Methods and compositions for treating nafld, hepatic steatosis, and sequelae thereof

Abstract

To provide oral pharmaceutical compositions for treating or reducing the incidence of nonalcoholic fatty liver disease (NAFLD), hepatic steatosis and sequelae thereof.SOLUTION: An oral pharmaceutical composition comprising insulin for inhibiting the progression of nonalcoholic steatohepatitis (NASH) or for treating NASH in a subject suffering from type II diabetes includes a liquid formulation, where the insulin is present in the liquid formulation. The liquid formulation is an oil-based liquid formulation free of water, and comprises: 8 to 16 mg of insulin; 100 to 200 mg of EDTA; 50 to 100 mg of soybean trypsin inhibitor (SBTI); 20 to 30 mg of aprotinin; and 0.4 to 0.7 mL of oil.SELECTED DRAWING: None

Description

Technical Field

[0001] This specification provides an oral pharmaceutical composition comprising a GLP-1 analog and / or insulin for treating non-alcoholic fatty liver disease (NAFLD), fatty liver, and its sequelae, and for reducing its incidence.

Background Art

[0002] The incretin hormone glucagon-like peptide 1 (GLP-1), which is secreted within minutes of food intake, is associated with the induction of insulin release. GLP-1 is used in the treatment of type 2 diabetes mellitus (T2DM).

[0003] The clinical use of native GLP-1 is limited due to its rapid enzymatic inactivation that results in a half-life of 2 - 3 minutes. To overcome this obstacle, natural or synthetic, long-acting and protease-resistant peptides, called GLP-1 mimetics or analogs, have been designed and used.

[0004] Non-alcoholic fatty liver disease (NAFLD) is a variety of chronic diseases including fatty liver or bland steatosis, and non-alcoholic steatohepatitis (NASH), lobular necroinflammation including fibrosis, and sequelae such as cirrhosis (Non-Patent Document 1, Non-Patent Document 2). NAFLD-related cirrhosis can lead to end-stage liver disease and hepatocellular carcinoma (HCC). As with other chronic end-stage liver diseases, the main option for affected individuals is liver transplantation. NAFLD seems to be associated with certain lipid abnormalities (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5). Important components of the Western diet contain both saturated and trans-saturated fatty acids. According to recent evidence, these fatty acids are involved, in part, in causing steatosis and exacerbating hepatocyte insulin resistance (Non-Patent Document 6).

[0005] Some studies have shown that GLP-1 may also reduce fat loading in hepatocytes and be beneficial for NAFLD, while other studies have demonstrated that GLP-1 increases hepatic glycogen synthase activity, which is expected to exacerbate NAFLD. Indeed, the mere presence of GLP-1 receptors in hepatocytes remains a subject of controversy (Non-Patent Documents 7, 8, 9, 10, 11). In order to fully understand the response of hepatocytes to such agents, a more rigorous evaluation of inflammation and fibrosis in response to GLP-1 is considered necessary (Non-Patent Document 12).

[0006] Alzheimer's disease (AD), also known as Alzheimer's dementia, is the most common form of dementia. There is no cure for this disease, which worsens over time and is fatal. AD is most frequently diagnosed in people over 65 years of age, although earlier onset of the less common Alzheimer's disease can occur much earlier. In 2006, 26.6 million people worldwide were suffering from this disease. The cause of AD is not well understood, but is generally associated with the accumulation of amyloid-β peptide and tau protein in the brain, and many scientists believe that these substances play a role in the cause.

[0007] Parkinson's disease (PD; also known as idiopathic or primary Parkinsonism, hypokinetic rigid syndrome (HRS), or paralysis agitans) is a neurodegenerative disease. The motor symptoms of Parkinson's disease are due to the death of dopamine-producing cells in the substantia nigra, a region of the midbrain, but the cause is unknown. During the course of the disease, movement-related symptoms, later-occurring thinking and behavioral problems, and dementia in advanced disease are commonly seen. Parkinson's disease is most common in the elderly and occurs most often after the age of 50.

[0008] Huntington's disease (HD) is a neurodegenerative genetic disorder that affects muscle coordination and causes cognitive decline and psychiatric problems. HD typically becomes prominent in middle age. The huntingtin gene provides the genetic information for a protein called "huntingtin". An expansion of the CAG triplet repeat stretch within huntingtin results in a mutant form of the protein, which gradually damages brain cells through a mechanism that is not fully understood.

[0009] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease and motor neuron disease, is an incurable neurodegenerative disorder of any motor system. ALS is characterized by the selective and progressive death of motor neurons in the brain and spinal cord, paralyzing voluntary muscles and leading to death within 5 years of clinical onset. Most cases of ALS can occur sporadically with unknown etiology (Non-Patent Document 13).

[0010] Traumatic brain injury (TBI) affects 1.7 million Americans every year and is the leading cause (30.5%) of all injury-related deaths in the United States. In particular, the elderly are vulnerable to TBI, and despite the low severity of the initial injury, the mortality rate is increasing and the functional outcome is deteriorating. Many survivors experience permanent changes in cognition, motor function, and personality, as well as long-term or incurable neurocognitive disorders (Non-Patent Document 14).

[0011] Mood disorders (e.g., bipolar disorder (BD) and major depressive disorder (MDD)) are widespread, affecting 10 - 15% of the general population at some point in their lifetime. Mood disorders are also associated with various cognitive impairments that often persist during the remission phases of mania and depression in both treated and untreated individuals. Cognitive impairments have been documented in most functional domains, including but not limited to deficits in learning, memory (e.g., working memory, episodic memory, and semantic memory), attention, executive function, processing speed, and social cognition. However, conventional drugs for mood disorders have not been proven to be sufficiently effective in treating objectively and / or subjectively measured cognitive impairments (Non - Patent Document 15).

[0012] A stroke is caused by the occlusion or rupture of blood vessels, which results in the disruption of blood flow to the surrounding tissue. The tissue in the area of the stroke can suffer irreversible damage and eventually necrose and die. This cell death and resulting brain injury may sometimes be unpreventable, but there are areas of tissue in the region surrounding the damaged infarcted area of the brain that may be receptive to treatment (Non - Patent Document 16).

[0013] Diabetic retinopathy, a form of retinal degeneration, is damage to the retina caused by complications of diabetes and can ultimately lead to blindness. Diabetic retinopathy is an eye manifestation of the systemic disease diabetes and affects up to 80% of all patients with diabetes for over 10 years (Non - Patent Document 17).

[0014] Peripheral neuropathy (PN) is damage or disease of the nerves in the peripheral nervous system. Depending on the type of nerve affected, PN can also affect sensation, movement, glands, or organ function and other aspects of health status. PN can be caused by systemic diseases such as diabetes or leprosy, vitamin deficiencies, drugs (e.g., chemotherapy), trauma, excessive alcohol consumption, immune system disorders, or infections, or it can be genetic (Non - Patent Document 18).

[0015] To date, GLP-1 analogs have only been available in injectable form. The inventors are developing an oral exenatide (GLP-1 analog) formulation for use in treating diabetes. A first-in-humans trial (n = 4) testing safety in healthy humans demonstrated the biological functionality of orally delivered exenatide (Non-Patent Document 19).

Prior Art Documents

Non-Patent Documents

[0016]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Summary of the Invention

Means for Solving the Problems

[0017] In this specification, an oral pharmaceutical composition containing a GLP-1 analog and / or insulin, and a method of using the same oral pharmaceutical composition are provided for treating and preventing non-alcoholic fatty liver disease (NAFLD), fatty liver, and its sequelae. Since oral dosage forms tend to utilize the portal route of absorption, it is not possible to estimate the results from subcutaneous, systemic, and other administration routes and apply them to oral administration. As far as the inventors know, GLP-1 analogs administered orally have not been tested for the treatment of NAFLD, its sequelae, or neurodegenerative diseases.

[0018] It is described herein that the described composition can treat and prevent the sequelae of NAFLD, as well as other therapeutic effects mediated by GLP-1 receptors outside the pancreas. Thus, the described composition can treat and prevent obesity, elevated total cholesterol, hypertriglyceridemia, elevated serum ApoB levels, elevated total cholesterol / HDL ratio, and elevated ApoB / ApoA1 ratio, atherosclerosis, asymptomatic inflammation, prothrombotic states, platelet activation, endothelial dysfunction, cardioembolic states, and worsening of insulin-induced potentiation of vasodilatory responses (Sung et al, 2012; Chatrath et al 2012; Nseir et al, 2011; Salcedo et al, 2012). It is also described that the described composition can treat various neurodegenerative disorders.

[0019] Similarly, this specification also provides a solid pharmaceutical composition for administering a therapeutic protein to a subject.

[0020] The terms "protein" and "peptide" are used interchangeably herein. Neither term is intended to give a limitation on the number of amino acids present, unless the limitation is clearly specified.

[0021] Unless otherwise indicated, all ranges recited herein include the endpoints.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] This specification provides a pharmaceutical composition for treating or reducing the incidence of human non-alcoholic fatty liver disease (NAFLD), said pharmaceutical composition comprising a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises GLP-1, an analog, insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition is administered to a subject for an extended period of time.

[0023] In some embodiments, the pharmaceutical compositions described herein are solid formulations. In other embodiments, the described pharmaceutical compositions include liquid formulations, said formulations being enclosed, in certain embodiments, by capsules and / or coatings that are resistant to degradation in the stomach. In some embodiments, the liquid formulation is an oil-based liquid formulation. Some embodiments of the liquid formulation include a GLP-1 analog, insulin, at least one protease inhibitor (examples of which are trypsin inhibitor, and chymotrypsin inhibitor), and a chelating agent for divalent cations. In other embodiments, the liquid formulation includes a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In other embodiments, the liquid formulation includes insulin, at least one protease inhibitor, and a chelating agent for divalent cations. Each of the described embodiments regarding the GLP-1 analog and its amount, insulin or its amount, protease inhibitor, chelating agent, coating, etc. is also intended to be a particular embodiment, among other things, in the context of a common oil-based liquid formulation, as well as in the context of the solid formulations described herein. One of ordinary skill in the art will recognize, in light of the present disclosure, that solid dosage forms may also include different lists of inert excipients from the described liquid formulations.

[0024] In some embodiments, the subject receiving the described pharmaceutical composition already has NAFLD. In other embodiments, the subject is at risk of developing NAFLD.

[0025] In yet another aspect, the use of the combination of ingredients described herein is provided in the preparation of a medicament for treating human NAFLD.

[0026] Yet another aspect provides a method of treating or reducing the incidence of human NAFLD, the method comprising the optional step of selecting a subject by diagnosing NAFLD, and thereafter administering to the subject in need of such treatment a pharmaceutical composition described herein, thereby treating or reducing the incidence of human NAFLD.

[0027] In certain embodiments, the disorder to be treated is selected from the group consisting of nonalcoholic steatosis, nonalcoholic steatohepatitis (NASH), and lobular necroinflammatory disease including fibrosis. In other embodiments, the disease is nonalcoholic steatosis, in other embodiments, the disease is nonalcoholic steatohepatitis (NASH), and in still other embodiments, the disease is lobular necroinflammatory disease including fibrosis.

[0028] One of ordinary skill in the art will recognize, in light of the present disclosure, that NAFLD and its sequelae may also be present in subjects with diabetes, subjects showing insulin resistance without overt diabetes, and subjects not showing insulin resistance. In some embodiments, the subjects treated by the methods and compositions described have insulin resistance but do not have overt diabetes. In other embodiments, the subject does not show insulin resistance. In other embodiments, the subject is obese but has an appropriately controlled plasma glucose level. In other embodiments, the treated subject is not obese (Younossi et al 2012).

[0029] Methods for diagnosing and measuring non-alcoholic steatohepatitis are well known in the art and include, for example, ultrasound (e.g., liver-kidney contrast, liver brightness, vascular obscurity under abdominal ultrasound (Saverymuttu et al)); measurement of liver enzyme values such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase, and γ-glutamyl transpeptidase (GGT); liver mRNA values of genes involved in lipid production; and liver diacylglycerol acyltransferase-2 (DGAT2) values; (Miyashita T et al 2012; Sung et al 2012; and Juurinen et al 2007); magnetic resonance imaging (Mazhar et al, 2009), and measurement of adiponectin values.

[0030] It will be appreciated by those skilled in the art that the various disorders disclosed herein are sequelae of NAFLD (see, for example, Sung et al, 2012; Chatrath et al 2012; Nseir et al, 2011; Salcedo et al, 2012). In other instances, it will also be appreciated by those skilled in the art that GLP-1 can have a therapeutic effect independent of its effect on NAFLD.

[0031] In another embodiment herein, a pharmaceutical composition is provided for treating an increase in total cholesterol in a human suffering from fatty liver or, in another aspect, for reducing its incidence, said pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a prolonged period of time.

[0032] In yet another aspect, the use of the combination of components described herein is provided in the preparation of a medicament for treating an increase in total cholesterol in a human suffering from fatty liver or, in another aspect, for reducing its incidence.

[0033] Yet another aspect provides a method of treating an increase in total cholesterol in a subject suffering from fatty liver, or alternatively, in another aspect, reducing the incidence thereof, the method comprising administering to a subject suffering from fatty liver a pharmaceutical composition described herein, thereby treating the increase in total cholesterol or reducing the incidence thereof. Herein, in another embodiment, a pharmaceutical composition is provided for treating hypertriglyceridemia in a human suffering from fatty liver, or alternatively, in another aspect, reducing the incidence thereof, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to the subject over an extended period of time.

[0034] In yet another aspect, the use of a combination of the components described herein is provided for the preparation of a medicament for treating hypertriglyceridemia in a human suffering from fatty liver, or alternatively, in another aspect, reducing the incidence thereof.

[0035] Yet another aspect provides a method of treating hypertriglyceridemia in a subject suffering from fatty liver, or alternatively, in another aspect, reducing the incidence thereof, the method comprising administering to a subject suffering from fatty liver a pharmaceutical composition described herein, thereby treating hypertriglyceridemia or reducing the incidence thereof.

[0036] In another embodiment herein, there is provided a pharmaceutical composition for treating an increase in serum apolipoprotein B (ApoB) levels in humans suffering from fatty liver, or in another aspect, for reducing its incidence, said pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a prolonged period of time.

[0037] In another aspect, the use of a combination of components described herein is provided for the preparation of a medicament for treating an increase in ApoB levels in humans suffering from fatty liver, or in another aspect, for reducing its incidence.

[0038] In yet another aspect, there is provided a method for treating an increase in ApoB levels in humans suffering from fatty liver, or in another aspect, for reducing its incidence, said method comprising administering to a subject suffering from fatty liver a pharmaceutical composition described herein, thereby treating the increase in ApoB levels or reducing its incidence.

[0039] In another embodiment herein, there is provided a pharmaceutical composition for treating an increase in the total cholesterol / HDL ratio in a human suffering from fatty liver or, in another aspect, for reducing its incidence, said pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0040] In another aspect, the use of the combination of components described herein is provided for the preparation of a drug for treating an increase in the total cholesterol / HDL ratio in a human suffering from fatty liver or, in another aspect, for reducing its incidence.

[0041] Another aspect provides a method for treating an increase in the total cholesterol / HDL ratio in a human suffering from fatty liver or, in another aspect, for reducing its incidence, said method comprising administering to a subject suffering from fatty liver a pharmaceutical composition described herein, thereby treating the increase in the total cholesterol / HDL ratio or reducing its incidence.

[0042] In another embodiment herein, there is provided a pharmaceutical composition for treating an elevated apolipoprotein B (ApoB) / apolipoprotein A1 (ApoA1) ratio in a human suffering from fatty liver, or in another aspect, reducing its incidence rate, said pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, wherein the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0043] In another aspect, the use of the combination of components described herein is provided for the preparation of a drug for treating an elevated ApoB / ApoA1 ratio in a human suffering from fatty liver, or in another aspect, reducing its incidence rate.

[0044] In yet another aspect, there is provided a method for treating an elevated ApoB / ApoA1 ratio in a human suffering from fatty liver, or in another aspect, reducing its incidence rate, said method comprising the step of administering to a subject suffering from fatty liver the pharmaceutical composition described herein, whereby treating the elevated ApoB / ApoA1 ratio or reducing its incidence rate.

[0045] Methods for measuring each of the aforementioned lipid parameters are well known to those skilled in the art. Typical methods are described, inter alia, by Chiquette E et al and Martinez-Colubi M et al.

[0046] In another embodiment herein, there is provided a pharmaceutical composition for treating an impairment of insulin-induced enhancement of the vasodilatory response in a subject or, in another aspect, for reducing the incidence thereof, said pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over an extended period of time.

[0047] In another aspect, the use of a combination of the components described herein is provided in the preparation of a drug for treating an impairment of insulin-induced enhancement of the vasodilatory response in a subject or, in another aspect, for reducing the incidence thereof.

[0048] In yet another aspect, there is provided a method for treating an impairment of insulin-induced enhancement of the vasodilatory response in a subject or, in another aspect, for reducing the incidence thereof, said method comprising administering to the subject a pharmaceutical composition as described herein, whereby treating an impairment of insulin-induced enhancement of the vasodilatory response or reducing the incidence thereof.

[0049] Another aspect provides a method of treating or, in another aspect, reducing the incidence of an impairment in the insulin-induced potentiation of the vasodilatory response in a subject suffering from NAFLD, the method comprising administering to a subject in need of such treatment a pharmaceutical composition as described herein, thereby treating or reducing the incidence of an impairment in the insulin-induced potentiation of the vasodilatory response in a subject suffering from NAFLD. Methods of measuring the insulin-induced vasodilatory response are known in the art and include, for example, measuring the blood flow response (e.g., in the forearm) to acetylcholine (ACh) and sodium nitroprusside (SNP) (Tesauro et al).

[0050] In another embodiment herein, there is provided a pharmaceutical composition for treating or, in another aspect, reducing the incidence of fat deposition in the liver, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over an extended period of time.

[0051] In yet another aspect, the use of a combination of components as described herein is provided in the preparation of a medicament for treating or, in another aspect, reducing the incidence of fat deposition in the liver.

[0052] Another aspect provides a method of treating fat deposition in the liver or, in another aspect, reducing its incidence, the method comprising administering to a subject a pharmaceutical composition described herein, thereby treating fat deposition in the liver or reducing its incidence.

[0053] In another embodiment herein, a pharmaceutical composition for treating cardiac embolism or, in another aspect, reducing its incidence is provided, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0054] In another aspect, the use of a combination of the components described herein is provided for the preparation of a drug for treating cardiac embolism or, in another aspect, reducing its incidence.

[0055] Another aspect provides a method of treating cardiac embolism or, in another aspect, reducing its incidence, the method comprising administering to a subject a pharmaceutical composition described herein, thereby treating cardiac embolism or reducing its incidence.

[0056] In another embodiment herein, a pharmaceutical composition is provided for reversing endothelial dysfunction or, in another aspect, reducing its incidence, said pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0057] In yet another aspect, the use of a combination of the components described herein is provided for the preparation of a drug for reversing endothelial dysfunction or, in another aspect, reducing its incidence.

[0058] Yet another aspect provides a method for reversing endothelial dysfunction or, in another aspect, reducing its incidence, said method comprising administering to a subject a pharmaceutical composition described herein, whereby endothelial dysfunction is reversed or its incidence is reduced.

[0059] In another embodiment herein, a pharmaceutical composition is provided for reversing a state selected from a thrombosis-promoting state and a platelet activation state, or in another aspect, for reducing its incidence, said pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0060] In another aspect, the use of a combination of the components described herein is provided in the preparation of a drug for reversing a state selected from a thrombosis-promoting state and a platelet activation state, or in another aspect, for reducing its incidence.

[0061] In yet another aspect, a method is provided for reversing a state selected from a thrombosis-promoting state and a platelet activation state, or in another aspect, for reducing its incidence, said method comprising administering to a subject a pharmaceutical composition described herein, whereby a state selected from a thrombosis-promoting state and a platelet activation state is reversed, or its incidence is reduced.

[0062] In another embodiment herein, a pharmaceutical composition is provided for reversing asymptomatic systemic inflammation or, in another aspect, reducing its incidence, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over an extended period of time.

[0063] In yet another aspect, the use of a combination of the components described herein is provided in the preparation of a drug for reversing asymptomatic systemic inflammation or, in another aspect, reducing its incidence.

[0064] Yet another aspect provides a method for reversing asymptomatic systemic inflammation or, in another aspect, reducing its incidence, the method comprising administering to a subject a pharmaceutical composition described herein, thereby reversing asymptomatic systemic inflammation or reducing its incidence.

[0065] In another embodiment herein, a pharmaceutical composition for reducing the incidence of atherosclerosis or, in another aspect, reversing atherosclerosis is provided, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0066] In yet another aspect, the use of the combinations of components described herein is provided for the preparation of a drug for reducing the incidence of atherosclerosis or, in another aspect, reversing atherosclerosis.

[0067] Yet another aspect provides a method for reducing the incidence of atherosclerosis or, in another aspect, reversing atherosclerosis, the method comprising administering to a subject a pharmaceutical composition described herein, thereby reducing the incidence of atherosclerosis or reversing atherosclerosis.

[0068] In another embodiment herein, there is provided a pharmaceutical composition for reducing the incidence of obesity (central obesity in one embodiment) or, in another aspect, for preventing obesity, said pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0069] In yet another aspect, the use of the combinations of components described herein is provided for the preparation of a medicament for reducing the incidence of obesity or, in another aspect, for reducing the occurrence of preventing obesity.

[0070] Yet another aspect provides a method of reducing obesity in a subject suffering from fatty liver, said method comprising administering to the subject suffering from fatty liver a pharmaceutical composition as described herein, thereby reducing obesity.

[0071] In another embodiment, the present specification provides a pharmaceutical composition for reducing hepatic insulin resistance in a subject suffering from fatty liver, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In yet another embodiment, the pharmaceutical composition comprises insulin, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings that are resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over an extended period of time.

[0072] In yet another aspect, the use of a combination of the components described herein is provided for the preparation of a medicament for reducing hepatic insulin resistance in a subject suffering from fatty liver.

[0073] Yet another aspect provides a method for reducing hepatic insulin resistance in a subject suffering from fatty liver, the method comprising administering to the subject suffering from fatty liver a pharmaceutical composition as described herein, thereby reducing hepatic insulin resistance in the subject suffering from fatty liver.

[0074] The oil, insulin, GLP-1 analog, protease inhibitor, chelating agent, coating, and any other optional components of the described methods and compositions may be any of those described herein, and the respective alternatives may be freely combined to form separate embodiments of the invention disclosed herein.

[0075] As used herein, "liquid" refers to a phase that flows freely and has a definite volume under ambient conditions. Fish oil, for example, is a liquid under ambient conditions. The term includes oil-based solutions, suspensions, and combinations thereof. In alternative embodiments, the term may refer to a composition having a viscosity in the range of 1-1000 millipascal seconds at 20°C.

[0076] In embodiments where both a GLP-1 analog and insulin are present, these two components are used for co-administration together, either in the same dosage form or in separate dosage forms. In the case of separate dosage forms, "co-administration" refers herein to co-administration at the same time or, in another embodiment, may refer to administration within 30 minutes of each other. In yet different embodiments, different components are used for administration in a specific order generally separated by a time interval of 30 minutes or less. For example, a dosage form containing insulin may be administered 2-10 minutes after a dosage form containing exenatide, 10-20 minutes after a dosage form containing exenatide in other embodiments, 20-30 minutes after a dosage form containing exenatide in other embodiments, and 30-60 minutes after a dosage amount containing exenatide in other embodiments. Oral dosage forms, such as those provided herein, are useful for continuous administration more so than injectable dosage forms, as regimens related to repeated injections are likely to be associated with low compliance rates.

[0077] References to "long-term" administration in various embodiments may refer to administration for 1 month or more, 6 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 12 months or more, 15 months or more, 18 months or more, 24 months or more, 30 months or more, or 36 months or more, 48 months or more, 60 months or more, 72 months or more, 96 months or more, 10 years or more, 15 years or more, or 20 years or more. In other embodiments, the term may refer to administration for 1 - 60 months, 2 - 60 months, 3 - 60 months, 4 - 60 months, 5 - 60 months, 6 - 60 months, 8 - 60 months, 10 - 60 months, 12 - 60 months, 1 - 36 months, 2 - 36 months, 3 - 36 months, 4 - 36 months, 5 - 36 months, 6 - 36 months, 8 - 36 months, 10 - 36 months, 12 - 36 months, 1 - 36 months, 2 - 24 months, 3 - 24 months, 4 - 24 months, 5 - 24 months, 6 - 24 months, 8 - 24 months, 10 - 24 months, 12 - 24 months, 1 - 120 months, 2 - 120 months, 3 - 120 months, 4 - 120 months, 5 - 120 months, 6 - 120 months, 8 - 120 months, 10 - 120 months, 12 - 120 months, 1 - 240 months, 2 - 240 months, 3 - 240 months, 4 - 240 months, 5 - 240 months, 6 - 240 months, 8 - 240 months, 10 - 240 months, or 12 - 240 months.

[0078] In further specific embodiments, 8 - 16 mg of insulin is administered once daily for 1 month or more, 6 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 12 months or more, 15 months or more, 18 months or more, 24 months or more, 30 months or more, or 36 months or more, 48 months or more, 60 months or more, 72 months or more, 96 months or more, 10 years or more, 15 years or more, or 20 years or more. In other embodiments, the term may refer to administration for 1 - 60 months, 2 - 60 months, 3 - 60 months, 4 - 60 months, 5 - 60 months, 6 - 60 months, 8 - 60 months, 10 - 60 months, 12 - 60 months, 1 - 36 months, 2 - 36 months, 3 - 36 months, 4 - 36 months, 5 - 36 months, 6 - 36 months, 8 - 36 months, 10 - 36 months, 12 - 36 months, 1 - 36 months, 2 - 24 months, 3 - 24 months, 4 - 24 months, 5 - 24 months, 6 - 24 months, 8 - 24 months, 10 - 24 months, 12 - 24 months, 1 - 120 months, 2 - 120 months, 3 - 120 months, 4 - 120 months, 5 - 120 months, 6 - 120 months, 8 - 120 months, 10 - 120 months, 12 - 120 months, 1 - 240 months, 2 - 240 months, 3 - 240 months, 4 - 240 months, 5 - 240 months, 6 - 240 months, 8 - 240 months, 10 - 240 months, or 12 - 240 months.

[0079] In other embodiments, 8 - 16 mg of insulin is administered twice daily for 1 month or more, 6 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 12 months or more, 15 months or more, 18 months or more, 24 months or more, 30 months or more, or 36 months or more, 48 months or more, 60 months or more, 72 months or more, 96 months or more, 10 years or more, 15 years or more, or 20 years or more. In other embodiments, the term may refer to administration for 1 - 60 months, 2 - 60 months, 3 - 60 months, 4 - 60 months, 5 - 60 months, 6 - 60 months, 8 - 60 months, 10 - 60 months, 12 - 60 months, 1 - 36 months, 2 - 36 months, 3 - 36 months, 4 - 36 months, 5 - 36 months, 6 - 36 months, 8 - 36 months, 10 - 36 months, 12 - 36 months, 1 - 36 months, 2 - 24 months, 3 - 24 months, 4 - 24 months, 5 - 24 months, 6 - 24 months, 8 - 24 months, 10 - 24 months, 12 - 24 months, 1 - 120 months, 2 - 120 months, 3 - 120 months, 4 - 120 months, 5 - 120 months, 6 - 120 months, 8 - 120 months, 10 - 120 months, 12 - 120 months, 1 - 240 months, 2 - 240 months, 3 - 240 months, 4 - 240 months, 5 - 240 months, 6 - 240 months, 8 - 240 months, 10 - 240 months, or 12 - 240 months.

[0080] In yet other embodiments, exenatide at 300 - 600 mcg is administered once daily for 1 month or more, 6 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 12 months or more, 15 months or more, 18 months or more, 24 months or more, 30 months or more, or 36 months or more, 48 months or more, 60 months or more, 72 months or more, 96 months or more, 10 years or more, 15 years or more, or 20 years or more. In other embodiments, the term may refer to administration for 1 - 60 months, 2 - 60 months, 3 - 60 months, 4 - 60 months, 5 - 60 months, 6 - 60 months, 8 - 60 months, 10 - 60 months, 12 - 60 months, 1 - 36 months, 2 - 36 months, 3 - 36 months, 4 - 36 months, 5 - 36 months, 6 - 36 months, 8 - 36 months, 10 - 36 months, 12 - 36 months, 1 - 36 months, 2 - 24 months, 3 - 24 months, 4 - 24 months, 5 - 24 months, 6 - 24 months, 8 - 24 months, 10 - 24 months, 12 - 24 months, 1 - 120 months, 2 - 120 months, 3 - 120 months, 4 - 120 months, 5 - 120 months, 6 - 120 months, 8 - 120 months, 10 - 120 months, 12 - 120 months, 1 - 240 months, 2 - 240 months, 3 - 240 months, 4 - 240 months, 5 - 240 months, 6 - 240 months, 8 - 240 months, 10 - 240 months, or 12 - 240 months.

[0081] In still other embodiments, exenatide at 300 - 600 mcg is administered twice daily for 1 month or more, 6 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 12 months or more, 15 months or more, 18 months or more, 24 months or more, 30 months or more, or 36 months or more, 48 months or more, 60 months or more, 72 months or more, 96 months or more, 10 years or more, 15 years or more, or 20 years or more. In other embodiments, the term may refer to administration for 1 - 60 months, 2 - 60 months, 3 - 60 months, 4 - 60 months, 5 - 60 months, 6 - 60 months, 8 - 60 months, 10 - 60 months, 12 - 60 months, 1 - 36 months, 2 - 36 months, 3 - 36 months, 4 - 36 months, 5 - 36 months, 6 - 36 months, 8 - 36 months, 10 - 36 months, 12 - 36 months, 1 - 36 months, 2 - 24 months, 3 - 24 months, 4 - 24 months, 5 - 24 months, 6 - 24 months, 8 - 24 months, 10 - 24 months, 12 - 24 months, 1 - 120 months, 2 - 120 months, 3 - 120 months, 4 - 120 months, 5 - 120 months, 6 - 120 months, 8 - 120 months, 10 - 120 months, 12 - 120 months, 1 - 240 months, 2 - 240 months, 3 - 240 months, 4 - 240 months, 5 - 240 months, 6 - 240 months, 8 - 240 months, 10 - 240 months, or 12 - 240 months.

[0082] In yet other embodiments, exenatide at 300 - 600 mcg is administered three times a day for 1 month or more, 6 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 12 months or more, 15 months or more, 18 months or more, 24 months or more, 30 months or more, or 36 months or more, 48 months or more, 60 months or more, 72 months or more, 96 months or more, 10 years or more, 15 years or more, or 20 years or more. In other embodiments, this term may refer to administration for 1 - 60 months, 2 - 60 months, 3 - 60 months, 4 - 60 months, 5 - 60 months, 6 - 60 months, 8 - 60 months, 10 - 60 months, 12 - 60 months, 1 - 36 months, 2 - 36 months, 3 - 36 months, 4 - 36 months, 5 - 36 months, 6 - 36 months, 8 - 36 months, 10 - 36 months, 12 - 36 months, 1 - 36 months, 2 - 24 months, 3 - 24 months, 4 - 24 months, 5 - 24 months, 6 - 24 months, 8 - 24 months, 10 - 24 months, 12 - 24 months, 1 - 120 months, 2 - 120 months, 3 - 120 months, 4 - 120 months, 5 - 120 months, 6 - 120 months, 8 - 120 months, 10 - 120 months, 12 - 120 months, 1 - 240 months, 2 - 240 months, 3 - 240 months, 4 - 240 months, 5 - 240 months, 6 - 240 months, 8 - 240 months, 10 - 240 months, or 12 - 240 months.

[0083] As used herein, administration once a day may refer to administration at any time of day, or in other embodiments, to daily administration at a specific time. In certain embodiments, administration once a day may be before bedtime.

[0084] (Insulin protein and GLP-1 analog) As used herein, insulin proteins and GLP-1 analogs are, in some embodiments, isolated prior to being introduced into the described pharmaceutical compositions. In this context, "isolated" excludes the preparation of insulin and / or GLP-1 analogs as homogenized tissue specimens or other forms containing a substantial amount of contaminating protein. An example of an isolated protein or peptide is a recombinant protein or peptide. Alternative embodiments are synthetic proteins or peptides.

[0085] One of ordinary skill in the art will recognize, in light of the present disclosure, that various types of insulin are suitable for the described methods and compositions. Exemplary insulin proteins include, but are not limited to, wild-type and mutant insulin proteins, including synthetic human insulin, synthetic bovine insulin, synthetic porcine insulin, synthetic whale insulin, as well as metal complexes of insulin such as zinc complexes of insulin, protamine zinc insulin, and globin zinc.

[0086] Insulins of various classifications may also be utilized, such as rapid-acting insulin, long-acting insulin, intermediate-acting insulin, ultra-long-acting insulin, NPH insulin, glargine insulin, lispro insulin, aspart insulin, or combinations of two or more of the above types of insulin.

[0087] In certain embodiments, the insulin for the described methods and compositions is wild-type human insulin (Uniprot ID P01308). In some embodiments, human insulin is produced as a recombinant protein in bacterial cells. In other embodiments, human insulin is produced synthetically.

[0088] GLP-1 analogs are also referred to as GLP-1 mimetics in the art. One of ordinary skill in the art will recognize that the described compositions may also include at least one of the following GLP-1 analogs: exenatide (Byetta™; CAS no. 141732-76-5; SEQ ID No: 4), 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 conjugated to recombinant human albumin manufactured by ConjuChem™). All CAS records were accessed on December 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. One of ordinary skill in the art will recognize, in light of the findings described herein, that other GLP-1 analogs may be utilized.

[0089] Therapeutic insulin and GLP-1 proteins suitable for use in the present invention include derivatives that are modified (i.e., by covalent attachment of non-amino acid residues to the protein). For example, but not limited to, the protein includes modified proteins, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, or derivatization with existing protecting / blocking groups. High MWPEG can be attached to therapeutic proteins, with or without multifunctional linkers, via site-specific conjugation of PEG to the N or C terminus, or via the epsilon amino group present on lysine residues. Further, the derivative may include one or more non-classical amino acids, such as D-isomers of common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, A-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, and, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids, and other designer amino acids.

[0090] (Emulsifier) In certain embodiments, the oil-based liquid formulations utilized in the described methods and pharmaceutical compositions further include an emulsifier. One of ordinary skill in the art will recognize that, in light of the present disclosure, a variety of pharmaceutically compatible emulsifiers can be utilized.

[0091] In certain embodiments, the emulsifier is a component provided as a mixture of (a) a monoacylglycerol (monoglyceride), a diacylglycerol (diglyceride), a triacylglycerol (triglyceride), or a mixture thereof, and (b) a mixture of polyethylene glycol (PEG) esters of fatty acids. In this regard, each of the terms "monoacylglycerol", "diacylglycerol", and "triacylglycerol" need not refer to a single compound, but rather can include a mixture of compounds, for example, a mixture of monoacylglycerols, diacylglycerols, or triacylglycerols having fatty acids of various lengths. In certain preferred embodiments, the monoacylglycerol, diacylglycerol, or triacylglycerol used in the described methods and compositions, such as those used in common PEG esters, are from generally recognized as safe (GRAS) oil sources. Examples of GRAS oil sources are coconut oil, corn oil, peanut oil, soybean oil, Myvacet 9-45 (diacetylated monoglyceride of C-18 fatty acids).

[0092] Even more specific lengths of the PEG moiety used in some embodiments of the described compositions and methods include 5 - 100 monomers. In specific embodiments, the PEG may include 15 - 50 monomers. In even more specific embodiments, the PEG may include 25 - 40 monomers. In specific embodiments, the PEG may include 32 monomers.

[0093] Examples of components that meet the above specific examples are Gelucire™ 44 / 14, Gelucire™ 53 / 10, and Gelucire™ 50 / 13. A specific example 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 the polyglycolysis of hydrogenated coconut (medium-chain and long-chain triacylglycerol with PEG-32). It has a hydrophilic / lipophilic balance of 14. It consists of a defined mixture of C8-C18 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 consisting of a PEG ester fraction under a lamellar phase of 120 Å, including a helical three-dimensional structure and an acylglycerol fraction under hexagonal packing. The main products of simulated gastrointestinal lipolysis of Gelucire™ 44 / 14 are PEG-32 mono- and diesters.

[0094] In some embodiments, the HLB of the self-emulsifying component utilized in the described methods and compositions is 10 or greater. In other embodiments, it is between 11 and 19. In other embodiments, it is between 12 and 18. In other embodiments, it is between 12 and 17. In other embodiments, it is between 12 and 16, indicating an oil-in-water (O / W) emulsifier. In other embodiments, it is between 13 and 15. In other embodiments, it is 14. Even more specific embodiments of the self-emulsifying component have an HLB of 12-16 and include PEG, free triacylglycerol, and medium-chain and long-chain triacylglycerols conjugated to free PEG. In other embodiments, the self-emulsifying component consists of a mixture of PEG, free triacylglycerol, and medium-chain and long-chain triacylglycerols conjugated to free PEG and has an HLB of 12-16. In other embodiments, the self-emulsifying component has an HLB of 14 and includes PEG, free triacylglycerol, and medium-chain and long-chain triacylglycerols conjugated to free PEG. In other embodiments, the self-emulsifying component consists of a mixture of PEG, free triacylglycerol, and medium-chain and long-chain triacylglycerols conjugated to free PEG and has an HLB of 14.

[0095] In certain embodiments, in particular embodiments, the aforementioned emulsifier, which is a self-emulsifying component, constitutes 8-16% weight / weight of the oil-based liquid formulation. In specific embodiments, the amount is 9-15%. In specific embodiments, the amount is 10-14%. In specific embodiments, the amount is 11-13%. In specific embodiments, the amount is 12%.

[0096] (Non-ionic detergent) In certain embodiments, the oil-based liquid formulations used in the described methods and pharmaceutical compositions further comprise a polysorbate-based detergent. Examples of polysorbate-based detergents are detergents derived from covalently bonding polyethoxylated sorbitan to fatty acids. Specific embodiments of polysorbate-based detergents are polysorbate-20, polysorbate-40, and polysorbate 80.

[0097] For example, polysorbate 80 (Tween 80) is a mild nonionic detergent derived from polyethoxylated sorbitan and oleic acid and has the following structure.

[0098] [Chemical formula] In the case of polysorbate 80, the portion shown on the right is a mixture of fatty acids containing 60 - 70% oleic acid (as depicted), the balance being mainly linoleic acid, palmitic acid, and stearic acid.

[0099] In a specific embodiment, polysorbate 80 constitutes 3 - 10% weight / weight of the oil-based liquid formulation used in the described methods and compositions. In a specific embodiment, the proportion is 4 - 8%. In a specific embodiment, the proportion is 4.5 - 6%. In a specific embodiment, the proportion is 5%.

[0100] (Dosage) In specific embodiments, the amount of insulin in the dosage forms of the described methods and compositions is 6 - 64 mg. In other embodiments, the amount is 6 - 56 mg. In other embodiments, the amount is 6 - 48 mg. In other embodiments, the amount is 6 - 40 mg. In other embodiments, the amount is 6 - 36 mg. In other embodiments, the amount is 6 - 32 mg. In other embodiments, the amount is 6 - 28 mg. In other embodiments, the amount is 6 - 24 mg. In other embodiments, the amount is 6 - 20 mg. In other embodiments, the amount is 6 - 14 mg. In other embodiments, the amount is 6 - 12 mg. In other embodiments, the amount is 6 - 10 mg. In other embodiments, the amount is 10 - 64 mg. In other embodiments, the amount is 10 - 56 mg. In other embodiments, the amount is 10 - 48 mg. In other embodiments, the amount is 10 - 40 mg. In other embodiments, the amount is 10 - 36 mg. In other embodiments, the amount is 10 - 32 mg. In other embodiments, the amount is 10 - 28 mg. In other embodiments, the amount is 16 - 64 mg. In other embodiments, the amount is 16 - 56 mg. In other embodiments, the amount is 16 - 48 mg. In other embodiments, the amount is 16 - 40 mg. In other embodiments, the amount is 16 - 36 mg. In other embodiments, the amount is 16 - 32 mg. In other embodiments, the amount is 16 - 28 mg. In certain embodiments, the above dosage is a daily dosage.

[0101] In other embodiments, the amount is 8 mg. In other embodiments, the amount is 12 mg. In other embodiments, the amount is 16 mg. In other embodiments, the amount is 20 mg. In other embodiments, the amount is 24 mg. In other embodiments, the amount is 32 mg. In other embodiments, the amount is 40 mg. In other embodiments, the amount is 40 mg. In other embodiments, the amount is 56 mg. In other embodiments, the amount is 64 mg. In other embodiments, the amount is 8 - 16 mg. In other embodiments, the amount is 8 - 14 mg. In other embodiments, the amount is 8 - 12 mg. In other embodiments, the amount is 8 - 10 mg. In other embodiments, the amount is 16 mg. In other embodiments, the amount is 10 - 16 mg. In other embodiments, the amount is 10 - 14 mg. In other embodiments, the amount is 10 - 18 mg. In certain embodiments, the above dosage is a daily dosage.

[0102] In other embodiments, the amount of insulin in the dosage forms of the described methods and compositions is 0.06 - 0.64 mg / kg (milligrams per kilogram of body weight). In other embodiments, the amount is 0.06 - 0.56 mg / kg. In other embodiments, the amount is 0.06 - 0.48 mg / kg. In other embodiments, the amount is 0.06 - 0.40 mg / kg. In other embodiments, the amount is 0.06 - 0.32 mg / kg. In other embodiments, the amount is 0.06 - 0.28 mg / kg. In other embodiments, the amount is 0.06 - 0.24 mg / kg. In other embodiments, the amount is 0.06 - 0.20 mg / kg. In other embodiments, the amount is 0.10 - 0.16 mg / kg. In other embodiments, the amount is 0.10 - 0.64 mg / kg. In other embodiments, the amount is 0.10 - 0.56 mg / kg. In other embodiments, the amount is 0.10 - 0.48 mg / kg. In other embodiments, the amount is 0.10 - 0.40 mg / kg. In other embodiments, the amount is 0.10 - 0.32 mg / kg. In other embodiments, the amount is 0.10 - 0.28 mg / kg. In other embodiments, the amount is 0.10 - 0.24 mg / kg. In other embodiments, the amount is 0.10 - 0.20 mg / kg. In other embodiments, the amount is 0.06 - 0.14 mg / kg. In other embodiments, the amount is 0.06 - 0.12 mg / kg. In other embodiments, the amount is 0.06 - 0.10 mg / kg. In other embodiments, the amount is 0.08 mg / kg. In other embodiments, the amount is 0.12 mg / kg. In other embodiments, the amount is 0.16 mg / kg. In other embodiments, the amount is 0.08 - 0.16 mg / kg. In other embodiments, the amount is 0.08 - 0.14 mg / kg. In other embodiments, the amount is 0.08 - 0.12 mg / kg. In other embodiments, the amount is 0.08 - 0.10 mg / kg. In other embodiments, the amount is 0.10 - 0.16 mg / kg. In other embodiments, the amount is 0.10 - 0.18 mg / kg. In other embodiments, the amount is 0.10 - 0.14 mg / kg. In certain embodiments, the above dosage is a daily dosage.

[0103] In yet other embodiments, the amount of insulin in the dosage form is an amount corresponding to one of the above amounts or ranges for adults adjusted per kilogram of body weight of the pediatric patient. This type of adjustment referred to herein employs 62 kilograms as the adult body weight. In other embodiments, insulin is present in an amount adjusted for the pediatric patient, and the GLP-1 analog is also present in an amount adjusted for the pediatric patient. In certain embodiments, the above dosage is a daily dosage.

[0104] The dosages described herein for insulin may be for wild-type human insulin, or, in another embodiment, may be for one of the other types of insulin known in the art.

[0105] In other embodiments, the amount of the GLP-1 analog in the dosage form of the described method and composition is 150 micrograms (mcg), 200 mcg, 250 mcg, 300 mcg, 350 mcg, 400 mcg, 500 mcg, or 600 mcg. In other embodiments, the amount of the GLP-1 analog is 100 - 1600 mcg in adult patients. In other embodiments, the amount is 100 - 1400 mcg. In other embodiments, the amount is 100 - 1200 mcg. In other embodiments, the amount is 100 - 1000 mcg. In other embodiments, the amount is 100 - 800 mcg. In other embodiments, the amount is 100 - 700 mcg. In other embodiments, the amount is 100 - 600 mcg. In other embodiments, the amount is 100 - 500 mcg. In other embodiments, the amount is 100 - 400 mcg. In other embodiments, the amount is 200 - 1400 mcg. In other embodiments, the amount is 200 - 1400 mcg. In other embodiments, the amount is 200 - 1200 mcg. In other embodiments, the amount is 200 - 1000 mcg. In other embodiments, the amount is 200 - 800 mcg. In other embodiments, the amount is 200 - 700 mcg. In other embodiments, the amount is 200 - 600 mcg. In other embodiments, the amount is 200 - 500 mcg. In other embodiments, the amount is 200 - 400 mcg. In other embodiments, the amount is 100 - 300 mcg. In other embodiments, the amount is 100 - 250 mcg. In other embodiments, the amount is 100 - 200 mcg. In other embodiments, the amount is 100 - 150 mcg. In other embodiments, the amount is 100 mcg. In other embodiments, the amount is 150 mcg. In other embodiments, the amount is 200 mcg. In other embodiments, the amount is 250 mcg. In other embodiments, the amount is 300 mcg. In other embodiments, the amount is 150 - 400 mcg. In other embodiments, the amount is 150 - 300 mcg. In other embodiments, the amount is 150 - 250 mcg. In other embodiments, the amount is 150 - 200 mcg. In certain embodiments, the above dosage is a daily dosage.

[0106] In other embodiments, the GLP-1 analog is exenatide present in one of the above amounts. In other embodiments, the amount of the GLP-1 analog in the dosage forms of the described methods and compositions is 0.100 - 1.60 mcg / kg in adult patients. In other embodiments, the amount is 0.100 - 1.40 mcg / kg. In other embodiments, the amount is 0.100 - 1.20 mcg / kg. In other embodiments, the amount is 0.100 - 1.0 mcg / kg. In other embodiments, the amount is 0.100 - 0.800 mcg / kg. In other embodiments, the amount is 0.100 - 0.700 mcg / kg. In other embodiments, the amount is 0.100 - 0.600 mcg / kg. In other embodiments, the amount is 0.100 - 0.500 mcg / kg. In other embodiments, the amount is 0.100 - 0.400 mcg / kg. In other embodiments, the amount is 0.200 - 1.40 mcg / kg. In other embodiments, the amount is 0.200 - 1.40 mcg / kg. In other embodiments, the amount is 0.200 - 1.20 mcg / kg. In other embodiments, the amount is 0.200 - 1.0 mcg / kg. In other embodiments, the amount is 0.200 - 0.800 mcg / kg. In other embodiments, the amount is 0.200 - 0.700 mcg / kg. In other embodiments, the amount is 0.200 - 0.600 mcg / kg. In other embodiments, the amount is 0.200 - 0.500 mcg / kg. In other embodiments, the amount is 0.200 - 0.400 mcg / kg. In other embodiments, the amount is 0.100 - 0.300 mcg / kg. In other embodiments, the amount is 0.100 - 0.250 mcg / kg. In other embodiments, the amount is 0.100 - 0.200 mcg / kg. In other embodiments, the amount is 0.100 - 0.150 mcg / kg. In other embodiments, the amount is 0.100 mcg / kg. In other embodiments, the amount is 0.150 mcg / kg. In other embodiments, the amount is 0.200 mcg / kg. In other embodiments, the amount is 0.250 mcg / kg. In other embodiments, the amount is 0.300 mcg / kg. In other embodiments, the amount is 0.150 - 0.400 mcg / kg.In other embodiments, the amount is 0.150 - 0.300 mcg / kg. In other embodiments, the amount is 0.150 - 0.250 mcg / kg. In other embodiments, the amount is 0.100 - 0.200 mcg / kg. In certain embodiments, the above dosage is a daily dosage. In still other embodiments, the GLP-1 analog is exenatide present in one of the above amounts.

[0107] In other embodiments, the amount of the GLP-1 analog in the dosage form is an amount corresponding to one of the above amounts or ranges for adults adjusted per kilogram of body weight of the pediatric patient. In other embodiments, the GLP-1 analog is present in an amount adjusted for the pediatric patient and the insulin is present in an amount adjusted for the pediatric patient, for example, an amount corresponding to 4 - 12 mg for adults adjusted with respect to the body weight of the pediatric patient. In certain embodiments, the above dosage is a daily dosage. In still other embodiments, the GLP-1 analog is exenatide present in one of the above amounts.

[0108] In various embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 8 - 16 mg of insulin. In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg or 150 - 250 mcg, together with 8 - 12 mg of insulin. In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 12 - 16 mg of insulin. In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 16 - 24 mg of insulin. In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 24 - 32 mg of insulin. In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 12 - 16 mg of insulin.In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 8 mg of insulin. In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 12 mg of insulin. In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 16 mg of insulin. In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 20 mg of insulin. In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 24 mg of insulin. In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 28 mg of insulin.In other embodiments, the described dosage forms contain exenatide in an amount of 100 - 600 mcg, 100 - 500 mcg, 100 - 400 mcg, 100 - 300 mcg, 200 - 600 mcg, 200 - 500 mcg, 200 - 400 mcg, 200 - 300 mcg, 150 - 300 mcg, or 150 - 250 mcg, together with 32 mg of insulin. In certain embodiments, the above dosages are daily dosages.

[0109] In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg, together with 150 - 300 mcg of exenatide. In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg, together with 300 - 450 mcg of exenatide. In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg, together with 450 - 600 mcg of exenatide. In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg, together with 100 - 150 mcg of exenatide. In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg, together with 150 - 200 mcg of exenatide. In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg, together with 200 - 250 mcg of exenatide.In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg, together with 250 - 300 mcg of exenatide. In certain embodiments, the above dosages are daily dosages.

[0110] In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg together with 100 mcg of exenatide. In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg together with 200 mcg of exenatide. In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg together with 250 mcg of exenatide. In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg together with 300 mcg of exenatide. In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg together with 400 mcg of exenatide. In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg together with 500 mcg of exenatide.In other embodiments, the described dosage forms contain insulin in an amount of 8 - 32 mg, 8 - 28 mg, 8 - 24 mg, 8 - 20 mg, 8 - 16 mg, 8 - 12 mg, 12 - 32 mg, 16 - 32 mg, 20 - 32 mg, 24 - 32 mg, 12 - 24 mg, 16 - 24 mg, 12 - 20 mg, or 16 - 20 mg together with 600 mcg of exenatide. In certain embodiments, the above dosages are daily dosages.

[0111] In other embodiments, the described dosage forms contain 8 - 16 mg of insulin and 150 - 300 mcg of exenatide. In other embodiments, the described dosage forms contain 8 - 12 mg of insulin and 150 - 300 mcg of exenatide. In other embodiments, the described dosage forms contain 12 - 16 mg of insulin and 150 - 300 mcg of exenatide. In other embodiments, the described dosage forms contain 6 - 16 mg of insulin and 150 - 300 mcg of exenatide. In certain embodiments, the above dosages are daily dosages.

[0112] In other embodiments, the described dosage forms contain 8 - 16 mg of insulin and 100 - 400 mcg of exenatide. In other embodiments, the described dosage forms contain 8 - 12 mg of insulin and 100 - 400 mcg of exenatide. In other embodiments, the described dosage forms contain 12 - 16 mg of insulin and 100 - 400 mcg of exenatide. In other embodiments, the described dosage forms contain 6 - 16 mg of insulin and 100 - 400 mcg of exenatide. In certain embodiments, the above dosages are daily dosages.

[0113] In other embodiments, the described dosage form contains 8 - 16 mg of insulin and 100 - 200 mcg of exenatide. In other embodiments, the described dosage form contains 8 - 12 mg of insulin and 100 - 200 mcg of exenatide. In other embodiments, the described dosage form contains 12 - 16 mg of insulin and 100 - 200 mcg of exenatide. In other embodiments, the described dosage form contains 6 - 16 mg of insulin and 100 - 200 mcg of exenatide. In certain embodiments, the above dosages are daily dosages.

[0114] In other embodiments, the described dosage form contains 8 - 16 mg of insulin and 200 - 400 mcg of exenatide. In other embodiments, the described dosage form contains 8 - 12 mg of insulin and 200 - 400 mcg of exenatide. In other embodiments, the described dosage form contains 12 - 16 mg of insulin and 200 - 400 mcg of exenatide. In other embodiments, the described dosage form contains 6 - 16 mg of insulin and 200 - 400 mcg of exenatide. In certain embodiments, the above dosages are daily dosages.

[0115] In other embodiments, the described dosage form contains 8 - 16 mg of insulin and 150 - 300 mcg of exenatide. In certain embodiments, the above dosages are daily dosages.

[0116] (Protease inhibitor) One skilled in the art will recognize that, in light of the present disclosure, various protease inhibitors may be used to protect the GLP-1 analog and / or insulin in the described formulations. In certain embodiments, the protease inhibitor present in the described composition is selected from a trypsin inhibitor and a chymotrypsin inhibitor. In more specific embodiments, the inhibitor is a trypsin inhibitor, or in other embodiments, a chymotrypsin inhibitor. In yet other embodiments, the pharmaceutical composition includes one or more inhibitors that inhibit both trypsin and chymotrypsin together. In more specific embodiments, both a trypsin inhibitor and a chymotrypsin inhibitor are present, or in other embodiments, the protease inhibitor is a single inhibitor that inhibits both trypsin and chymotrypsin. An example of a single inhibitor that inhibits both trypsin and chymotrypsin, although not intended to be limiting, is BBI (described herein).

[0117] As used herein, the term "chymotrypsin inhibitor" refers to any agent that can inhibit the action of chymotrypsin on a substrate. The ability of this agent to inhibit chymotrypsin can be measured using assays well known in the art. 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, for example, K. Ozawa, M. Laskowski, 1966, J. Biol. Chem. 241:3955; and Y. Birk, 1976, Meth. Enzymol. 45:700. Unless otherwise specified, the anti-chymotrypsin 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 the protein being tested. BTEE refers to N-benzoyl-L-tyrosine ethyl ester (see instructions for Sigma-Aldrich Product No. B6125).

[0118] As used herein, the term "trypsin inhibitor" refers to any agent that can inhibit the action of trypsin on a substrate. The ability of an agent to inhibit trypsin can be measured using assays well known in the art. 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, for example, K. Ozawa, M. Laskowski, 1966, J. Biol. Chem. 241:3955;￥ and Y. Birk, 1976, Meth. Enzymol. 45:700. An example of an inhibitor that inhibits trypsin is, but is not intended to be limiting, KTI3 (described herein).

[0119] Unless otherwise specified, the anti-trypsin activity referred to herein is measured using trypsin having an activity of 10,000 BAEE units per mg of trypsin and is expressed in mg of trypsin inhibited per mg of the protein being tested. BAEE refers to a solution of Na-benzoyl-L-arginine ethyl ester (see instructions for Sigma-Aldrich Product No. 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 by 0.001 per minute at 253 nm at pH 7.6 and 25°C. See, for example, K. Ozawa, M. Laskowski, 1966, J. Biol. Chem. 241:3955; and Y. Birk, 1976, Meth. Enzymol. 45:700.

[0120] Some of the known trypsin inhibitors of the art are specific for trypsin, while other trypsin inhibitors inhibit trypsin and other proteases such as chymotrypsin. Trypsin inhibitors can be derived from animal or plant sources: for example, soybeans, corn, lima beans, and other legumes, squash, sunflowers, the pancreas and lungs of cows and other animals, chicken, and the egg white of turkeys, soy-based infant formula, and mammalian blood. Trypsin inhibitors can also be of microbial origin, such as antipain. See, for example, H. Umezawa, 1976, Meth. Enzymol. 45, 678. Trypsin inhibitors can be arginine or lysine mimetics or other synthetic compounds, such as arylguanidine, benzamidine, 3,4-dichloroisocoumarin, diisopropylfluorophosphate, gabexate mesylate, or phenylmethanesulfonyl fluoride. As used herein, an arginine or lysine mimetic is a compound that can bind to the P1 pocket of trypsin and / or interfere with trypsin active site function.

[0121] In certain embodiments, when a trypsin inhibitor is utilized in the methods and compositions of the invention, the trypsin inhibitor is selected from the group consisting of lima bean trypsin inhibitor, aprotinin (also known as pancreatic trypsin inhibitor or basic pancreatic trypsin inhibitor [BPTI]), Uniprot No. P00974 [database accessed on January 2, 2013], Kazal inhibitor (pancreatic secretory trypsin inhibitor), ovomucoid, alpha1-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, Clan A serpins [e.g., Serpina3 (NCBI gene ID: 12; database accessed on December 27, 2012), Serpina6 (NCBI gene ID: 866; database accessed on December 27, 2012), Serpinal2 (NCBI gene ID: 145264; database accessed on December 27, 2012), Serpina10 (NCBI gene ID: 51156; database accessed on December 27, 2012); Serpina7 (NCBI gene ID: 6906; database accessed on December 27, 2012); Serpina9 (NCBI gene ID: 327657; database accessed on December 27, 2012); Serpina11 (NCBI gene ID: 256394; database accessed on December 27, 2012); Serpina13 (NCBI gene ID: 388007; database accessed on December 27, 2012); Serpina2 (NCBI gene ID: 390502; database accessed on December 27, 2012); and, Serpina4 (NCBI gene ID: 5104; database accessed on December 27, 2012)] Yukopin (SerpinB12; gene ID: 89777; database accessed on December 27, 2012), antipain, benzamidine, 3,4-dichloroisocoumarin, and, gabexate mesylate. In other embodiments, one or more, such as 2, 3, or 4 of the above inhibitors are selected.

[0122] Typical precursor sequences of aprotinin are as follows:

[0123] [Chemical formula]

[0124] Of these 100 residues, residues 1-21 are the signal peptide, 22-35 and 94-100 are the propeptide, and the aprotinin chain, which is the mature chain, consists of residues 36-93 (58AA).

[0125] In certain embodiments, when a chymotrypsin inhibitor is used in the methods and compositions of the invention, the chymotrypsin inhibitor is selected from the group consisting of soybean trypsin inhibitor, Bowman-Birk inhibitor, aprotinin, N-acetyl-eglin C from hirudin (Sigma-Aldrich cat.no.E7888), chymostatin (Sigma-Aldrich cat.No.C7268), 1-antitrypsin (Sigma-Aldrich cat.No.A9024), al-antichymotrypsin (Sigma-Aldrich cat.No.A9285), potato type I protease inhibitor, and potato type II protease inhibitor.

[0126] In other embodiments, the trypsin inhibitor and / or chymotrypsin inhibitor is derived from soybeans. Inhibitors of chymotrypsin and trypsin derived from soybeans (Glycine max) are readily available and are considered safe for human consumption. These include SBTI (soybean trypsin inhibitor), which in its native form consists of KTI3 (Kunitz soybean trypsin inhibitor 3), which inhibits trypsin, and BBI (Bowman - Birk inhibitor; Uniprot number P01055 [database accessed on January 3, 2013]), which inhibits both chymotrypsin and trypsin. The term "SBTI" as used herein, unless otherwise specified, refers to the combination of KTI3 and BBI, which may be a naturally occurring combination or an artificial combination of KTI3 and BBI. In accordance with the approved uses in the art, SBTI is often referred to herein as a single protease inhibitor despite the fact that it comprises two separate protease components.

[0127] Inhibitors of chymotrypsin and trypsin are available, for example, from Sigma - Aldrich, St. Louis, Missouri, USA.

[0128] In certain embodiments, SBTI is present in an amount of 25-125 mg per dosage unit when present with chymotrypsin and / or another inhibitor of trypsin, and in an amount of 75-200 mg per dosage unit when present as the inhibitor of chymotrypsin and / or trypsin alone. In various embodiments, SBTI is present in an amount of 30-180 mg, 35-170 mg, 40-160 mg, 45-150 mg, 50-140 mg, 50-130 mg, 50-120 mg, 50-100 mg, 55-95 mg, 60-90 mg, 65-85 mg, 70-80 mg, 50-150 mg, 60-140 mg, 70-130 mg, 80-120 mg, 90-100 mg, 100-150 mg, 110-140 mg, 120-130 mg, 100-200 mg, 110-190 mg, 120-180 mg, 130-170 mg, or 140-160 mg per dosage unit. All of the foregoing ranges are included. In other embodiments, SBTI is present in an amount of 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 75 mg, 90 mg, 100 mg, 110 mg, 125 mg, 150 mg, 175 mg, or 200 mg per dosage unit.

[0129] Methods for preparing BBI are described, for example, in U.S. Patent No. 7,404,973 and PCT International Application No. WO 2013 / 10289 by Avraham Hershko, filed January 31, 2013, the contents of which are incorporated herein by reference.

[0130] A representative precursor sequence of BBI is as follows:

[0131]

Chemical formula

[0132] Of these 110 residues, residues 1-19 are the signal peptide, 20-39 are the propeptide, and the mature BBI chain consists of residues 40-110 (71 AA).

[0133] In certain embodiments, SBTI is present in an amount of 25 - 125 mg per dosage unit when present with another inhibitor of chymotrypsin and / or trypsin, and in an amount of 75 - 200 mg per dosage unit when present as the inhibitor of chymotrypsin and / or trypsin only. In various embodiments, BBI is present in an amount of 30 - 180 mg, 35 - 170 mg, 40 - 160 mg, 45 - 150 mg, 50 - 140 mg, 50 - 130 mg, 50 - 120 mg, 50 - 100 mg, 55 - 95 mg, 60 - 90 mg, 65 - 85 mg, 70 - 80 mg, 50 - 150 mg, 60 - 140 mg, 70 - 130 mg, 80 - 120 mg, 90 - 100 mg, 100 - 150 mg, 110 - 140 mg, 120 - 130 mg, 100 - 200 mg, 110 - 190 mg, 120 - 180 mg, 130 - 170 mg, or 140 - 160 mg per dosage unit. All of the foregoing ranges are included. In other embodiments, BBI is present in an amount of 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 75 mg, 90 mg, 100 mg, 110 mg, 125 mg, 150 mg, 175 mg, or 200 mg per dosage unit.

[0134] In one aspect, the BBI utilized in the described methods and compositions is at least 85% pure as measured in various embodiments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie Brilliant Blue staining, or imaging device quantification. In certain embodiments, the soy product is soy flour.

[0135] In yet another aspect, the protein content of BBI is 95% or more by the BCA (bicinchoninic acid) assay.

[0136] In yet another aspect, the BBI utilized in the described methods and compositions contains less than 0.1% high MW contaminants as evaluated, for example, by SDS-PAGE and imaging device quantification.

[0137] In other embodiments, the isolated BBI inhibits the anti-chymotrypsin activity of chymotrypsin by at least 0.8, 0.9, 1.0, 1.1, 1.2, or 1.4 mg of chymotrypsin per mg of inhibitor. In other embodiments, the anti-chymotrypsin activity is in the range of 0.8 - 1.8, 0.9 - 1.8, 1.0 - 1.8, 1.1 - 1.8, 1.2 - 1.8, or 1.4 - 1.8 mg of inhibited chymotrypsin per mg of inhibitor. In a specific embodiment, the activity is 0.8 - 1.8 mg of inhibited chymotrypsin per mg of inhibitor. In other embodiments, the activity is in the range of 0.8 - 1.5 mg of inhibited chymotrypsin per mg of inhibitor. In the context of a pharmaceutical composition, this value refers to the characteristics of the BBI before it is mixed with one or more other components of the pharmaceutical composition. One of ordinary skill in the art will understand that each of the purity requirements above is generally evaluated for the BBI before it is mixed with any of the other components of the pharmaceutical composition, with respect to its protein content, contaminant values, or potency. Various purified types of BBI are described in PCT International Application No. WO 2013 / 10289 of Avraham Hershko, filed Jan. 31, 2013, the contents of which are incorporated herein by reference.

[0138] In other embodiments, the isolated BBI in the described methods and compositions is a recombinant BBI, e.g., a BBI produced by a microorganism such as a bacterium that is engineered to express and then isolated. In still other embodiments, the BBI is a synthetic BBI. An example of a synthetic BBI is a BBI produced in a cell-free device such as a peptide synthesizer. Peptide synthesizers, e.g., automated peptide synthesizers, are well known in the art and commercially available. Pharmaceutical compositions comprising recombinant BBI are also provided herein. Pharmaceutical compositions comprising synthetic BBI are also provided herein.

[0139] In certain embodiments, the described BBI is the only protease inhibitor of the described methods and compositions. Ineffective SBTI requires additional protease inhibitors (e.g., aprotinin) to efficiently protect specific therapeutic proteins in the human gastrointestinal tract, whereas the isolated BBI described can, in some embodiments, reduce the need for additional protease inhibitors in this regard.

[0140] The method for preparing KTI3 is described in PCT International Application No. WO 2013 / 102899 of Avraham Hershko, filed on January 31, 2013, the content of which is incorporated herein by reference.

[0141] KTI3 has Uniprot number P01070 (database accessed on January 3, 2013). A representative precursor sequence of KTI3 is as follows:

[0142]

Chemical formula

[0143] Of the above sequences, residues 1-24 are the signal peptide, 206-216 are the propeptide, and the mature KTI3 chain consists of residues 25-205 (181 AA).

[0144] In certain embodiments, KTI3 is present in an amount of 25-125 mg per dosage unit and is present in the chymotrypsin inhibitor. In various embodiments, KTI3 is present in an amount of 30-120 mg, 35-115 mg, 40-110 mg, 45-105 mg, 50-100 mg, 55-95 mg, 60-90 mg, 65-85 mg, 70-80 mg, 25-100 mg, 30-90 mg, 35-80 mg, 35-70 mg, 40-60 mg, 45-55 mg, 80-120 mg, 85-115 mg, 90-100 mg, or 95-105 mg per dosage unit. All of the foregoing ranges are included. In other embodiments, KTI3 is present in an amount of 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 75 mg, 90 mg, 100 mg, 110 mg, or 120 mg per dosage unit.

[0145] In additional aspects, the KTI3 utilized in the described methods and compositions is at least 85% pure as measured in various embodiments by SDS-PAGE, Coomassie Blue staining, or imaging device quantification.

[0146] In yet another aspect, the protein content of KTI3 is 95% or greater as measured by the BCA assay. In yet another aspect, KTI3 contains less than 0.1% high MW contaminants as evaluated, for example, by SDS-PAGE and imaging device quantification.

[0147] In other embodiments, the isolated KTI3 has an anti-trypsin activity of at least 0.8, 0.9, 1.0, 1.1, 1.2, or 1.3 mg of inhibited trypsin per mg of inhibitor. In other embodiments, the activity of KTI3 is in the range of 0.8-1.8, 0.9-1.8, 1.0-1.8, 1.1-1.8, 1.2-1.8, or 1.3-1.8 mg of inhibited trypsin per mg of inhibitor. In specific embodiments, the activity of KTI3 is 0.8-1.7 mg of inhibited trypsin per mg of inhibitor. In other embodiments, the activity is in the range of 0.8-1.4 mg of inhibited trypsin per mg of inhibited.

[0148] In other preferred embodiments, the isolated KTI is a recombinant KTI, for example, a KTI produced by a microorganism such as a bacterium engineered to express it. In yet other embodiments, the KTI is a synthetic KTI. An example of a synthetic KTI is a KTI produced by a cell-free device such as a peptide synthesizer.

[0149] One of ordinary skill in the art will understand that each of the purity requirements above is generally evaluated with respect to its protein content, contaminant values, or potency before KTI3 is mixed with any of the other components of the pharmaceutical composition. Various types of purified KTI are described in PCT International Application No. WO 2013 / 10289 to Avraham Hershko, filed January 31, 2013, the contents of which are incorporated herein by reference.

[0150] Other embodiments relate to the ratio of anti-chymotrypsin activity present in the described pharmaceutical composition to the anti-trypsin activity of the pharmaceutical composition. In some embodiments, this parameter is from 1.5:1 to 1:1. In specific embodiments, this ratio is from 1.4:1 to 1.1:1. In specific embodiments, this ratio is from 1.35:1 to 1.2:1.

[0151] In still further other embodiments, the described purified SBTI, BBI, and / or KTI are present, in some embodiments, in combination with at least one of the described nonionic detergents and together with at least one of the described emulsifiers. Each of the described embodiments of the purified SBTI, BBI, and / or KTI may be freely combined with the described embodiments of the emulsifier and / or nonionic detergent.

[0152] (Combination of Protease Inhibitors) In certain embodiments, the pharmaceutical compositions utilized in the described methods and compositions comprise a combination of isolated BBI and another protease inhibitor, such as a trypsin inhibitor. In other embodiments, the pharmaceutical compositions comprise a combination of isolated KTI3 and another protease inhibitor. One of ordinary skill in the art will recognize, in light of the present disclosure, that a variety of protease inhibitors, including those inhibitors mentioned above, may be employed. In the case of protease inhibitors that are proteins, the size is generally up to 100 kDa at most.

[0153] In other embodiments, the pharmaceutical compositions utilized in the described methods and compositions comprise both isolated BBI and isolated KTI, in certain embodiments, both isolated BBI and isolated KTI3. The terms “isolated KTI3” and “isolated BBI” as used herein refer to preparations that are enriched in the specified component relative to other components of SBTI (i.e., BBI or KTI3, respectively). In various embodiments, the preparation of KTI utilized in the described methods and compositions is at least 85% pure as evaluated by SDS-PAGE, Coomassie Blue staining, or imager quantification (e.g., according to the procedures described herein). In other embodiments, the protein content of the KTI preparation is 95% or greater by BCA assay. In the context of a pharmaceutical composition, such values refer to the characteristics of the KTI prior to being mixed with one or more other components of the pharmaceutical composition. In other embodiments, the KTI preparation comprises 5% or less BBI as evaluated by SDS-PAGE. In other embodiments, the KTI preparation comprises less than 0.1% high MW contaminants (i.e., substances having an MW greater than 30,000).

[0154] In other embodiments, the weight / weight (w / w) ratio of BBI to KTI3 in the pharmaceutical composition is from 1.5:1 to 2.5:1, in other embodiments from 1.6:1 to 2.4:1, in other embodiments from 1.7:1 to 2.3:1, in other embodiments from 1.8:1 to 2.2:1, in other embodiments from 1.9:1 to 2.1:1, in other embodiments from 1.95:1 to 2.05:1, and in other embodiments is 2:1.

[0155] In yet more specific embodiments, the described methods and compositions contain the described BBI and KTI, and in even more specific embodiments BBI and KTI3, as the only protease inhibitor. In other embodiments, the described methods and compositions contain KTI and aprotinin, and in specific embodiments KTI3 and aprotinin, as the only protease inhibitor. In other embodiments, isolated BBI, isolated KTI, and aprotinin are all present in the pharmaceutical composition.

[0156] In other embodiments, BBI and KTI3 are present in amounts of 50 - 100 and 25 - 50 mg per dosage form, respectively, and are the only protease inhibitors in the composition. In other embodiments, the amounts are 55 - 90 and 25 - 45 mg, respectively, 60 - 90 and 25 - 45 mg, respectively, 60 - 85 and 25 - 45 mg, respectively, 60 - 80 and 25 - 45 mg, respectively, 65 - 75 and 30 - 40 mg, respectively, 68 - 72 and 33 - 37 mg, respectively, or 70 and 35 mg, respectively. In other embodiments, the aforementioned amounts of BBI and KTI3 are present together with another trypsin inhibitor, such as aprotinin.

[0157] In other embodiments, the described combinations of protease inhibitors are present in combination with the described nonionic detergent and, in some embodiments, with the described nonionic emulsifier. The described combinations of protease inhibitors may each be freely combined with the described embodiments of the emulsifier and / or nonionic detergent.

[0158] (Chelating agents for divalent cations) The chelating agents for divalent cations utilized in the described methods and compositions are, in one embodiment, any physiologically acceptable compound having a high affinity for at least one of the ions of calcium, magnesium, and manganese. In another embodiment, the chelating agent is selected from the group consisting of citrate or a salt thereof; ethylenediaminetetraacetic acid (EDTA) or a salt thereof (e.g., disodium EDTA and 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 above-listed chelating agents is utilized. In a specific embodiment, the chelating agent is EDTA.

[0159] (Oil) Certain embodiments of the pharmaceutical compositions and methods described herein include a liquid phase, and one or more oils are utilized as the basis of the liquid phase. In a specific embodiment, the oil may be any physiologically acceptable oil that is liquid at ambient temperature.

[0160] References to a component being “in oil” or “in liquid” mean that each of the designated components is dissolved, suspended, and / or emulsified in the oil or oil phase. References to an “oil-based” composition mean that all solid components of the composition (i.e., components that are normally provided as solids in the tableting process) are dissolved, suspended, and / or emulsified in the oil or oil phase. In some embodiments, the oil phase is the only liquid component of the pharmaceutical composition. In a specific embodiment, the oil phase is the only component of the pharmaceutical composition, excluding capsules and any other coatings.

[0161] In other embodiments, the oil-based liquid phase of the pharmaceutical composition is free of water, or in other embodiments, free of alcohol, or in other embodiments, free of both alcohol and water. In other embodiments, the oil or oil mixture is the only liquid component of the oil phase, excluding any optional emulsifiers or surfactants. In yet other embodiments, the oil or oil mixture is the only liquid component of the oil phase. In yet other embodiments, the oil phase consists of an oil or oil mixture and solid components dissolved, suspended, and / or emulsified therein. In yet other embodiments, the oil phase consists of an oil or oil mixture and one or more therapeutic proteins, one or more protease inhibitors, and a chelating agent for divalent cations dissolved, suspended, and / or emulsified therein. In yet other embodiments, the oil phase consists of an oil or oil mixture, one or more therapeutic proteins, one or more protease inhibitors, and divalent cations dissolved, suspended, and / or emulsified therein; and one or more optional emulsifiers or surfactants. Trace liquid components such as water absorbed from the atmosphere are not considered to be present for the purpose of classifying a composition having an oil or oil mixture as "free of water" as the "only liquid component" and the like.

[0162] In a specific embodiment, the oil contains omega-3 fatty acids. In other embodiments, the omega-3 fatty acids are omega-3 polyunsaturated fatty acids. In another embodiment, the omega-3 fatty acid is DHA, an omega-3, polyunsaturated 22-carbon fatty acid, also known as 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 (6,9,12,15-octadecatetraenoic 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 eicosapentaenoic acid (5,7,9,11,14,17-eicosapentaenoic 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).

[0163] In other embodiments, the oil is a naturally occurring oil containing omega-3 fatty acids. In other embodiments, the oil is a generally recognized as safe (GRAS) naturally occurring oil. In other embodiments, the oil is a naturally occurring GRAS vegetable oil. In certain embodiments, the oil may be selected from cardamom oil, carrot seed oil, Roman chamomile oil, coriander oil, black cumin oil, Aquilegia oil, mandarin oil, lemon balm oil, frankincense oil, spearmint oil, olive oil, linseed oil, sesame oil, avocado oil, walnut oil, canola oil, cottonseed oil, corn oil, safflower oil, sunflower oil, soybean oil, grape seed oil, almond oil, and fish oil (non-limiting examples of this are salmon oil and tuna oil). In specific embodiments, the oil may be selected from olive oil, linseed oil, sesame oil, avocado oil, walnut oil, canola oil, and fish oil. In even more specific embodiments, the oil is selected from fish oil, canola oil, and linseed oil. Alternatively, the oil is selected from the group consisting of fish oil, canola oil, linseed oil, algal oil, and hemp seed oil. In certain embodiments, the oil is fish oil. Several types of fish oil have been tested in the compositions described herein and have all been found to work equally well.

[0164] (Neurological disorder) It will be understood by those skilled in the art that GLP- may have a therapeutic effect on neurological disorders independent of its effect on NAFLD. Those skilled in the art will recognize that neurological disorders can be diagnosed by many known methods, including MRI (magnetic resonance imaging) of the central nervous system.

[0165] In another embodiment herein, a pharmaceutical composition for treating Alzheimer's disease or, in another aspect, for reducing its incidence rate is provided, said pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings that are resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0166] In yet another aspect, a method for treating Alzheimer's disease or, in another aspect, for reducing its incidence rate is provided, the method comprising the step of administering to a subject a pharmaceutical composition as described herein, thereby treating Alzheimer's disease or reducing its incidence rate.

[0167] In another embodiment herein, a pharmaceutical composition for treating Parkinson's disease or, in another aspect, for reducing its incidence rate is provided, said pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings that are resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0168] Another aspect provides a method of treating Parkinson's disease or, in another aspect, reducing its incidence, the method comprising administering to a subject a pharmaceutical composition described herein, thereby treating Parkinson's disease or reducing its incidence.

[0169] In another embodiment herein, a pharmaceutical composition for treating Huntington's disease or, in another aspect, reducing its incidence is provided, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over an extended period of time.

[0170] Another aspect provides a method of treating Huntington's disease or, in another aspect, reducing its incidence, the method comprising administering to a subject a pharmaceutical composition described herein, thereby treating Huntington's disease or reducing its incidence.

[0171] In another embodiment herein, a pharmaceutical composition for treating ALS or, in another aspect, reducing its incidence is provided, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0172] Yet another aspect provides a method for treating ALS or, in another aspect, reducing its incidence, the method comprising administering to a subject a pharmaceutical composition described herein, thereby treating ALS or reducing its incidence.

[0173] In another embodiment herein, a pharmaceutical composition for treating traumatic brain injury (TBI) or, in another aspect, reducing its incidence is provided, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0174] Another aspect provides a method of treating TBI or, in another aspect, reducing its incidence rate, the method comprising administering to a subject a pharmaceutical composition described herein, thereby treating TBI or reducing its incidence rate.

[0175] In another embodiment herein, a pharmaceutical composition for treating mood disorder or, in another aspect, reducing its incidence rate is provided, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0176] Another aspect provides a method of treating mood disorder or, in another aspect, reducing its incidence rate, the method comprising administering to a subject a pharmaceutical composition described herein, thereby treating mood disorder or reducing its incidence rate.

[0177] In another embodiment herein, a pharmaceutical composition for reducing tissue damage from stroke is provided, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0178] Yet another aspect provides a method for reducing tissue damage from stroke, the method comprising administering to a subject a pharmaceutical composition as described herein, thereby reducing tissue damage from stroke.

[0179] In another embodiment herein, a pharmaceutical composition for treating retinal degeneration or, in another aspect, for reducing its incidence is provided, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over a long period of time.

[0180] Another aspect provides a method of treating retinal degeneration or, in another aspect, reducing its incidence, the method comprising administering to a subject a pharmaceutical composition described herein, thereby treating retinal degeneration or reducing its incidence.

[0181] In another embodiment herein, a pharmaceutical composition for treating peripheral neuropathy (PN) or, in another aspect, reducing its incidence is provided, the pharmaceutical composition comprising insulin, a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In another embodiment, the pharmaceutical composition comprises a GLP-1 analog, at least one protease inhibitor, and a chelating agent for divalent cations. In certain embodiments, the pharmaceutical composition comprises one of the oil-based liquid formulations described herein, where the pharmaceutical composition may further comprise, in some embodiments, capsules and / or coatings resistant to degradation in the stomach. In other embodiments, the pharmaceutical composition comprises one of the solid formulations described herein. In certain embodiments, the pharmaceutical composition is administered to a subject over an extended period of time.

[0182] Another aspect provides a method of treating PN or, in another aspect, reducing its incidence, the method comprising administering to a subject a pharmaceutical composition described herein, thereby treating PN or reducing its incidence.

[0183] (Representative specific formulations) In certain embodiments, the formulations utilized in the described methods and compositions are liquid formulations comprising insulin, a GLP-1 analog, and at least one trypsin inhibitor, all of which are, for example, dissolved, suspended, and / or emulsified in oil. In specific embodiments, the GLP-1 analog may be exenatide. In other embodiments, the described liquid formulations comprise insulin, a GLP-1 analog, and at least one chymotrypsin inhibitor. In yet another embodiment, the described liquid formulations comprise insulin, a GLP-1 analog, at least one trypsin inhibitor, and at least one chymotrypsin inhibitor. In yet another embodiment, the described liquid formulations comprise insulin, a GLP-1 analog, one trypsin inhibitor, and one chymotrypsin inhibitor. In more specific embodiments, the protease inhibitors are SBTI and aprotinin. Alternatively, purified KTI3 and aprotinin are utilized. In other embodiments, purified BBI is the only protease inhibitor present. In other embodiments, a chelating agent for divalent cations is also present in the liquid formulation, in a specific embodiment, EDTA. Alternatively, or in addition, the liquid formulation is in gelatin capsules. In specific embodiments, the gelatin capsules are coated with an enteric coating.

[0184] References to trypsin inhibitors in combination with chymotrypsin inhibitors encompass instances where trypsin inhibitors such as KTI3 are present together with inhibitors of both chymotrypsin and trypsin.

[0185] In certain embodiments, the formulations utilized in the described methods and compositions are liquid formulations that include insulin and at least one trypsin inhibitor, both / all of which are dissolved, suspended, and / or emulsified, for example, in oil. In other embodiments, the described liquid formulations include insulin and at least one chymotrypsin inhibitor. In still other embodiments, the described liquid formulations include insulin, at least one trypsin inhibitor, and at least one chymotrypsin inhibitor. In still other embodiments, the described liquid formulations include insulin, one trypsin inhibitor, and one chymotrypsin inhibitor. In more specific embodiments, the protease inhibitors are SBTI and aprotinin. Alternatively, purified KTI3 and aprotinin are utilized. In still other embodiments, purified BBI is the only protease inhibitor present. In other embodiments, a divalent cation chelating agent is also present in the liquid formulation, in a specific embodiment, EDTA. Alternatively, or in addition, the liquid formulation is in gelatin capsules. In a specific embodiment, the gelatin capsules are coated with an enteric coating.

[0186] In certain embodiments, the formulations utilized in the described methods and compositions are liquid formulations comprising a GLP-1 analog and at least one trypsin inhibitor, both / all of which are dissolved, suspended, and / or emulsified, for example, in oil. In a specific embodiment, the GLP-1 analog may be exenatide. In other embodiments, the described liquid formulation comprises a GLP-1 analog and at least one chymotrypsin inhibitor. In still other embodiments, the described liquid formulation comprises a GLP-1 analog, at least one trypsin inhibitor, and at least one chymotrypsin inhibitor. In still other embodiments, the described liquid formulation comprises a GLP-1 analog, one trypsin inhibitor, and one chymotrypsin inhibitor. In more specific embodiments, the protease inhibitors are SBTI and aprotinin. Alternatively, purified KTI3 and aprotinin are utilized. In still other embodiments, purified BBI is the only protease inhibitor present. In other embodiments, a chelating agent for divalent cations is also present in the liquid formulation, in a specific embodiment EDTA. Alternatively, or in addition, the liquid formulation is contained in a gelatin capsule. In a specific embodiment, the gelatin capsule is coated with an enteric coating.

[0187] In other embodiments, the liquid formulation utilized in the described method or composition includes insulin, exenatide, a surfactant, EDTA, SBTI, aprotinin, and an oil. In some embodiments, the surfactant cited in this paragraph may be Gelucire 44 / 14. In other embodiments, the liquid formulation consists essentially of insulin, exenatide, a surfactant, EDTA, SBTI, aprotinin, and an oil. "Consists essentially of" in this paragraph and the following paragraphs indicates that the liquid formulation does not contain any other components that would significantly affect its physiological characteristics. In other embodiments, the liquid formulation consists of insulin, exenatide, a surfactant, EDTA, SBTI, aprotinin, and an oil. In yet more specific embodiments, the amounts of insulin, exenatide, EDTA, SBTI, aprotinin, and oil per dosage form are 8 - 16 mg, 150 - 300 mcg, 100 - 200 mg, 50 - 100 mg, 20 - 30 mg, and 0.4 - 0.7 ml, respectively, and the amount of the surfactant, e.g., Gelucire 44 / 14, is 8 - 16%. In even more specific embodiments, the amounts of insulin, exenatide, EDTA, SBTI, aprotinin, and oil per dosage form are 8 - 16 mg, 150 - 300 mcg, 150 mg, 65 - 85 mg, 22 - 26 mg, and 0.5 - 0.7 ml, respectively, and the amount of the surfactant, e.g., Gelucire 44 / 1444 / 14, is 8 - 16%. Even more specific embodiments include 75 mg of SBTI and 24 mg of aprotinin. In other embodiments, the above composition further includes polysorbate 80. In some embodiments, polysorbate 80 constitutes 3 - 10% weight / weight, including the oil-based liquid formulation. In certain embodiments, all of the aforementioned components (other than the oil) are provided in the oil, e.g., dissolved or suspended in one of the oils described herein in some embodiments. Alternatively, or in addition, the above composition is covered by a coating that resists degradation in the stomach. Alternatively, or in addition, purified KTI3 is present in one of the above amounts in place of SBTI.In various embodiments, the purified BBI is the sole protease inhibitor and is present in an amount of 50 - 200 mg, in other embodiments 75 - 200 mg, in other embodiments 75 - 180 mg, in other embodiments 75 - 150 mg, in other embodiments 75 - 125 mg, in other embodiments 90 - 110 mg, in other embodiments 100 - 150 mg, in other embodiments 110 - 140 mg, in other embodiments 120 - 130 mg, in other embodiments 120 - 160 mg, in other embodiments 130 - 150 mg, in other embodiments 75 mg, in other embodiments 100 mg, in other embodiments 125 mg, in other embodiments 150 mg, in other embodiments 175 mg, in other embodiments 200 mg.

[0188] In a specific embodiment, the liquid formulation utilized in the described method or composition comprises insulin, exenatide, a self-emulsifying component, for example, (a) monoacylglycerol, diacylglycerol, triacylglycerol, or a mixture thereof, and (b) a PEG ester of a fatty acid; EDTA; SBTI; aprotinin; and an oil, provided as a mixture of components. In other embodiments, the liquid formulation consists essentially of insulin, exenatide, a self-emulsifying component, EDTA, SBTI, aprotinin, and an oil. In other embodiments, the liquid formulation consists of insulin, exenatide, a self-emulsifying component, EDTA, SBTI, aprotinin, and an oil. In yet more specific embodiments, the amount of insulin, exenatide, EDTA, SBTI, aprotinin, or oil per dosage form is respectively 8 - 16 mg, 150 - 300 mcg, 100 - 200 mg, 50 - 100 mg, 20 - 30 mg, and 0.4 - 0.7 ml, and the amount of the self-emulsifying component is 8 - 16%. In even more specific embodiments, the amount of insulin, exenatide, EDTA, SBTI, aprotinin, and oil per dosage form is respectively 8 - 16 mg, 150 - 300 mcg, 150 mg, 65 - 85 mg, 22 - 26 mg, and 0.5 - 0.7 ml, and the amount of the self-emulsifying component is 8 - 16%. Even more specific embodiments include 75 mg of SBTI and 24 mg of aprotinin. In other embodiments, the above composition further comprises polysorbate 80. In certain embodiments, polysorbate 80 constitutes 3 - 10% (inclusive) weight / weight of the oil-based liquid formulation. In certain embodiments, all of the aforementioned components other than the oil are provided in the oil, for example, dissolved or suspended in the oil, which in some embodiments is one of the oils described above herein. Alternatively, or in addition, the above composition is covered by a coating resistant to degradation in the stomach. Alternatively, or in addition, purified KTI3 is present in place of SBTI in one of the amounts mentioned above. In other embodiments, purified BBI is present in one of the amounts mentioned above for SBTI and is the only protease inhibitor present.

[0189] In other embodiments, the liquid formulation utilized in the described method or composition comprises exenatide, a surfactant, EDTA, SBTI, aprotinin, and an oil. In some embodiments, the surfactant referred to in this paragraph may be Gelucire 44 / 14. In other embodiments, the liquid formulation consists essentially of exenatide, a surfactant, EDTA, SBTI, aprotinin, and an oil. In other embodiments, the liquid formulation consists of exenatide, a surfactant, EDTA, SBTI, aprotinin, and an oil. In other specific embodiments, the amounts of exenatide, EDTA, SBTI, aprotinin, and oil per dosage form are 150 - 300 mcg, 100 - 200 mg, 50 - 100 mg, 20 - 30 mg, and 0.4 - 0.7 ml, respectively, and the amount of the surfactant, e.g., Gelucire 44 / 14, is 8 - 16%. In further specific embodiments, the amounts of exenatide, EDTA, SBTI, aprotinin, and oil per dosage form are 150 - 300 mcg, 150 mg, 65 - 85 mg, 22 - 26 mg, and 0.5 - 0.7 ml, respectively, and the amount of the surfactant, e.g., Gelucire 44 / 1444 / 14, is 8 - 16%. Further specific embodiments contain 75 mg of SBTI and 24 mg of aprotinin. In other embodiments, the above composition further comprises polysorbate 80. In some embodiments, polysorbate 80 constitutes 3 - 10% weight / weight, including the oil-based liquid formulation. In certain embodiments, all of the aforementioned components other than the oil are provided in the oil and are dissolved or suspended in an oil, e.g., one of the oils described herein. Alternatively, or in addition, the above composition is covered by a coating resistant to degradation in the stomach. Alternatively, or in addition, purified KTI3 is present in place of SBTI in one of the amounts mentioned above.In various embodiments, the purified BBI is the only protease inhibitor present and is present in an amount of 50 - 200 mg, in other embodiments 75 - 200 mg, in other embodiments 75 - 180 mg, in other embodiments 75 - 150 mg, in other embodiments 75 - 125 mg, in other embodiments 90 - 110 mg, in other embodiments 100 - 150 mg, in other embodiments 110 - 140 mg, in other embodiments 120 - 130 mg, in other embodiments 120 - 160 mg, in other embodiments 130 - 150 mg, in other embodiments 75 mg, in other embodiments 100 mg, in other embodiments 125 mg, in other embodiments 150 mg, in other embodiments 175 mg, in other embodiments 200 mg.

[0190] In further specific embodiments, the liquid formulation utilized in the described method or composition comprises exenatide, a self-emulsifying component, EDTA, SBTI, aprotinin, and an oil. In other embodiments, the liquid formulation consists essentially of exenatide, a self-emulsifying component, EDTA, SBTI, aprotinin, and an oil. In other embodiments, the liquid formulation consists of exenatide, a self-emulsifying component, EDTA, SBTI, aprotinin, and an oil. In other specific embodiments, the amounts of exenatide, EDTA, SBTI, aprotinin, and oil per dosage form are respectively, 150 - 300 mcg, 100 - 200 mg, 50 - 100 mg, 20 - 30 mg, and 0.4 - 0.7 ml, and the amount of the self-emulsifying component is 8 - 16%. In further specific embodiments, the amounts of exenatide, EDTA, SBTI, aprotinin, and oil per dosage form are respectively, 150 - 300 mcg, 150 mg, 65 - 85 mg, 22 - 26 mg, and 0.5 - 0.7 ml, and the amount of the self-emulsifying component is 8 - 16%. Further specific embodiments include 75 mg of SBTI and 24 mg of aprotinin. In other embodiments, the above composition further comprises polysorbate 80. In certain embodiments, polysorbate 80 constitutes 3 - 10% weight / weight including the oil-based liquid formulation. In certain embodiments, all of the aforementioned components other than the oil are provided in the oil and are dissolved or suspended in an oil, for example, one of the oils described herein in some embodiments. Alternatively, or in addition, the above composition is covered by a coating resistant to degradation in the stomach. Alternatively, or in addition, purified KTI3 is present in place of SBTI in one of the amounts mentioned above.In various embodiments, the purified BBI is the only protease inhibitor present and is present in an amount of 50 - 200 mg, in other embodiments 75 - 200 mg, in other embodiments 75 - 180 mg, in other embodiments 75 - 150 mg, in other embodiments 75 - 125 mg, in other embodiments 90 - 110 mg, in other embodiments 100 - 150 mg, in other embodiments 110 - 140 mg, in other embodiments 120 - 130 mg, in other embodiments 120 - 160 mg, in other embodiments 130 - 150 mg, in other embodiments 75 mg, in other embodiments 100 mg, in other embodiments 125 mg, in other embodiments 150 mg, in other embodiments 175 mg, in other embodiments 200 mg.

[0191] In other embodiments, the liquid formulation utilized in the described method or composition comprises insulin, Gelucire 44 / 14, EDTA, SBTI, aprotinin, and oil. In other embodiments, the liquid formulation consists essentially of insulin, Gelucire 44 / 14, EDTA, SBTI, aprotinin, and oil. In other embodiments, the liquid formulation consists of insulin, Gelucire 44 / 14, EDTA, SBTI, aprotinin, and oil. In other specific embodiments, the amounts of insulin, EDTA, SBTI, aprotinin, and oil per dosage form are 8 - 16 mg, 100 - 200 mg, 50 - 100 mg, 20 - 30 mg, and 0.4 - 0.7 ml, respectively, and the amount of Gelucire 44 / 14 is 8 - 16%. In further specific embodiments, the amounts of insulin, EDTA, SBTI, aprotinin, and oil per dosage form are 8 - 16 mg, 150 mg, 65 - 85 mg, 22 - 26 mg, and 0.5 - 0.7 ml, respectively, and the amount of Gelucire 44 / 14 is 8 - 16%. Further specific embodiments include 75 mg of SBTI and 24 mg of aprotinin. In other embodiments, the above composition further comprises polysorbate 80. In some embodiments, polysorbate 80 constitutes 3 - 10% weight / weight including the oil-based liquid formulation. In certain embodiments, all of the foregoing components other than oil are provided in the oil and are dissolved or suspended in an oil, for example, one of the oils described herein in some embodiments. Alternatively, or in addition, the above composition is covered by a coating resistant to degradation in the stomach. Alternatively, or in addition, purified KTI3 is present in place of SBTI in one of the amounts mentioned above.In various embodiments, the purified BBI is the only protease inhibitor present and is present in an amount of 50 - 200 mg, in other embodiments 75 - 200 mg, in other embodiments 75 - 180 mg, in other embodiments 75 - 150 mg, in other embodiments 75 - 125 mg, in other embodiments 90 - 110 mg, in other embodiments 100 - 150 mg, in other embodiments 110 - 140 mg, in other embodiments 120 - 130 mg, in other embodiments 120 - 160 mg, in other embodiments 130 - 150 mg, in other embodiments 75 mg, in other embodiments 100 mg, in other embodiments 125 mg, in other embodiments 150 mg, in other embodiments 175 mg, in other embodiments 200 mg.

[0192] In further specific embodiments, the liquid formulation utilized in the described method or composition comprises insulin, a self-emulsifying component, EDTA, SBTI, aprotinin, and oil. In other embodiments, the liquid formulation consists essentially of insulin, a self-emulsifying component, EDTA, SBTI, aprotinin, and oil. In other embodiments, the liquid formulation consists of insulin, a self-emulsifying component, EDTA, SBTI, aprotinin, and oil. In other specific embodiments, the amounts of insulin, EDTA, SBTI, aprotinin, and oil per dosage form are respectively 8 - 16 mg, 100 - 200 mg, 50 - 100 mg, 20 - 30 mg, and 0.4 - 0.7 ml, and the amount of the self-emulsifying component is 8 - 16%. In further specific embodiments, the amounts of insulin, EDTA, SBTI, aprotinin, and oil per dosage form are respectively 8 - 16 mg, 150 mg, 65 - 85 mg, 22 - 26 mg, and 0.5 - 0.7 ml, and the amount of the self-emulsifying component is 8 - 16%. Even more specific embodiments include 75 mg of SBTI and 24 mg of aprotinin. In some embodiments, the above composition further comprises polysorbate 80. In certain embodiments, polysorbate 80 constitutes 3 - 10% weight / weight of the oil-based liquid formulation. In certain embodiments, all of the aforementioned components other than oil are provided in the oil and are dissolved or suspended in an oil, such as one of the oils described herein in some embodiments. Alternatively, or in addition, the above composition is covered by a coating resistant to degradation in the stomach. Alternatively, or in addition, purified KTI3 is present in place of SBTI in one of the amounts mentioned above.In various embodiments, the purified BBI is the only protease inhibitor present and is present in an amount of 50 - 200 mg, in other embodiments 75 - 200 mg, in other embodiments 75 - 180 mg, in other embodiments 75 - 150 mg, in other embodiments 75 - 125 mg, in other embodiments 90 - 110 mg, in other embodiments 100 - 150 mg, in other embodiments 110 - 140 mg, in other embodiments 120 - 130 mg, in other embodiments 120 - 160 mg, in other embodiments 130 - 150 mg, in other embodiments 75 mg, in other embodiments 100 mg, in other embodiments 125 mg, in other embodiments 150 mg, in other embodiments 175 mg, in other embodiments 200 mg.

[0193] As used herein in the context of emulsifiers and detergents, the "weight / weight" ratio refers to the amount of oil (e.g., fish oil) - based in the formulation as the denominator. Thus, for example, 60 mg of Gelucire in 500 mg of oil is considered to be 12% w / w, regardless of the weight of other components. Similarly, 50 mg of Tween 80 mixed with 500 mg of oil is considered 10% Tween 80.

[0194] In other embodiments, the liquid formulations utilized in the described methods or compositions are water-free. If two or more liquid formulations are present, for example, in a multi-component composition, each liquid formulation is water-free. In certain embodiments, "water-free" refers to formulations in which no aqueous components have been deliberately added. This term does not exclude the presence of trace amounts of water absorbed from the atmosphere into the components. In other embodiments, the liquid formulations are free of aqueous components. If two or more liquid formulations are present, for example, in a multi-component composition, each liquid formulation may be free of aqueous components. In still other embodiments, one or more oils selected from olive oil, linseed oil, sesame oil, avocado oil, walnut oil, canola oil, and fish oil are the sole liquid component of each of one or more liquid formulations. In yet another embodiment, fish oil is the sole liquid component of each of one or more liquid formulations. In yet another embodiment, the sole liquid component of one or more liquid formulations is selected from the group consisting of fish oil, canola oil, and linseed oil.

[0195] In other embodiments, the formulations utilized in the described methods and compositions are solid formulations that include insulin, a GLP-1 analog, EDTA, and at least one trypsin inhibitor. In a specific embodiment, the GLP-1 analog may be exenatide. In other embodiments, the formulation includes insulin, a GLP-1 analog, EDTA, and at least one chymotrypsin inhibitor. In other embodiments, the formulation includes insulin, a GLP-1 analog, EDTA, at least one trypsin inhibitor, and at least one chymotrypsin inhibitor. In other embodiments, the formulation includes insulin, a GLP-1 analog, EDTA, one trypsin inhibitor, and one chymotrypsin inhibitor. In a more specific embodiment, the protease inhibitors are SBTI and aprotinin. Alternatively, purified KTI3 and aprotinin are utilized. In various embodiments, purified BBI is the only protease inhibitor present and is present in an amount of 50 - 200 mg, in other embodiments 75 - 200 mg, in other embodiments 75 - 180 mg, in other embodiments 75 - 150 mg, in other embodiments 75 - 125 mg, in other embodiments 90 - 110 mg, in other embodiments 100 - 150 mg, in other embodiments 110 - 140 mg, in other embodiments 120 - 130 mg, in other embodiments 120 - 160 mg, in other embodiments 130 - 150 mg, in other embodiments 75 mg, in other embodiments 100 mg, in other embodiments 125 mg, in other embodiments 150 mg, in other embodiments 175 mg, in other embodiments 200 mg. Alternatively, or in addition, the solid formulation is coated with an enteric coating.

[0196] The term "solid formulation" as used herein refers to tablets, capsules, and other dosage forms that have a non-liquid structure throughout the dosage form. In certain non-limiting embodiments, the therapeutic peptide, EDTA, and protease inhibitor (all in crystalline form, or in other embodiments amorphous) are combined and subjected to a tableting process known in the art.

[0197] In other embodiments, the formulations utilized in the described methods and compositions are solid formulations comprising a GLP-1 analog, EDTA, and at least one trypsin inhibitor. In a specific embodiment, the GLP-1 analog may be exenatide. In other embodiments, the formulation comprises a GLP-1 analog, EDTA, and at least one chymotrypsin inhibitor. In other embodiments, the formulation comprises a GLP-1 analog, EDTA, at least one trypsin inhibitor, and at least one chymotrypsin inhibitor. In other embodiments, the formulation comprises a GLP-1 analog, EDTA, one trypsin inhibitor, and one chymotrypsin inhibitor. In a more specific embodiment, the protease inhibitors are SBTI and aprotinin. Alternatively, purified KTI3 and aprotinin are utilized. In still other embodiments, purified BBI is the only protease inhibitor present. Alternatively, or in addition, the liquid formulation is in gelatin capsules. In a specific embodiment, the gelatin capsules are coated with an enteric coating.

[0198] In other embodiments, the formulations utilized in the described methods and compositions are solid formulations comprising insulin, EDTA, and at least one trypsin inhibitor. In other embodiments, the formulation comprises insulin, EDTA, and at least one chymotrypsin inhibitor. In other embodiments, the formulation comprises insulin, EDTA, at least one trypsin inhibitor, and at least one chymotrypsin inhibitor. In other embodiments, the formulation comprises insulin, EDTA, one trypsin inhibitor, and one chymotrypsin inhibitor. In a more specific embodiment, the protease inhibitors are SBTI and aprotinin. Alternatively, purified KTI3 and aprotinin are utilized. In still other embodiments, purified BBI is the only protease inhibitor present. Alternatively, or in addition, the liquid formulation is in gelatin capsules. In a specific embodiment, the gelatin capsules are coated with an enteric coating.

[0199] (Diabetes and Other Metabolic Disorders) Similarly, provided herein is a solid pharmaceutical formulation described herein for treating type 1 diabetes mellitus, which in some embodiments is type 1 DM, and in other embodiments is type 2 DM. Similarly, provided is a method for treating diabetes by administering to a subject a solid pharmaceutical formulation described herein. In certain embodiments, the pharmaceutical composition is administered to the subject one or more times per day, depending on blood glucose levels. In other embodiments, the pharmaceutical composition is administered to the subject over an extended period of time.

[0200] In other embodiments, provided herein is a solid pharmaceutical formulation described herein comprising, as an active ingredient, in various embodiments, insulin, a GLP-1 analog, or a combination thereof, for inhibiting or reducing the progression of type 2 diabetes mellitus (T2DM) in a subject at risk of such progression. In certain embodiments, such subjects at risk are considered "prediabetic" and / or exhibit impaired glucose tolerance (IGT). IGT is generally considered to be present when the fasting plasma glucose (FPG) level is between 100 and 126 milligrams per deciliter (mg / dl) and / or the two-hour value in an oral glucose tolerance test (OGTT) is between 140 and 200 mg / dl, and T2DM is considered to be present when the FPG level > 126 or the two-hour OGTT value > 200 mg / dl. However, one of ordinary skill in the art will recognize that the exact criteria for determining IGT and T2DM are not critical and that other tests, such as a frequently sampled intravenous glucose tolerance test (FSIVGTT) and HbAlC levels, may be utilized. In other embodiments, such subjects at risk exhibit insulin resistance, obesity, and / or a family history of diabetes. In certain embodiments, the pharmaceutical composition is administered over an extended period of time. In specific embodiments, the pharmaceutical composition may be administered over an extended period of time, for example, once or twice a day. Non-limiting examples of daily administration are a single administration before bedtime.

[0201] Similarly, the present specification provides a solid pharmaceutical preparation described herein for reducing food intake. Similarly, a method for reducing food intake by a subject is provided by administering to the subject the solid pharmaceutical preparation described herein.

[0202] Similarly, the present specification provides a solid pharmaceutical preparation described herein for enhancing insulin secretion. Similarly, a method for enhancing insulin secretion is provided by administering to a subject the solid pharmaceutical preparation described herein. In certain embodiments, the pharmaceutical composition is administered to the subject one or more times a day, depending on the blood glucose level. In other embodiments, the pharmaceutical composition is administered to the subject over an extended period of time.

[0203] Similarly, the present specification provides a solid pharmaceutical preparation described herein for prophylactically reducing the occurrence of hyperglycemia. Similarly, a method for prophylactically reducing hyperglycemia is provided by administering to a subject the solid pharmaceutical preparation described herein. In certain embodiments, the pharmaceutical composition is administered to the subject one or more times a day, depending on the blood glucose level. In other embodiments, the pharmaceutical composition is administered to the subject over an extended period of time.

[0204] Similarly, the present specification provides a solid pharmaceutical preparation described herein for prophylactically reducing glucagon secretion. Similarly, a method for prophylactically reducing glucagon secretion is provided by administering to a subject the solid pharmaceutical preparation described herein.

[0205] Similarly, in the present specification, for treating true diabetes (type I diabetes in some embodiments and type II diabetes in other embodiments), in various embodiments, pharmaceutical formulations having a specific w / w ratio of BBI:KTI in a pharmaceutical composition containing insulin, a GLP-1 analog, or a combination thereof as an active ingredient are provided. Similarly, a method for treating DM is provided by administering to a subject a pharmaceutical formulation having a specific BBI:KTI w / w ratio. In certain embodiments, the pharmaceutical composition is administered to the subject one or more times a day, depending on blood glucose levels. In other embodiments, the pharmaceutical composition is administered to the subject over an extended period of time. The pharmaceutical composition is, in various embodiments, the liquid pharmaceutical formulation described herein or the solid pharmaceutical formulation described herein. The aforementioned w / w ratio of BBI to KTI is, in some embodiments, from 1.5:1 to 2.5:1, in other embodiments, from 1.6:1 to 2.4:1, in other embodiments, from 1.7:1 to 2.3:1, in other embodiments, from 1.8:1 to 2.2:1, in other embodiments, from 1.9:1 to 2.1:1, in other embodiments, from 1.95:1 to 2.05:1, and in other embodiments, 2:1.

[0206] In other embodiments, pharmaceutical formulations having a specific BBL:KTI w / w ratio are provided, which include, as active ingredients, insulin, GLP-1 analogs, or combinations thereof, in various embodiments, for inhibiting or reducing the progression of type 2 diabetes mellitus (T2DM) in subjects at risk of such progression. In certain embodiments, such at-risk subjects are considered "prediabetic", exhibit obesity, and / or have a family history of diabetes. In certain embodiments, the pharmaceutical composition is administered over a long period of time. In a specific embodiment, the pharmaceutical composition may be administered once a day or twice a day, for example, over a long period of time. A non-limiting example of daily administration is a single administration before bedtime. The aforementioned w / w ratio of BBI to KTI is 1.5:1 to 2.5:1 in some embodiments, 1.6:1 to 2.4:1 in other embodiments, 1.7:1 to 2.3:1 in other embodiments, 1.8:1 to 2.2:1 in other embodiments, 1.9:1 to 2.1:1 in other embodiments, 1.95:1 to 2.05:1 in other embodiments, and 2:1 in other embodiments.

[0207] (Coating) In certain embodiments, the described liquid formulations are placed within capsules, such as gelatin capsules, and in more specific embodiments, within capsules coated with a pH-sensitive coating. In other embodiments, the solid formulations may be directly coated, for example, with a pH-sensitive coating. One of ordinary skill in the art will understand, in view of the present disclosure, that various 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. Generally, such coatings do not dissolve in human gastric juice within 2 hours and dissolve within 30 minutes in duodenal fluid.

[0208] In other embodiments, the coating comprises a biodegradable polysaccharide. In other embodiments, a hydrogel is used. In other embodiments, the coating comprises one of the following excipients: chitosan, Aquacoat ECD coating, azo-crosslinked polymer, cellulose acetate phthalate, cellulose acetate trimellitate (CAT), cellulose acetate butyrate, hydroxypropyl methylcellulose phthalate, or polyvinyl acetate phthalate.

[0209] In other embodiments, a sustained release system such as Pulsincap™ is used.

[0210] In a preferred embodiment, the coated dosage form described herein releases the core (including oil-based formulations) when the pH reaches a region found in the intestine, which is alkaline relative to that in the stomach. In a specific embodiment, the coating comprises a pH-sensitive polymer. In various embodiments, either a single-layer or multi-layer coating may be utilized.

[0211] In one embodiment, the coating is an enteric coating. Methods for enteric coatings are well known in the art (see, for example, Siepmann F et al 2005). In a specific embodiment, an Eudragit™ coating is utilized as the enteric coating. An Eudragit™ coating is an acrylic polymer, and its use is well known in the art.

[0212] In another embodiment, microencapsulation is used as a gastric-resistant coating in the compositions described herein. Methods for microencapsulation are well known in the art and are particularly described in US Patent Application Publication No. 2011 / 0305768, which is incorporated herein by reference.

[0213] In other embodiments, the described pharmaceutical composition is in the form of a capsule. Gelatin capsules are most preferred and may be soft gelatin capsules or, in other embodiments, hard gelatin capsules. Methods for inserting oil-based formulations into gelatin capsules are well known in the art. In a specific embodiment, the gelatin capsule is itself coated with an enteric coating.

[0214] Various oral dosage forms such as tablets, capsules, pills, powders, and granules may be used in the described solid compositions. Suitable excipients include, but are not limited to, solid powder carriers such as those described in the US Pharmacopeia and the Handbook of Pharmaceutical Excipients, such as mannitol, microcrystalline cellulose, calcium hydrogen phosphate, calcium sulfate, and starch; binders such as polyvinylpyrrolidone, starch, and hydroxypropylmethylcellulose; disintegrants such as croscarmellose sodium, sodium starch glycolate, and polyvinylpyrrolidone; and lubricants such as magnesium stearate, sodium stearyl fumarate, talc, and hydrogenated vegetable oils such as Sterotex NF. In some embodiments, lactose-free compositions contain the active ingredient, binder / filler, and lubricant in compatible amounts. Specific embodiments of lactose-free dosage forms contain the active ingredient, microcrystalline cellulose, pregelatinized starch, and magnesium stearate. The mixture is then processed into tablets or granules for capsules in certain embodiments. In certain embodiments, rapidly disintegrating tablets are utilized, which in some embodiments are useful for elderly or physically weak individuals who have difficulty swallowing. In other embodiments, the oral dosage form is enteric coated.

[0215] Whenever alternatives of a single separable feature, such as, for example, an insulin protein or its dosage, a GLP-1 analog or its dosage, a protease inhibitor or its amount, a chelating agent or its amount, an emulsifier or its amount, a nonionic detergent or its amount, or a capsule and / or coating, are described herein as “embodiments,” it will be understood that such alternatives may be freely combined to form separate embodiments of the invention disclosed herein.

[0216] With respect to the jurisdiction that enables this, all patents, patent applications, and publications described herein above and below are incorporated herein by reference.

[0217] The present invention is further illustrated by the following examples and drawings, from which additional embodiments and advantages can be drawn. These examples are for illustrative purposes of the present invention and do not limit the scope of the present invention.

Examples

[0218] (Details of the experiment) In all animal experiments described herein, the liquid dosage form administered to humans in enteric-coated capsules may be administered directly to the digestive system of the animal, for example, by a cannula. Generally, liquid and solid dosage forms may be administered to animals by enteral nutrition.

[0219] Example 1: Test of an Oral GLP-1 Analogue and / or Insulin Preparation for Treating NAFLD Subjects with NAFLD are continuously administered with one or more dosage forms having a pH-sensitive coating and / or capsule, comprising one or more protease inhibitors; EDTA; and a GLP-1 analog, for a period of, for example, 1 - 24 months. In other experiments, the dosage form comprises insulin and a GLP-1 analog. In yet another experiment, subjects at risk of developing NAFLD are treated with the composition. To test the effectiveness of the composition, the NAFLD status of the subjects is observed over the experimental period.

[0220] Example 2: Testing of Oral GLP-1 Analogs and / or Insulin Formulations for Other Metabolic Markers Subjects with metabolic disorders (Grundy et al, 2004) are continuously administered with one or more dosage forms having a pH-sensitive coating and / or capsule, comprising one or more protease inhibitors; EDTA; and a GLP-1 analog, for a period of, for example, 1 - 24 months. In other experiments, the dosage form comprises insulin and a GLP-1 analog. In yet another experiment, subjects at risk of developing metabolic disorders are treated with the composition. The subjects are observed over the experimental period for obesity (e.g., by measuring waist circumference), total cholesterol level, hypertriglyceridemia, serum ApoB level, total cholesterol / HDL ratio, ApoB / ApoA1 ratio, atherosclerosis, asymptomatic inflammation (which can be measured, for example, by measuring the value of C-reactive protein), prothrombotic state (which can be measured, for example, by measuring the value of plasminogen activator inhibitor-1 [PAI-1]), presence of platelet activation, presence of endothelial dysfunction, presence of cardiac embolism, and / or enhancement of insulin-induced vasodilatory response (Sung et al, 2012; Chatrath et al 2012; Nseir et al, 2011).

[0221] Example 3: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Alzheimer's Disease in an Animal Model One or more dosage forms containing one or more protease inhibitors, EDTA, and a GLP-1 analog are continuously administered to experimental animals in the context of an animal model of Alzheimer's disease, for example, for 1 - 24 weeks. In other experiments, the formulation contains insulin and a GLP-1 analog as active agents. In some experiments, the animal model is the streptozotocin (STZ)-induced rat model of AD. Intracerebral injection of STZ results in hyperphosphorylation of tau protein, causing a disease that mimics AD. Some experiments use sham-injected animals with CSF instead of STZ as a control group.

[0222] After fully recovering from the surgery (generally several months after induction), the animals are divided into several groups and treated orally with GLP-1 or an empty carrier (e.g., normal saline) for several days and in some experiments for at least 30 days.

[0223] The dose-dependent and time-dependent effects of oral GLP-1 on memory maintenance are measured during the treatment. After treatment, the animals are sacrificed and the GLP-1 levels, amyloid-beta (Αβ) load, phosphorylation of tau, and inflammatory markers in the hippocampus and cortex are evaluated using brain tissue.

[0224] In some experiments, the cognitive state of the subjects is observed during the experimental period, and the effectiveness of the composition is tested by evaluating the state of neurodegenerative diseases according to Salcedo et al. or according to the literature cited herein. The following are some representative protocols that can be used.

[0225] (Radial arm maze (RAM) task) The working memory of the animals is tested using a RAM device (with an alarm) and the described test method including training. The test is generally performed several months after STZ induction.

[0226] (Hole board (HB) task) Examine animals with learning disabilities using a complex HB device motivated by food. The device consists of an open field containing many holes surrounded by a plexiglass wall. Each hole contains a metal cup with a perforated bottom, and a food pellet agent is placed beneath it. The examination records the number of times the animal comes to the hole with food (hits), the number of times it comes to the hole without food (errors), and the time until the test is completed.

[0227] (Biochemical research) After completion of the behavioral study, euthanize the animals and perform biochemical research and histological examination on the fixed brains. In some experiments, the hippocampus and prefrontal cortex from one hemisphere are dissected and used to perform the following listed biochemical tests.

[0228] (Evaluation of active GLP-1 value) Homogenize the isolated hippocampus and cortex samples in 10 volumes of cold 50 mM phosphate buffered saline (pH 7.8). Divide the homogenate into four equal parts and use them for the evaluation of GLP-1, Αβ42, tau, and inflammatory markers. The values of active GLP-1 and Αβ42 can be examined using commercially available ELISA kits.

[0229] (Measurement of phosphorylated tau (p-tau)) Collect the hippocampus and cortex tissues and subject them to Western blot analysis.

[0230] (Measurement of TNF-α and IL-Ιβ values) The TNF-α and IL-Ιβ mp values in the hippocampus and cortex homogenates can be measured using available ELISA kits. (Histological examination and number of neurons) Separate the hippocampus and cortex, stain them with cresyl violet acetate (CV), and analyze the stained neurons using an image analyzer. Compare the number of CV-positive neurons with the sham control group and obtain the average cell number of the separated parts from each animal.

[0231] Example 4: Examination of an Oral GLP-1 Analogue Preparation for Treating and Preventing Stroke in an Animal Model The preventive and therapeutic effects of an oral GLP-1 analogue are examined in an animal stroke model. One such model is the rat reperfusion injury model, where the middle cerebral artery of the rat is temporarily occluded for 90 minutes. The GLP-1 analogue or carrier is administered to the rats for several days before and after reperfusion. Evaluations are made regarding neurological outcomes, biochemical changes, and / or infarct size. Neurological function may be determined using the modified Bederson test at one or more time points after occlusion, after which the rats may be euthanized for histological studies. In some experiments, peripheral blood is collected for measurement of blood glucose levels and oxidative stress, and / or brain tissue is collected to measure vascular endothelial growth factor (VEGF) levels (Sato et al.).

[0232] Example 5: Examination of an Oral GLP-1 Analogue Preparation for Treating and Preventing Parkinson's Disease Patients with moderate Parkinson's disease (PD) are randomly assigned to ingest either an oral GLP-1 analogue or placebo for 12 months. In some cases, the progression of PD is measured after discontinuing conventional PD medications. For example, the Movement Disorders Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) may be used at multiple time points during treatment, in some cases after a washout period, and in some cases together with one or more non-motor tests (Aviles-Olmos et al.).

[0233] Example 6: Examination of an Oral GLP-1 Analogue Preparation for Treating and Preventing Traumatic Brain Injury (TBI) in an Animal Model Evaluate the ability of an oral GLP-1 analog formulation to prevent TBI and promote recovery from TBI. In some experiments, the GLP-1 analog is administered for several days after injury. One model that may be used is the fluid percussion injury model in vivo (Eakin et al.). Markers of cell death and means of cognitive function may also be utilized. Examples of the latter are the Morris water maze and other cognitive ability tests described herein.

[0234] Example 7: Examination of an oral GLP-1 analog formulation for treating and preventing peripheral nerve injury in an animal model Evaluate the ability of an oral GLP-1 analog formulation to prevent peripheral neuropathy and promote recovery from peripheral neuropathy. One model that may be used is sciatic nerve crush nerve injury. The GLP-1 analog or an empty carrier is administered immediately after the crush injury and continued for several days or weeks. Rats subjected to sciatic nerve crush may exhibit significant loss of function of the tibialis anterior muscle (TA), electrophysiological dysfunction, and atrophy. Recovery can be observed by measuring neuronal function, electrophysiological function, muscle atrophy, and / or morphological parameters several days or weeks after nerve crush (Yamamoto et al.).

[0235] Example 8: Examination of an oral GLP-1 analog formulation for treating and preventing cognitive impairment and mood disorders In other experiments, evaluate the ability of an oral GLP-1 analog formulation to treat bipolar disorder, major depressive disorder, schizophrenia, and / or schizoaffective disorder. Individuals suffering from or at risk of developing such diseases are continuously treated with the GLP-1 analog, for example, over 1 - 24 months, and clinical overall improvement of psychiatric symptoms and / or manic symptoms, depressive symptoms, or schizophrenic symptoms is measured. In other experiments, cognitive function is determined using tests known to those skilled in the art (Mclntyre et al.).

[0236] Example 9: Examination of an oral GLP-1 analog formulation for treating and preventing amyotrophic lateral sclerosis (ALS) in an animal model In other experiments, the ability of an oral GLP-1 analog formulation to treat and prevent ALS is evaluated. The GLP-1 analog is administered continuously, for example, for 1 - 18 weeks. In some experiments, animal models such as SOD1 G93A mutant mice are utilized (Li et al.). Disease progression can be observed by measuring activity levels, such as running behavior, spinal cord structure, and by using neuronal density and specific disease progression markers in brain tissue, glial fibrillary acidic protein (GFAP), caspase-3, choline acetyltransferase (ChAT), and neuronal cell neurofilament protein (SMI-32) (Li et al.).

[0237] Example 10: Examination of an oral GLP-1 analog formulation for treating and preventing Huntington's disease in an animal model In other experiments, the ability of an oral GLP-1 analog formulation to treat and prevent Huntington's disease is evaluated. The GLP-1 analog is administered continuously, for example, for 1 - 18 weeks. In some experiments, animal models such as those described by Brooks and Dunnett and in the literature cited herein are employed.

[0238] Example 11: Examination of an oral GLP-1 analog formulation for treating and preventing diabetic neuropathy in an animal model In other experiments, the ability of an oral GLP-1 analog formulation to treat and prevent diabetic neuropathy is evaluated. The GLP-1 analog is administered continuously for 1 - 18 weeks. In some experiments, animal models such as those described by Lai and Lo and in the literature cited herein are employed.

[0239] Example 12: Examination of an oral GLP-1 analog formulation for treating and preventing Alzheimer's disease, Huntington's disease, stroke, TBI, peripheral nerve injury, AL, and diabetic neuropathy in humans For example, one or more dosage forms comprising one or more protease inhibitors, EDTA, and a GLP-1 analog are continuously administered to a subject at risk of developing Alzheimer's disease, Huntington's disease, stroke, TBI, peripheral nerve injury, ALS, or diabetic neuropathy, for example, over a period of 1 - 48 months. In other experiments, the formulation comprises insulin and a GLP-1 analog. To test the effectiveness of the composition, the disease state of the subject is observed over the course of the experiment, optionally in combination with brain imaging diagnostics. The disease state may be observed, for example, by performing physiological tests and / or determining the cognitive state or nerve function according to Salcedo et al. and the references cited herein, or according to other tests known in the art.

[0240] Example 13: Testing of an oral GLP-1 analog formulation for treating and preventing diabetic neuropathy in an animal model In other experiments, the ability of a solid pharmaceutical formulation comprising insulin, a GLP-1 analog, or a combination thereof to inhibit or prevent the progression of type 2 diabetes (T2DM) in subjects with impaired glucose tolerance (IGT) is evaluated. The pharmaceutical composition is continuously administered, for example, over a period of 1 - 240 weeks, and in some experiments daily at bedtime. The patients are observed for signs of DM known in the art, such as fasting plasma glucose (FPG) values, frequently sampled intravenous glucose tolerance test (FSIVGTT), oral glucose tolerance test (OGTT), and HbAlC values, to monitor the progression of T2DM. Alternatively, or in addition, insulin sensitivity and β-cell function are measured.

[0241] In the claims, the words "comprise", "comprises", "comprising", and the like, do not exclude other components although they indicate the inclusion of the listed components.

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Claims

1. An oral pharmaceutical composition for inhibiting the progression of non-alcoholic fatty liver disease (NAFLD) or treating NAFLD, which comprises insulin, a GLP-1 analog, or a combination thereof, a protease inhibitor, and a chelating agent for divalent cations.

2. The oral pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered for 1 month or longer.

3. The oral pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered once or twice a day.

4. The oral pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises a liquid preparation, and the insulin, the GLP-1 analog, or a combination thereof, the protease inhibitor, and the chelating agent for divalent cations are in the liquid preparation.

5. The oral pharmaceutical composition according to claim 4, wherein the liquid preparation is inside a capsule.

6. The oral pharmaceutical composition according to claim 5, wherein the capsule is surrounded by a coating resistant to degradation in the stomach.

7. The oral pharmaceutical composition according to claim 4, wherein the liquid preparation is an oil-based liquid preparation.

8. The oral pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a solid preparation.

9. The oral pharmaceutical composition according to any one of claims 1 to 8, wherein the GLP-1 analog is present in an amount of 100 - 600 micrograms (inclusive) per dose in adult patients or in a corresponding amount per body weight in pediatric patients.

10. The oral pharmaceutical composition according to claim 9, wherein the GLP-1 analog is present in an amount of 100 - 300 micrograms (inclusive) per dose in adult patients or in a corresponding amount per body weight in pediatric patients.

11. The oral pharmaceutical composition according to claim 9 or 10, wherein the GLP-1 analog is exenatide.

12. The oral pharmaceutical composition according to any one of claims 1 to 8, wherein the insulin is present in an amount of 8 - 32 mg (inclusive) per dose in adult patients or in a corresponding amount per body weight in pediatric patients.

13. The oral pharmaceutical composition according to any one of claims 1 to 8, wherein the insulin is present in an amount of 4 - 12 mg (inclusive) per dose in adult patients or in a corresponding amount per body weight in pediatric patients.

14. The oral pharmaceutical composition according to claim 13, wherein, in addition to the insulin, the GLP-1 analog is present in an amount of 100 - 300 micrograms (inclusive) per dose in adult patients, or in a corresponding amount per body weight in pediatric patients.

15. The oral pharmaceutical composition according to claim 14, wherein the GLP-1 analog is exenatide.

16. The oral pharmaceutical composition according to any one of claims 1 to 15, wherein the protease inhibitor is selected from the group consisting of soybean trypsin inhibitor (SBTI), Bowman-Birk inhibitor (BBI), Kunitz soybean trypsin inhibitor 3 (KTI3), and aprotinin, and these may be present individually or in combination with another protease inhibitor.

17. The oral pharmaceutical composition according to any one of claims 1 to 15, wherein one or more protease inhibitors are present, and the protease inhibitor inhibits trypsin and chymotrypsin collectively.

18. The oral pharmaceutical composition according to any one of claims 1 to 15, wherein two protease inhibitors are present in the pharmaceutical composition, and the two protease inhibitors are SBTI and aprotinin.

19. The oral pharmaceutical composition according to any one of claims 1 to 15, wherein two protease inhibitors are present in the pharmaceutical composition, and the two protease inhibitors are isolated KTI3 and isolated BBI.

20. The oral pharmaceutical composition according to any one of claims 1 to 15, wherein two protease inhibitors are present in the pharmaceutical composition, and the two protease inhibitors are (i) isolated KTI3 and (ii) aprotinin.

21. The oral pharmaceutical composition according to any one of claims 1 to 15, wherein one protease inhibitor is present in the pharmaceutical composition, and the one protease inhibitor is isolated BBI.

22. The oral pharmaceutical composition according to any one of claims 1 to 21, wherein the chelating agent is EDTA.

23. The oral pharmaceutical composition according to any one of claims 7 - 22, wherein the oil is selected from olive oil, linseed oil, sesame oil, avocado oil, walnut oil, canola oil, and fish oil.

24. The oral pharmaceutical composition according to claim 23, wherein the oil is fish oil.

25. The oral pharmaceutical composition according to any one of claims 7 - 24, wherein the oil-based liquid formulation does not contain water.

26. The oral pharmaceutical composition according to any one of claims 1-10, 12-14, and 16-25, wherein the GLP-1 analog is exenatide.

27. A method for inhibiting the progression of NAFLD or treating NAFLD in a human subject, comprising the step of administering to the subject an oral pharmaceutical composition according to any one of claims 1-26, thereby inhibiting the progression of NAFLD or treating NAFLD.

28. The method according to claim 27, wherein the pharmaceutical composition is administered for 1 month or longer.

29. A method for inhibiting the development of NAFLD or treating NAFLD in a non-human subject, comprising the step of administering to the non-human subject an oral pharmaceutical composition according to any one of claims 1-26, thereby inhibiting the development of NAFLD or treating NAFLD.

30. The method according to claim 29, wherein the pharmaceutical composition is administered for 1 month or longer.

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