HLB of 12-16 and include medium and long chain triacylglycerols that are bound to PEG, free triacylglycerols, and free PEG. In other embodiments, the self-emulsifying component has an HLB of 12-16 and consists of a mixture of medium and long chain triacylglycerols that are bound to PEG, free triacylglycerols, and free PEG. In another embodiment, the self-emulsifying component comprises medium and long chain triacylglycerols having an HLB of 14 and conjugated to PEG, free triacylglycerols, and free PEG, In another embodiment, the self-emulsifying component consists of a mixture of medium and long chain triacylglycerols having an HLB of 14 and conjugated to PEG, free triacylglycerols, and free PEG.

[0081] Certain more specific embodiments utilize a self-emulsifying component comprising: (a) a monoacylglycerol, a diacylglycerol, a triacylglycerol, or a mixture thereof; and (b) a polyethylene glycol (PEG) ester of a fatty acid. In this regard, each of the terms "monoacylglycerol," "diacylglycerol," and "triacylglycerol" need not refer to a single compound, but rather may include a mixture of compounds, such as, for example, a mixture of monoacylglycerols, diacylglycerols, or triacylglycerols having fatty acids of various lengths. More specific embodiments utilize a self-emulsifying component comprising: 8 -C 18 A more specific embodiment of component (b) is a mixture of monoacylglycerol, diacylglycerol, and triacylglycerol. 8 -C 18It is a mixture of PEG mono- and diesters of a mixture of fatty acids.

[0082] In other, more specific embodiments, the self-emulsifying component further comprises free PEG.

[0083] Certain PEG moieties for use in the described compositions and methods contain between 5-100 monomers. In more specific embodiments, the PEG may contain between 15-50 monomers. In more specific embodiments, the PEG may contain between 25-40 monomers. In more specific embodiments, the PEG may contain 32 monomers.

[0084] In still more specific embodiments of the methods and compositions described, the self-emulsifying component used therein is: 8 -C 18 (b) a mixture of monoacylglycerols, diacylglycerols, and triacylglycerols; (b) C 8 -C 18 PEG-32 mono- and diesters of a mixture of fatty acids; and (c) free PEG-32, and the weight / weight ratio of component (a) to components (b)+(c) is 20:80. In certain embodiments, such components constitute an 8-16% weight / weight ratio oil-based liquid formulation. In more specific embodiments, the amount is 9-15%. In more specific embodiments, the amount is 10-14%. In more specific embodiments, the amount is 11-13%. In more specific embodiments, the amount is 12%.

[0085] Examples of self-emulsifying ingredients that meet the above specifications are Gelucire™ 44 / 14, Gelucire™ 53 / 10, and Gelucire™ 50 / 13. A more specific embodiment is Gelucire™ 44 / 14. The suffixes 44 and 14 refer to its melting point and its hydrophilic / lipophilic balance (HLB), respectively. Gelucire™ 44 / 14 (Gattefosse SAS, Saint-Priest, France) is obtained by polyglycolization of hydrogenated coconut oil (medium and long chain triacylglycerols with PEG-32). It has a hydrophilic / lipophilic balance of 14. It is a C 8 -C 18 It is composed of a defined mixture of mono-, di-, and triacylglycerols (20% w / w); PEG-32 mono- and diesters, and free PEG-32 (80% w / w). The main fatty acid present is lauric acid, which accounts for an average of 45% of the total fatty acid content. It is a solid dispersion composed of a PEG ester fraction under a 120 Å lamellar phase in a helical structure and an acylglycerol fraction under a hexagonal packing. The main products of the simulated gastrointestinal lipolysis of Gelucire™ 44 / 14 are PEG-32 mono- and diesters. In a more specific embodiment, the described composition comprises about 12% Gelucire 44 / 14 as the sole emulsifier, or in other embodiments, with another emulsifier. In other embodiments, the described composition comprises about 12% Gelucire 44 / 14 and about 10% Tween 80.

[0086] Nonionic Surfactants

[0087] In some embodiments, the oil-based liquid formulations utilized in the described methods and pharmaceutical compositions further comprise a non-ionic surfactant in addition to the self-emulsifying component. In some embodiments, the non-ionic surfactant is selected from the group consisting of polysorbate-20, polysorbate-40, polysorbate 80, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid esters, methylcellulose, carboxymethylcellulose, n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton®-X-100, Triton®-X-114, Thesit®, Isotridecypoly(ethylene glycol ether) n , 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and N-dodecyl=N,N-dimethyl-3-ammonio-1-propanesulfonate. In other embodiments, one of the nonionic surfactants listed above is selected.

[0088] In some more specific embodiments, the non-ionic surfactant used in the described methods and compositions is a polysorbate-based surfactant. Examples of polysorbate-based surfactants are surfactants derived from polyethoxylated sorbitan covalently bound to fatty acids. More specific embodiments of polysorbate-based surfactants are polysorbate-20, polysorbate-40, and polysorbate-80.

[0089] For example, polysorbate 80 (Tween 80) is a nonionic surfactant derived from polyethoxylated sorbitan and oleic acid and has the following structure:

[0090] [ka]

[0091] In the case of polysorbate 80, the portion shown on the right is a mixture of fatty acids, containing 60-70% oleic acid (as shown) with the remainder being primarily linoleic, palmitic, and stearic acids.

[0092] In more specific embodiments, polysorbate 80 comprises 3-15% weight / weight of the oil-based liquid formulation used in the described methods and compositions. In more specific embodiments, the percentage is 10-14%. In more specific embodiments, the percentage is 7-12%. In more specific embodiments, the percentage is 10%, or alternatively, 5%.

[0093] Chelating Agents for Divalent Cations

[0094] The divalent cation chelator utilized in the described methods and compositions, in one embodiment, is any physiologically acceptable compound that has a high affinity for at least one of calcium, magnesium, and manganese ions. In another embodiment, the chelator is selected from the group consisting of citrate, or a salt thereof; ethylenediaminetetraacetic acid (EDTA), or a salt thereof (e.g., disodium EDTA or calcium disodium EDTA); EGTA (ethylene glycol tetraacetic acid), or a salt thereof; diethylenetriaminepentaacetic acid (DTPA), or a salt thereof; and BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), or a salt thereof. In other embodiments, one of the chelators listed above is utilized. In a more specific embodiment, the chelator is ETDA.

[0095] oil

[0096] In light of this observation, one of skill in the art will appreciate that a variety of oils may be utilized as a base for the liquid phase of the described compositions, in certain embodiments, the oil may be any physiologically acceptable oil that is liquid at ambient temperature.

[0097] In a more specific embodiment, the oil comprises an omega-3 fatty acid. In another embodiment, the omega-3 fatty acid is an omega-3 polyunsaturated fatty acid. In another embodiment, the omega-3 fatty acid is DHA, an omega-3, polyunsaturated, 22-carbon fatty acid, also called 4,7,10,13,16,19-docosahexaenoic acid. In another embodiment, the omega-3 fatty acid is -linolenic acid (9,12,15-octadecatrienoic acid). In another embodiment, the omega-3 fatty acid is stearidonic acid (9,12,15-octadecatrienoic acid). In another embodiment, the omega-3 fatty acid is eicosatrienoic acid (ETA; 11,14,17-eicosatrienoic acid). In another embodiment, the omega-3 fatty acid is eicosatetraenoic acid (8,11,14,17-eicosatetraenoic acid). In one embodiment, the omega-3 fatty acid is eicosapentaenoic acid (EPA; 5,8,11,14,17-eicosapentaenoic acid). In another embodiment, the omega-3 fatty acid is eicosahexaenoic acid (also called 5,7,9,11,14,17-eicosahexaenoic acid). In another embodiment, the omega-3 fatty acid is docosapentaenoic acid (DPA; 7,10,13,16,19-docosapentaenoic acid). In another embodiment, the omega-3 fatty acid is tetracosahexaenoic acid (6,9,12,15,18,21-tetracosahexaenoic acid).

[0098] In other embodiments, the oil is a natural oil that contains omega-3 fatty acids. In more specific embodiments, the oil is selected from the group consisting of fish oil, canola oil, flaxseed oil, algal oil, and hemp seed oil. In more specific embodiments, the oil is fish oil. Several types of fish oil have been tested with the described compositions and all have been found to work equally well.

[0099] In other embodiments, the liquid formulation utilized in the described method or composition is free of water. "Free of water" refers in some embodiments to a formulation to which no aqueous components have been intentionally added. This formulation does not exclude the presence of trace amounts of water absorbed into the composition from the atmosphere. In another embodiment, the liquid formulation is free of aqueous components. In yet other embodiments, one or more oils are the only liquid components of the liquid formulation. In more specific embodiments, fish oil is the only liquid component of the liquid formulation.

[0100] coating

[0101] In light of this finding, one of skill in the art will appreciate that a variety of pH-sensitive coatings may be utilized in the described methods and compositions. In certain embodiments, any coating that inhibits digestion of the composition in the subject's stomach may be utilized.

[0102] In other embodiments, the coating comprises a biodegradable polysaccharide. In other embodiments, a hydrogel is utilized. In other embodiments, the coating comprises one of the following excipients: chitosan, an aquacoat ECD coating, an azo-crosslinked polymer, cellulose acetate phthalate, cellulose acetate trimellitate (CAT), cellulose acetate butyrate, hydroxypropyl methylcellulose phthalate, or polyvinyl acetate phthalate.

[0103] In other embodiments, a sustained release system such as Pulsincap™ is utilized.

[0104] In a preferred embodiment, the coated dosage forms described release the core (containing an oil-based formulation) when the pH reaches the range found in the intestine, which is alkaline relative to the range in the stomach. In a more specific embodiment, the coating contains a pH-sensitive polymer. In various embodiments, either a single layer or a multi-layer coating may be utilized.

[0105] In one embodiment, the coating is an enteric coating. Methods for enteric coating are well known in the art (see, for example, Siepmann F et al 2005). In a more specific embodiment, Eudragit™ coating is utilized as the enteric coating. Eudragit™ coating is an acrylic polymer, the use of which is well known in the art.

[0106] In another embodiment, microencapsulation is used as a gastroresistant coating with the described compositions. Methods for microencapsulation are well known in the art.

[0107] In another embodiment, the coating is a capsule, of which gelatin capsules are a more specific embodiment. Methods for inserting oil-based formulations into gelatin capsules are well known in the art. In yet another embodiment, the coating is a soft gel, enteric coated capsule.

[0108] In another embodiment, an oral pharmaceutical composition is provided comprising an oil-based liquid formulation, wherein the oil-based liquid formulation comprises a therapeutic protein of up to 100 kilodaltons, a chelator of divalent cations, an isolated chymotrypsin / trypsin inhibitor, an isolated trypsin inhibitor, and a PEG ester of a fatty acid. In another embodiment, the liquid formulation has an antichymotrypsin activity such that at least 50 mg of chymotrypsin is inhibited per ml of liquid formulation. In another embodiment, the liquid formulation has both an antichymotrypsin activity such that at least 50 mg of chymotrypsin is inhibited per ml of liquid formulation, and an antitrypsin activity such that at least 25 mg of trypsin is inhibited per ml of liquid formulation. In another embodiment, free PEG is also present. In another embodiment, a non-ionic surfactant is also present. In another embodiment, the liquid formulation consists essentially of a therapeutic protein of up to 100 kilodaltons, a chelator of divalent cations, a chymotrypsin / trypsin inhibitor, a trypsin inhibitor, and a PEG ester of a fatty acid. In other embodiments, the liquid formulation consists essentially of a therapeutic protein of up to 100 kilodaltons, a chelator of divalent cations, a chymotrypsin / trypsin inhibitor, a trypsin inhibitor, a PEG ester of a fatty acid, and free PEG. In other embodiments, the liquid formulation consists essentially of a therapeutic protein of up to 100 kilodaltons, a chelator of divalent cations, a chymotrypsin / trypsin inhibitor, a trypsin inhibitor, a PEG ester of a fatty acid, free PEG, and a non-ionic surfactant.

[0109] Representative preparations

[0110] A representative insulin formulation contains insulin, Gelucire 44 / 14, EDTA, SBTI, and aprotinin in fish oil and coated in a capsule. A representative exenatide formulation contains exenatide, Gelucire 44 / 14, EDTA, SBTI, and aprotinin in fish oil and coated in a capsule.

[0111] In some embodiments, the present invention provides a formulation having the following ingredients: 8-20% Gelucire 44 / 14; 25-50 mg rBBI, 25-50 mg isolated KTI per capsule; 100-200 mg EDTA; and a therapeutic protein, which may be insulin 2-32 mg per capsule, and / or exenatide 150-600 mcg per capsule, all combined in 0.5-0.7 ml of fish oil.

[0112] In some embodiments, the present invention provides a formulation having the following ingredients: 8-20% Gelucire 44 / 14; 12.5-25 mg rBBI, 12.5-25 mg isolated KTI per capsule; 100-200 mg EDTA; and a therapeutic protein, which may be insulin 2-32 mg per capsule, and / or exenatide 150-600 mcg per capsule, all combined in 0.5-0.7 ml of fish oil.

[0113] In some embodiments, the present invention provides a formulation having the following ingredients: 12.5-25 mg rBBI, 12.5-25 mg isolated KTI per capsule; 100-200 mg EDTA; and a therapeutic protein, which may be insulin, 2-32 mg per capsule, and / or exenatide, 150-600 mcg per capsule, all combined in 0.5-0.7 ml of fish oil.

[0114] In some embodiments, the present invention provides a formulation having the following ingredients: 25-50 mg of rBBI, 25-50 mg of isolated KTI per capsule; 100-200 mg of EDTA; and a therapeutic protein, which may be 2-32 mg of insulin per capsule, and / or 150-600 mcg of exenatide per capsule, all combined in 0.5-0.7 ml of fish oil.

[0115] In some embodiments, the present invention provides a formulation having the following ingredients: 50-100 mg of rBBI, 50-100 mg of isolated KTI per capsule; 100-200 mg of EDTA; and a therapeutic protein, which may be insulin, 2-32 mg per capsule, and / or exenatide, 150-600 mcg per capsule, all combined in 0.5-0.7 ml of fish oil.

[0116] In some embodiments, the present invention provides a formulation having the following ingredients: 75-125 mg of rBBI, 75-125 mg of isolated KTI per capsule; 100-200 mg of EDTA; and a therapeutic protein, which may be insulin, 2-32 mg per capsule, and / or exenatide, 150-600 mcg per capsule, all combined in 0.5-0.7 ml of fish oil.

[0117] In some embodiments, formulations comprising recombinant BBI and insulin as described demonstrate a more rapid and qualitatively greater reduction in circulating blood glucose levels, an effect that is sustained for a longer period of time than that achieved with formulations comprising BBI chemically purified from a soy preparation.

[0118] In some embodiments, formulations comprising recombinant BBI as described herein and a therapeutic protein or peptide based therapeutic agent provide a greater therapeutic framework in terms of sustained efficacy over an extended period of time than that achieved with formulations comprising BBI chemically purified from soy preparations, which may be evidenced in local effects following administration, or, in some embodiments, in circulating protein / peptide levels following administration.

[0119] In some embodiments, formulations comprising recombinant BBI as described herein and a therapeutic protein or peptide-based therapeutic provide comparable or longer sustained efficacy following administration of recombinant BBI, or provide comparable or greater circulating protein / peptide levels, with a lower recombinant BBI concentration than the concentration of BBI chemically purified from soy preparations in an otherwise identical formulation.

[0120] According to this aspect, and in some embodiments, the concentration of recombinant BBI provided in the formulations of the invention will be at least 30%, or in some embodiments at least 35%, or in some embodiments at least 40%, or in some embodiments at least 45%, or in some embodiments at least 50%, or in some embodiments at least 55%, or in some embodiments at least 60%, or in some embodiments at least 65%, or in some embodiments at least 70%, or in some embodiments at least 75% lower than the concentration of BBI chemically purified from soy preparations used in an otherwise comparable or identical formulation.

[0121] References to the term "otherwise similar or identical" with respect to the formulations of the invention contemplate the same major components, including proteins / peptides, BBI or KTI protease inhibitors, EDTA, and / or fish oil, or omega-3 fatty acids derived from fish oil, although other components of the formulation may be equivalent as would be recognized by one of skill in the art.

[0122] In one embodiment, a formulation is provided having the following ingredients: 8-20% Gelucire 44 / 14; 50-100 mg rBBI or a mixture of rBBI / isolated or recombinant KTI per capsule; 20-30 mg aprotinin per capsule; and 100-200 mg EDTA; with a therapeutic protein, which may be insulin 8-32 mg per capsule and / or exenatide 150-600 mcg per capsule, all combined in 0.5-0.7 ml of fish oil.

[0123] In another embodiment, a representative liquid insulin formulation is provided that includes insulin, Gelucire 44 / 14, EDTA, rBBI, KTI, and aprotinin in fish oil.

[0124] In another embodiment, the liquid formulation consists essentially of insulin, Gelucire 44 / 14, EDTA, rBBI, KTI, aprotinin, and fish oil.

[0125] In some embodiments, the formulation comprises 8 mg insulin, 12% Gelucire 44 / 14, 150 mg EDTA, 75 mg total rBBI and KTI, and 24 mg aprotinin in 0.5-0.7 ml of fish oil; or in some embodiments, 16 mg insulin, 12% Gelucire 44 / 14, 150 mg EDTA, 75 mg total rBBI and KTI, and 24 mg aprotinin in 0.5-0.7 ml of fish oil; or in some embodiments, 16 mg insulin, 12% Gelucire 44 / 14, 150 mg EDTA, 150 mg total rBBI and KTI, and 24 mg aprotinin in 0.5-0.7 ml of fish oil.

[0126] In some embodiments, the invention provides formulations, wherein the ratio of antitrypsin activity to antichymotrypsin activity of the composition is about 1.28: 1. In some embodiments, a formulation containing only rBBI in the amounts described above is sufficient for protease inhibitor activity, and does not include additional proteases.

[0127] In other embodiments, the composition further comprises a non-ionic surfactant. In more specific embodiments, the non-ionic surfactant is a polysorbate-based surfactant. In even more specific embodiments, the polysorbate-based surfactant is polysorbate 80. Preferably, polysorbate 80 comprises 3-10% weight / weight of the oil-based liquid formulation.

[0128] The liquid formulation may be a softgel or an enteric coated capsule. The specific formulations described in this paragraph include, in certain embodiments, up-scaled and down-scaled formulations containing the same ingredients in the same ratios.

[0129] Some representative oral exenatide formulations include exenatide, EDTA, rBBI, KTI, and aprotinin in fish oil. In other embodiments, the liquid formulation consists essentially of exenatide, Gelucire 44 / 14, EDTA, rBBI, KTI, aprotinin, and fish oil. More specific formulations include: 150 micrograms (mcg) of exenatide, 150 mg EDTA, 75 mg total of rBBI and KTI, and 24 mg of aprotinin in 0.5-0.7 ml of fish oil; 300 mcg of exenatide, 150 mg EDTA, 75 mg total of rBBI and KTI, and 24 mg of aprotinin in 0.5-0.7 ml of fish oil; and 300 mcg of exenatide, 150 mg EDTA, 150 mg total of rBBI and KTI, and 24 mg of aprotinin in 0.5-0.7 ml of fish oil. In some embodiments, formulations containing only the aforementioned amounts of rBBI are sufficient for protease inhibitor activity, which does not include additional proteases.

[0130] In a more specific formulation, the ratio of antitrypsin activity to antichymotrypsin activity of the composition is from 1.5:1 to 1:1. In an even more specific embodiment, the ratio is about 1.28:1. The liquid formulation may be a softgel or may be an enteric coated capsule. In another embodiment, the composition further comprises a non-ionic surfactant. In a more specific embodiment, the non-ionic surfactant is a polysorbate-based surfactant. In an even more specific embodiment, the polysorbate-based surfactant is polysorbate 80. Preferably, polysorbate 80 comprises 3-10% weight / weight of the oil-based liquid formulation. The liquid formulation may be a softgel or may be an enteric coated capsule.

[0131] therapeutic indicators

[0132] In other aspects, uses of the rBBI described herein are provided in the preparation of a medicament for oral administration of an active ingredient to a subject.

[0133] In yet another aspect, a method for producing a pharmaceutical composition is provided, comprising the steps of: (a) purifying a mixture comprising rBBI as described herein and an active ingredient; and (b) formulating the mixture into a pharma- ceutically acceptable formulation.

[0134] As referred to herein, the process of formulating optionally includes steps such as adding excipients, milling, coating, etc., appropriate to the desired pharmaceutical composition. These steps are well within the capabilities of one of ordinary skill in the art.

[0135] In certain embodiments, the active ingredients referred to herein are susceptible to degradation or inactivation in the human digestive tract.

[0136] In another aspect, an oral pharmaceutical composition is provided as described herein for orally administering an active ingredient to a subject. In certain preferred embodiments, the active ingredient is susceptible to degradation or inactivation in the human digestive tract. In more specific embodiments, the active ingredient may be a therapeutic protein. In other embodiments, the active ingredient is a non-protein molecule that is susceptible to degradation or inactivation in the human digestive tract.

[0137] In another aspect, there is provided a use of an oral pharmaceutical composition described herein in the preparation of a medicament for oral administration of a therapeutic protein to a subject.

[0138] In another aspect, a method is provided for orally administering a therapeutic protein to a subject, the method comprising administering to the subject an oral pharmaceutical composition described herein, thereby orally administering the therapeutic protein to the subject.

[0139] In another aspect, there is provided a pharmaceutical composition as described herein for treating diabetes in a human, wherein in some embodiments the therapeutic protein is, in various embodiments, insulin, exenatide, or a combination thereof.

[0140] In yet another aspect, the use of a combination of components described herein for treating diabetes in a human is provided in the preparation of a drug, wherein in some embodiments the therapeutic protein is, in various embodiments, insulin, exenatide, or a combination thereof.

[0141] In yet another aspect, methods are provided for treating diabetes, comprising administering to a subject in need of such treatment a pharmaceutical composition described herein, where in some embodiments the therapeutic protein is, in various embodiments, insulin, exenatide, or a combination thereof, thereby treating diabetes. In certain embodiments, the subject is a human subject, while in other embodiments, the subject is a non-human mammal.

[0142] In an additional aspect, a pharmaceutical composition as described herein is provided for treating unstable diabetes in a human. In another aspect, a pharmaceutical composition as described herein is provided for treating high fasting blood glucose levels in a human.

[0143] In yet another aspect, the use of the combination of ingredients described herein is provided in the preparation of a medicament for treating labile diabetes in a human.Furthermore, the use of the combination of ingredients described herein is provided in the preparation of a medicament for treating high fasting blood glucose levels in a human.

[0144] Further provided is a method for treating unstable diabetes, comprising administering to a subject in need of such treatment a pharmaceutical composition as described herein, thereby treating the unstable diabetes. Further provided is a method for reducing high fasting blood glucose levels, comprising administering to a subject in need of such treatment a pharmaceutical composition as described herein, thereby reducing the high fasting blood glucose. In a preferred embodiment, the subject is a human subject.

[0145] unstable diabetes

[0146] A skilled practitioner in the art will appreciate that unstable diabetes, also known as glycemic instability, can be diagnosed by a number of acceptable standard procedures, one such procedure being found in Ryan et al, 2004. In this procedure, subjects are asked to monitor their blood glucose levels with a minimum of two capillary blood glucose readings per day. The patient records on a sheet all measured blood glucose readings, and details regarding hypoglycemic episodes, over a four-week period. In any case where blood glucose is recorded as <3.0 mmol / l, the subject is asked to fill in a questionnaire with details regarding the incident, including what symptoms occurred and whether external assistance from a third party was obtained to either recognize or treat the hypoglycemic reaction. A reaction is considered severe if the individual loses control of the situation and requires external assistance to treat the hypoglycemic event. Other such methods include calculation of the MAGE index (Service et al 1970), or "M value" (Schlichtkrull et al), or utilize continuous glucose monitoring systems (Kessler et al.) Unstable diabetes is generally associated with high fasting glucose and / or episodes of hypoglycemia.

[0147] It is to be understood that the present invention contemplates oral compositions for treating a variety of diseases / disorders as described herein, including any form of diabetes, as would be recognized by one of skill in the art.

[0148] In one embodiment, the term "treating" or "treatment" includes prophylactic as well as disorder ameliorating treatment. The terms "reducing", "suppressing" and "inhibiting" have their commonly understood meaning of reducing, or in another embodiment, decreasing, in another embodiment, delaying, or in another embodiment, reducing the occurrence, severity, or pathogenesis of a disease, disorder, or condition. In an embodiment, the term treatment refers to a delay in progression, long-term remission, reduced occurrence, or amelioration of symptoms associated with a disease, disorder, or condition. In one embodiment, the terms "treating", "reducing", "suppressing", or "inhibiting" refer to a reduction in morbidity, mortality, or a combination thereof, associated with the indicated disease, disorder, or condition. In one embodiment, the term "progression" refers to an increase in extent or severity, progression, progression, progression, or worsening. In another embodiment, the term "recurrence" refers to a recurrence of a disease. In one embodiment, the method of treatment of the invention reduces the severity of the disease, or in another embodiment, reduces symptoms associated with the disease, or in another embodiment, reduces the number of biomarkers expressed during the disease.

[0149] In one embodiment, the term "treating" and its inclusive aspects refers to administration to a subject having the indicated disease, disorder, or condition, or, in some embodiments, to a subject predisposed to the indicated disease, disorder, or condition. The term "predisposed to" should be considered to refer, among other things, to a genetic profile or family relationship associated with a trend or statistical increase in the incidence, severity, etc., of the indicated disease. In some embodiments, the term "predisposed to" should be considered to refer, among other things, to a lifestyle associated with an elevated risk of the indicated disease. In some embodiments, the term "predisposed to" should be considered to refer, among other things, to the presence of a biomarker associated with the indicated disease, e.g., in cancer, the term "predisposed to" cancer can include the presence of a precancerous precursor for the indicated cancer.

[0150] In some embodiments, the term "reducing pathogenic mechanisms" is understood to encompass reducing tissue or organ damage associated with a particular disease, disorder, or condition. In another embodiment, the term "reducing pathogenic mechanisms" is understood to encompass reducing the incidence or severity of an associated disease, disorder, or condition. In another embodiment, the term "reducing pathogenic mechanisms" is understood to encompass reducing the number of associated diseases, disorders, or conditions, or symptoms related thereto, for the disease, disorder, or condition indicated.

[0151] Method for purifying a pharmaceutical composition

[0152] Also provided herein is a method for purifying a pharmaceutical composition. In an embodiment, the method includes the steps of optionally combining dissolved Gelucire (e.g., Gelucire 44 / 14) with fish oil, cooling the mixture, and adding EDTA, SBTI, aprotinin, and a therapeutic protein or peptide, such as insulin, or in another embodiment, exenatide, in powder form, although those skilled in the art will understand in light of this finding that other therapeutic proteins or peptides can also be used. In another embodiment, the method includes the steps of optionally combining dissolved Gelucire (e.g., Gelucire 44 / 14) with fish oil, cooling the mixture, and adding EDTA, SBTI, and a therapeutic protein or peptide in powder form. In an embodiment, the powder components are added in the order listed. In another embodiment, the resulting mixture is optionally mixed and / or homogenized.

[0153] As used herein, "consisting essentially of" limits the scope of the invention to particular materials or steps, and those that do not materially affect the basic and novel characteristics of the invention as asserted in the claims.

[0154] In one embodiment, the term "about" refers to a variation of 1-10%, or in another embodiment, 5-15%, or in another embodiment, up to 10%, or in another embodiment, up to 25% from the indicated value, unless the context indicates that the variation should not result in a value of more than 100%.

[0155] In some embodiments of the invention, "weight" refers to "dry weight." In other embodiments, "weight" refers to total weight.

[0156] In some embodiments, the term "comprising" or grammatical forms thereof refers to the inclusion of the indicated components of the present invention, as well as the inclusion of other active agents, and pharma- ceutically acceptable carriers, excipients, emollients, stabilizers, and the like, known in the pharmaceutical industry.

[0157] In one embodiment, the present invention provides a combined preparation. In one embodiment, the term "combined preparation" specifically defines a "kit of parts" in the sense that the combination partners defined above can be used independently or in different combinations, i.e. simultaneously, in parallel, separately or sequentially.

[0158] In some embodiments, the compositions of the invention consist essentially of the components as described herein in any form or embodiment described herein. In some embodiments, the term "comprising" refers to the inclusion of the indicated components as described herein, such as the rBBI of the invention and a therapeutic peptide or protein, as well as the inclusion of other active agents, and pharma- ceutically acceptable carriers, excipients, emollients, stabilizers, and the like, known in the pharmaceutical industry. In some embodiments, the term "consisting essentially of" refers to a composition whose only active ingredients are the indicated therapeutic peptide and protein of the invention, and rBBI. However, it is well known that other compounds may be included that are intended to stabilize and preserve the formulation, etc., but are not directly involved in the therapeutic effect of the indicated active ingredients, and other protease inhibitors may also be included, but the inclusion of rBBI is an essential component of the composition to promote long-term sustained activity of the therapeutic peptide or protein of the invention. In some embodiments, the term "consisting essentially of" refers to a composition in which the only active ingredient having the same mechanism of action or the same molecular target is the indicated active ingredient, although other active ingredients may be incorporated, such secondary active ingredients acting on different targets or in a palliative capacity, and the like, also refer to a therapeutic peptide or protein of the invention, and such a composition further comprises rBBI as described. In some embodiments, the term "consisting essentially of" may refer to ingredients that aid in the release of the active ingredient in addition to rBBI, which helps to facilitate sustained delivery / prevention of degradation of the therapeutic peptide or protein of the invention, as described herein. In some embodiments, the term "consisting of" refers to a composition that includes a therapeutic peptide or protein of the invention, rBBI, as described herein, as the only active ingredient, and a pharmaceutically acceptable carrier or excipient.

[0159] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will occur to those skilled in the art, and it is therefore to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

[0160] It will be understood by those skilled in the art that various changes in form and detail may be made without departing from the spirit and scope of the invention as set forth in the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed within the scope of the claims.

[0161] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict between the specification and an incorporated reference, the specification shall control. When numerical ranges are given in this document, the endpoints are included within the range. Furthermore, unless otherwise indicated or otherwise evident from the context and the understanding of one of ordinary skill in the art, it is to be understood that values ​​expressed as ranges can be considered in different embodiments of the invention to be any particular value or subrange within the specified range, optionally including or excluding one or both endpoints, up to one tenth of the unit of the lower limit of the range, unless otherwise clearly indicated by the context. When percentages are given with reference to values ​​that inherently have units of integers, the resulting fraction may be rounded to the nearest integer.

[0162] In the claims, articles such as "a," "an," and "the" mean one or more unless otherwise indicated or clear from the context. A claim or description including "or" or "and / or" between members of a group is deemed to mean that one, more than one, or all of the members of the group are present in, used in, or otherwise relevant to a given product or process, unless otherwise indicated or clear from the context. The invention includes embodiments in which exactly one member of a group is present in, used in, or otherwise relevant to a given product or process. The invention also includes embodiments in which one or more, or all of the members of a group are present in, used in, or otherwise relevant to a given product or process. Moreover, it is to be understood that the present invention, in its various embodiments, provides for all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc., introduced from one or more of the enumerated claims into another claim dependent on the same base claim, unless otherwise indicated or unless a contradiction or inconsistency would be apparent to one of ordinary skill in the art. When elements are arranged in a list, such as in Markush group format, it is to be understood that each subgroup of elements is also disclosed and any element can be deleted from the group.

[0163] In general, when the invention or aspects of the invention are referred to as including certain elements, features, etc., it should be understood that an embodiment of the invention or aspect of the invention consists of, or consists essentially of, such elements, features, etc. For purposes of brevity, such embodiments have not been specifically set forth in these terms in every instance herein. Although certain claims are presented in dependent form for convenience, applicants reserve the right to rewrite a dependent claim in independent form to include elements or limitations of both the independent claim and any other claim from which such claim depends, and such rewritten claim, in whatever form (whether amended or not), shall be deemed equivalent in all respects to the dependent claim before it was rewritten in independent form.

[0164] The following examples illustrate certain embodiments of the invention and should not be construed in any way as limiting the scope of what is encompassed by the invention. EXAMPLES

[0165] Experimental Details Section Example 1: Generation of recombinant BBI Materials and methods The gene for expression of recombinant Bowman-Birk proteinase inhibitor (rBBI) was codon optimized and synthesized in the Pichia pastoris system. The plasmid used for secretory expression of BBI was the pPIC9K Pichia expression vector (Invitrogen) expressed in the host: Pichia pastoris GS115. The biased and optimized BBI DNA sequence was cloned in frame (STE13 cleavage site deleted) with the Saccharomyces cerevisiae alpha mating factor presequence of the pPIC9K plasmid, resulting in a clone expressing secreted BBI.

[0166] The nucleic acid sequence optimized in a Pichia pastoris host for BBI is as follows:

[0167] [ka]

[0168] The amino acid sequence of BBI isolated from Glycine max (Soybean) of UniProtKB-P01055 (IBB1_SOYBN) (110 aa) is as follows:

[0169] [ka]

[0170] The bold text represents the start codon, the underline represents the signal peptide, and the italics represent the propeptide. The residue designations in normal font correspond to the E00124 chain, the sequence of which was gene biased and optimized for expression in Pichia, and in silico translation of the optimized gene sequence yielded a sequence identical to that of SEQ ID NO:2.

[0171] Two stop codons were added to the 3' end of the final DNA sequence (italicized, bold, and underlined text in SEQ ID NO:1). The biased and optimized BBI DNA sequence was cloned in frame with the Saccharomyces cerevisiae alpha mating factor presequence (STE13 cleavage site deleted) in pPIC9K plasmid to obtain a clone expressing secreted BBI. The recombinant construct was then recharacterized by sequencing.

[0172] Expression of recombinant products was accomplished via standard techniques. Briefly, Pichia were grown in YPD medium at 30°C with shaking until an OD600 of 0.8-1.0 was reached, after which the cells were harvested, washed, suspended, washed in 100 mM LiCl, and transformed. Standard transformation protocols were used. Briefly, the LiCl cell solution was then washed and resuspended in a 50% PEG-4000 solution containing 1M LiCl, single-stranded salmon sperm DNA pieces and plasmid DNA pieces in sterile water (5-10 μg; 100-200 ng / μl). The cells were incubated at 30°C for 30 min, then heat shocked at 42°C for 20-25 min, after which the cells were washed and resuspended in YPD medium and incubated at 30°C before being plated on YNB-His selection plates and incubated at 30°C for an additional 2-3 days.

[0173] Approximately 1000 putative clones were screened to obtain a high-yielding clone. Approximately 5-6 clones were scaled up for 200 ml, 1 L, and 5 L fermenter batches for process optimization. When the purified secreted rBBI protein from clone B17 was subjected to N- and C-terminal sequencing by MALDI-TOF-MS, the observed N- and C-terminal sequences were identical to those expected. The spectral data confirmed that the observed mass was the same as the expected mass with 99% homogeneity. Thus, the processing of rBBI from clone B17 was intact without removing or adding extra residues from the signal peptide. Thus, clone B17 was selected as a potentially high-yielding clone and was used further for scale-up.

[0174] Example 2: Characterization of recombinant BBI products The B17 clone was further studied for expression, purification and material production at 200 ml, 5 L and 50 L scales and the purified protein was characterized using different analytical methods.

[0175] RP-HPLC analysis was performed using standard techniques and the results are shown in Figure 1, which overlays the RP-HPLC analysis with the available standards. The final purified BBI showed the same retention time as observed for the standard purified BBI.

[0176] A lyophilized powder was prepared from purified rBBI and BCA quantification of total protein content was assessed using standard techniques (Figure 2), SDS-PAGE analysis of 15% gels under reducing and non-reducing conditions, and staining with Coomassie blue to confirm protein size (Figure 3).

[0177] The enzymatic activity of the protease inhibitors was examined on the recombinant products and demonstrable inhibition of chymotrypsin activity (Figure 4) as well as trypsin activity (Figure 5) was achieved. Surprisingly, the results of these assays showed a much greater inhibition of enzymatic activity upon use of the recombinant proteins when compared to purified SBTI produced from soybean.

[0178] Example 3: Recombinant protease inhibitors provide surprising efficacy. Materials and methods

[0179] Fasting non-SPF commercial pigs (obtained from Ibelin Farm, Israel (n = 8)) weighing 25-30 kg and aged 3-4 months were incubated with isoflurane (2 L O per min). 2 The animals were anesthetized with 100% isoflurane (100% ethanol and 3-5% isoflurane), tagged for easy identification, and intubated. Access to water was free. The animals were placed on their left side and the formulations were administered under endoscopic guidance through the endoscopic catheter directly into the duodenum. The study was performed with approval by the Israeli Council of Animal Experimentation, Ministry of Health (protocol number IL-16-11-400).

[0180] Injection of 1 ml of test formulation was followed by injection of 150-180 ml of air to ensure delivery of the formulation to the duodenum. Blood samples were taken from a central venous catheter (inserted on the first day of the study and replaced as necessary) at the following times: 15 min before and immediately before dosing, and every 15 min for 3 h after dosing, and then 3 h 20 min (3:20), 3 h 40 min (3:40), 4 h (4:00), and 4 h 30 min (4:30) after dosing. A small blood sample (5 μl) was used to measure blood glucose concentration with a glucometer. If the reading was <10 mg / dL, the sample was retested and the two readings were averaged. There was a minimum of 2 days of washout period between treatments.

[0181] Test conditions were performed with 150 mg EDTA, 8 mg insulin, and either purified recombinant BBI produced as described herein or BBI purified from SBTI extracted from soy flour as described in US Patent No. 5,399,663.

[0182] The test conditions were as described in Table 1 below, and each test item was tested in four different pigs.

[0183] [Table 1]

[0184] All test items were prepared in 1 mL of fish oil and stored refrigerated. Prior to use, samples were allowed to reach room temperature and mixed thoroughly before administration.

[0185] Data analysis included assessment of circulating blood glucose levels as a function of time.

[0186] The animals were observed for the duration of the study; no abnormal clinical findings were observed during the study and the animal weights remained stable throughout the study.

[0187] result: All formulations resulted in a reduction in circulating blood glucose levels (Figure 6), however, use of recombinant BBI surprisingly resulted in a consistent and sustained reduction in blood glucose levels over a sustained period of time.

[0188] As previously shown, SBTI-containing formulations reduced circulating blood glucose levels from 50 mg / dl to approximately 35 mg / dl within 30 minutes, but circulating levels had returned substantially to baseline levels within 3 hours of dosing, and furthermore, the return to baseline levels was gradual, beginning as early as 1 hour after oral ingestion.

[0189] To our great surprise, all groups receiving recombinant BBI demonstrated a more rapid and qualitatively greater decline in circulating levels to approximately 20 mg / dl, an effect that persisted for at least 2 hours after dosing, with a gradual increase evident thereafter, whereas animals receiving 100 mg recombinant BBI (rBBI) with 25 mg KTI had not yet returned to baseline after 4 hours, and animals receiving 100 mg rBBI alone did not return to baseline until more than 3.5 hours after dosing. Surprisingly, animals receiving only 50 mg rBBI and KTI also did not return to baseline until more than 3 hours after dosing.

[0190] Thus, the results surprisingly confirmed that rBBI is more effective at lowering circulating blood glucose levels compared to purified BBI-containing compositions, with the superior effect potentially lasting for a longer period of time.

[0191] Example 4: Recombinant protease inhibitors provide surprising efficacy over time. Materials and methods

[0192] Fasting non-SPF commercial pigs (obtained from Ibelin Farm, Israel (n = 8)) weighing 25-30 kg and aged 3-4 months were incubated with isoflurane (2 L O per min). 2The animals were anesthetized with 100% isoflurane (100% ethanol and 3-5% isoflurane), tagged for easy identification, and intubated. Access to water was free. The animals were placed on their left side and the formulations were administered under endoscopic guidance through the endoscopic catheter directly into the duodenum. The study was performed with the approval of the Israeli Council of Animal Experimentation, Ministry of Health (protocol number IL-16-11-400).

[0193] Injection of 1 ml of test formulation was followed by injection of 150-180 ml of air to ensure delivery of the formulation to the duodenum. Blood samples were taken from a central venous catheter (inserted on the first day of the study and replaced as necessary) at the following times: 15 min before and immediately before dosing, and every 15 min for 3 h after dosing, and then 3 h 20 min (3:20), 3 h 40 min (3:40), 4 h (4:00), 4 h 30 min (4:30), and 5 h (5:00) after dosing. A small blood sample (5 μl) was used to measure blood glucose concentration with a glucometer. If the reading was <10 mg / dL, the sample was retested and the two readings were averaged. There was a minimum of 2 days of washout between treatments.

[0194] Test conditions included 150 mg EDTA, 4 mg insulin, and increasing amounts of recombinant BBI produced as described herein.

[0195] The test conditions were as described in Table 2 below, and each test item was tested in 7–8 different pigs over 8–10 sessions.

[0196] [Table 2]

[0197] All test items were prepared in 1 mL of fish oil and stored refrigerated. Prior to use, samples were allowed to reach room temperature and mixed thoroughly before administration.

[0198] Data analysis included evaluation of change from baseline; the percentage change from baseline blood glucose area under the curve (AUC) was determined for each treatment session. Additionally, the time from first decrease to first increase in blood glucose measurement was also determined for each treatment session.

[0199] The animals were observed for the duration of the study; no abnormal clinical findings were observed during the study and the animal weights remained stable throughout the study.

[0200] result: Example 3 demonstrated that rBBI provided enhanced glycemic control in that there was a greater reduction in circulating blood glucose levels in subjects administered 8 mg of insulin orally when the protease inhibitor was recombinant BBI rather than chemically purified. To further elaborate on the effects of rBBI and extend the results, it was of interest to optimize the rBBI and evaluate its effects over time. To this end, oral formulations containing only 4 mg of insulin but 12.5, 25, 50, and 75 mg of rBBI were similarly evaluated. Table 3 provides the results of these studies:

[0201] [Table 3]

[0202] The greatest decrease from baseline blood glucose ratio AUC occurred following treatment with ORMD-0801 formulated with 25 mg rBBI, followed by the formulation containing 75 mg rBBI. When examining the animals' response to treatment with formulations containing protease inhibitors and control treatments without protease inhibitors, again, the formulations containing 25 mg rBBI, followed by 75 mg rBBI, produced the greatest change from baseline AUC (-6,369 and -4,640, respectively) and correspondingly, the largest change from control session AUC calculations (-4,989 and -2,960, respectively).

[0203] With regard to durability of effect, quite unexpectedly, the rBBI-containing formulation provided a prolonged effect, increasing the time between the first sign of hypoglycemia and the first sign of return to baseline values ​​by an average of 140 minutes in test subjects, an effect that was 75% longer than the duration of effect measured in control animals.

[0204] Thus, surprisingly and unexpectedly, although the downstream purification steps for BBI, either from pure chemical extraction means or from chemical purification of recombinantly expressed protein, were essentially identical, greater and more sustained inhibitor activity was seen in these studies when the source material for BBI was rBBI.

[0205] Thus, quite unexpectedly, recombinant BBI, as a single protease inhibitor, was sufficient to enable sustainable and qualitatively significant glycemic control.

Claims

1. An oral pharmaceutical composition comprising a therapeutic peptide or protein of up to 100 kilodaltons, a chelator of divalent cations, and an isolated recombinantly expressed Bowman-Birk Inhibitor (BBI).

2. 2. The oral pharmaceutical composition of claim 1, wherein the recombinantly expressed BBI has a nucleotide sequence that shares at least 95% identity with that set forth in SEQ ID NO:

1.

3. 2. The oral pharmaceutical composition of claim 1, wherein the recombinantly expressed BBI has a nucleotide sequence that shares at least 97% identity with that set forth in SEQ ID NO:

1.

4. 2. The oral pharmaceutical composition of claim 1, wherein the recombinantly expressed BBI has a nucleotide sequence that shares at least 99% identity with that set forth in SEQ ID NO:

1.

5. 2. The oral pharmaceutical composition of claim 1, wherein the recombinantly expressed BBI has the nucleotide sequence set forth in SEQ ID NO:

1.

6. 2. The oral pharmaceutical composition of claim 1, wherein the recombinantly expressed BBI is expressed in a yeast-based expression system.

7. 7. The oral pharmaceutical composition of claim 6, wherein the yeast expression system is Pichia pastoris.

8. 2. The oral pharmaceutical composition of claim 1, wherein the formulation further comprises a trypsin inhibitor other than the BBI.

9. The oral pharmaceutical composition of claim 8, wherein the trypsin inhibitor is KTI3.

10. 10. The oral pharmaceutical composition of claim 9, wherein the KTI3 is chemically isolated and purified to a purity of at least 85% as measured by SDS-PAGE, or the KTI3 is purified to a protein content of greater than 95% as measured by BCA analysis.

11. The oral pharmaceutical composition of claim 9, wherein the KTI3 is recombinantly expressed.

12. The oral pharmaceutical composition of any one of claims 1-11, wherein the therapeutic protein is useful in treating a subject having a metabolic disease or disorder.

13. The therapeutic peptide or protein is selected from the group consisting of insulin, influenza hemagglutinin, influenza neuraminidase, glucagon, interferon gamma, interferon beta, interferon alpha, growth hormone, erythropoietin, GLP-1, GLP-1 analogs, leptin, granulocyte colony stimulating factor (G-CSF), renin, growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, a clotting factor, an anticoagulant, an anti-coagulant, an anti-inflammatory ... Solid factor, atrial natriuretic factor, surfactant protein A (SP-A), surfactant protein B (SP-B), surfactant protein C (SP-C), surfactant protein D (SP-D), plasminogen activator, bombesin, hematopoietic growth factors (colony stimulating factors, multiple), tumor necrosis factor (TNF) proteins, enkephalinase, RANTES (regulator of normal T cell expression and activation of secretion), human macrophage inflammatory protein (MIP-1-α), serum albumin amine, Mullerian inhibitory substance, relaxin, mouse gonadotropin releasing hormone, deoxyribonuclease, inhibin, activin, vascular endothelial growth factor (VEGF), neurotrophic factor, neurotrophic factor-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), nerve growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor, nerve growth factor, transforming growth factor (TGF), insulin-like growth factor-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain I 12. The oral pharmaceutical composition of claim 1, wherein the insulin-like growth factor binding protein 1 (IGFBP-1), IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, keratinocyte growth factor, osteoinductive factor, bone morphogenetic protein (BMP)-2, BMP-4, BMP-7, colony stimulating factor (CSF), interleukin (IL), superoxide dismutase, dissociation accelerating factor, chemokine family members, and complement factors.

14. 14. The oral pharmaceutical composition of claim 13, wherein the therapeutic peptide or protein is selected from the group consisting of insulin and a GLP-1 analogue.

15. 14. The oral pharmaceutical composition of claim 13, wherein the therapeutic peptide or protein is an immunomodulatory agent.

16. 16. The oral pharmaceutical composition of claim 15, wherein the therapeutic peptide or protein is glatiramer acetate.

17. 17. The oral pharmaceutical composition of any one of claims 1-16, wherein the chelating agent is EDTA.

18. The oral pharmaceutical composition of any one of claims 1-17, wherein the formulation is an oil-based liquid formulation.

19. 20. The oral pharmaceutical composition of claim 18, further comprising a polyethylene glycol (PEG) ester of a monoglyceride, diglyceride, triglyceride, or mixtures thereof.

20. 20. The oral pharmaceutical composition of claim 19, wherein the PEG ester is provided as a mixture of: (a) a monoacylglycerol, a diacylglycerol, a triacylglycerol, or a mixture thereof; and (b) a polyethylene glycol (PEG) ester of a fatty acid.

21. The portion (a) of the mixture is ８ -C １８ 21. The oral pharmaceutical composition of claim 20 comprising monoacylglycerol, diacylglycerol, and triacylglycerol.

22. The portion (b) of the mixture is ８ -C １８ 21. The method of claim 20, comprising PEG mono- and diesters of a mixture of fatty acids, genetically attached polypeptide tags, or a combination thereof.

23. The portion (a) of the mixture is ８ -C １８ and wherein portion (b) of said mixture comprises C ８ -C １８ 21. The oral pharmaceutical composition of claim 20, comprising PEG-32 mono- and diesters of a mixture of fatty acids; the oil-based liquid formulation comprises (c) free PEG-32; and the weight / weight ratio of part (a) of the mixture to the sum of parts (b) and (c) of the mixture is from 10:90 to 30:

70.

24. 24. The oral pharmaceutical composition of claim 23, wherein (a), (b) and (c) collectively comprise 8-16% weight / weight of the oil-based liquid formulation.

25. 20. The oral pharmaceutical composition of claim 18, wherein the oil-based liquid formulation further comprises free PEG.

26. 18. The oral pharmaceutical composition of any one of claims 1-17, wherein the formulation comprises gelatin and glycerol.

27. 27. The oral pharmaceutical composition of any one of claims 18-26, further comprising a non-ionic surfactant.

28. 28. The oral pharmaceutical composition of claim 27, wherein the non-ionic surfactant is a polysorbate-based surfactant.

29. 30. The oral pharmaceutical composition of claim 28, wherein the polysorbate-based surfactant is polysorbate 80.

30. 30. The oral pharmaceutical composition of claim 29, wherein the polysorbate 80 comprises 3-10% weight / weight of the oil-based liquid formulation.

31. 31. The oral pharmaceutical composition of any one of claims 1-30, wherein the oil is a fish oil.

32. 32. The oral pharmaceutical composition of any one of claims 1-31, wherein the oil-based liquid formulation is water-free.

33. 33. The oral pharmaceutical composition of any one of claims 1-32, further comprising a coating that resists degradation in the stomach.

34. 34. The oral pharmaceutical composition of claim 33, wherein the coating is a pH sensitive capsule.

35. 34. The oral pharmaceutical composition of claim 33, wherein the coating is a soft gelatin capsule.

36. An oral pharmaceutical composition according to any one of claims 1 to 35 for oral administration of a therapeutic protein to a subject.

37. 37. The oral pharmaceutical composition for use according to claim 36, wherein said oral pharmaceutical composition prevents rapid degradation of said therapeutic peptide or therapeutic protein in said subject.

38. 37. The oral pharmaceutical composition for use according to claim 36, wherein the oral pharmaceutical composition promotes sustained activity of an orally administered therapeutic peptide or protein in the subject, or wherein the oral pharmaceutical composition promotes sustained bioavailability of an orally administered therapeutic peptide or protein in the subject.

39. 39. The oral pharmaceutical composition for use according to claim 38, wherein the sustained activity or sustained bioavailability of an orally administered therapeutic peptide or therapeutic protein exceeds the sustained activity or sustained bioavailability obtained following use of an otherwise identical oral formulation comprising chemically purified BBI instead of the isolated recombinantly expressed BBI.

40. 37. An oral pharmaceutical composition for use as described in claim 36, wherein said use provides a therapeutically effective amount of said peptide or protein or said BBI, or said therapeutic peptide or protein up to 100 kilodaltons, or a combination thereof, is provided in said formulation at a lower concentration than is therapeutically effective in an otherwise identical oral formulation containing chemically purified BBI instead of said isolated recombinantly expressed BBI, and said use achieves a comparable therapeutic effect in said subject.

41. 11. A method for orally administering a therapeutic peptide or protein to a subject, the method comprising administering to the subject an oral pharmaceutical composition according to any one of claims 1-35, thereby administering the therapeutic protein to the subject.

42. 36. A method for preventing degradation of an orally administered therapeutic peptide or protein in a subject, said method comprising administering to the subject an oral pharmaceutical composition according to any one of claims 1-35.

43. 11. A method for promoting sustained activity of an orally administered therapeutic peptide or protein in a subject, the method comprising administering to the subject an oral pharmaceutical composition according to any one of claims 1-35.

44. 11. A method for promoting sustained bioavailability of an orally administered therapeutic peptide or protein in a subject, the method comprising administering to the subject an oral pharmaceutical composition according to any one of claims 1-35.

45. 1. A method of providing a therapeutically effective amount of a peptide or protein in an oral formulation to a subject, the method comprising the step of preparing an oral pharmaceutical composition comprising a therapeutic peptide or protein of up to 100 kilodaltons, a chelator of a divalent cation, and an isolated, recombinantly expressed Bowman-Birk Inhibitor (BBI), wherein the BBI, or the therapeutic peptide or protein of up to 100 kilodaltons, or a combination thereof, is provided in an otherwise identical oral formulation that contains chemically purified BBI instead of the isolated, recombinantly expressed BBI, and which achieves a comparable therapeutic effect in the subject.

46. 46. ​​The method of claim 45, wherein the recombinantly expressed BBI has a nucleotide sequence that shares at least 95-99% identity with that set forth in SEQ ID NO:

1.

47. 46. ​​The method of claim 45, wherein the recombinantly expressed BBI has the nucleotide sequence set forth in SEQ ID NO:

1.

48. 46. ​​The method of claim 45, wherein the recombinantly expressed BBI is expressed in a yeast expression system.

49. 49. The method of claim 48, wherein the yeast expression system is Pichia pastoris.

50. 46. ​​The method of claim 45, wherein the formulation further comprises a trypsin inhibitor other than the BBI.

51. 51. The method of claim 50, wherein the trypsin inhibitor is KTI3.

52. The method of claim 51, wherein the KTI3 is chemically isolated and purified to a purity of at least 85% as measured by SDS-PAGE, or the KTI3 is purified to a protein content of greater than 95% as measured by BCA analysis.

53. 52. The method of claim 51, wherein the KTI3 is recombinantly expressed.

54. 54. The method of any one of claims 45-53, wherein the therapeutic protein is useful in treating a subject having a metabolic disease or disorder.

55. The therapeutic peptide or protein may be insulin, influenza hemagglutinin, influenza neuraminidase, glucagon, interferon gamma, interferon beta, interferon alpha, growth hormone, erythropoietin, GLP-1, GLP-1 analogs, leptin, granulocyte colony stimulating factor (G-CSF), renin, growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, coagulation factors, antigens, antibodies ... Coagulation factors, atrial natriuretic factor, surfactant protein A (SP-A), surfactant protein B (SP-B), surfactant protein C (SP-C), surfactant protein D (SP-D), plasminogen activator, bombesin, hematopoietic growth factors (colony stimulating factors, multiple), tumor necrosis factor (TNF) proteins, enkephalinase, RANTES (regulator of normal T cell expression and activation), human macrophage inflammatory protein (MIP-1-α), serum α albumin, Mullerian inhibitory substance, relaxin, mouse gonadotropin releasing hormone, deoxyribonuclease, inhibin, activin, vascular endothelial growth factor (VEGF), neurotrophic factor, neurotrophic factor-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), nerve growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor, nerve growth factor, transforming growth factor (TGF), insulin-like growth factor-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I 55. The method of any one of claims 45-54, wherein the IGF-I is selected from the group consisting of insulin-like growth factor binding protein 1 (IGFBP-1), IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, keratinocyte growth factor, osteoinductive protein, bone morphogenetic protein (BMP)-2, BMP-4, BMP-7, colony stimulating factor (CSF), interleukin (IL), superoxide dismutase, dissociation accelerating factor, chemokine family members, and complement factors.

56. 57. The method of claim 56, wherein the therapeutic peptide or protein is selected from the group consisting of insulin and a GLP-1 analogue.

57. 57. The method of claim 56, wherein the therapeutic peptide or protein is an immunomodulatory agent.

58. 58. The method of claim 57, wherein the therapeutic peptide or protein is glatiramer acetate.

59. 59. The method of any one of claims 45-58, wherein the chelating agent is EDTA.

60. 60. The method of any one of claims 45-59, wherein the formulation is an oil-based liquid formulation.

61. 61. The method of claim 60, further comprising a polyethylene glycol (PEG) ester of a monoglyceride, a diglyceride, a triglyceride, or a mixture thereof.

62. 62. The method of claim 61 , wherein the PEG ester is provided as a mixture of: (a) a monoacylglycerol, a diacylglycerol, a triacylglycerol, or a mixture thereof; and (b) a polyethylene glycol (PEG) ester of a fatty acid.

63. The portion (a) of the mixture is ８ -C １８ 63. The method of claim 62, comprising monoacylglycerols, diacylglycerols, and triacylglycerols.

64. The portion (b) of the mixture is ８ -C １８ 64. The method of claim 63, comprising PEG mono- and diesters of a mixture of fatty acids, genetically attached polypeptide tags, or a combination thereof.

65. The portion (a) of the mixture is ８ -C １８ portion (b) of said mixture comprises C ８ -C １８ 64. The method of claim 63, wherein the oil-based liquid formulation comprises PEG-32 mono- and diesters of a mixture of fatty acids; the oil-based liquid formulation comprises (c) free PEG-32; and the weight / weight ratio of part (a) of the mixture to the sum of parts (b) and (c) of the mixture is from 10:90 to 30:

70.

66. 66. The method of claim 65, wherein (a), (b), and (c) collectively comprise 8-16% weight / weight of the oil-based liquid formulation.

67. 61. The method of claim 60, wherein the oil-based liquid formulation further comprises free PEG.

68. 68. The method of any one of claims 60-67, wherein the formulation comprises gelatin and glycerol.

69. The method of any one of claims 45-68, further comprising a non-ionic surfactant.

70. 70. The method of claim 69, wherein the non-ionic surfactant is a polysorbate-based surfactant.

71. 71. The method of claim 70, wherein the polysorbate-based surfactant is polysorbate 80.

72. 72. The method of claim 71, wherein the polysorbate 80 comprises 3-10% weight / weight of the oil-based liquid formulation.

73. 73. The method of any one of claims 45-72, wherein the oil is a fish oil.

74. 74. The method of any one of claims 60-73, wherein the oil-based liquid formulation is water-free.

75. 75. The method of any one of claims 45-74, further comprising a coating that resists degradation in the stomach.

76. 76. The method of claim 75, wherein the coating is a pH sensitive capsule.

77. 76. The method of claim 75, wherein the coating is a soft gelatin capsule.

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