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

Abstract

Provided herein are oral pharmaceutical compositions containing a GLP-1 analogue and/or insulin for treating and reducing the incidence of nonalcoholic fatty liver disease (NAFLD), hepatic steatosis, and sequelae thereof, and methods of utilizing same.

Claims

1. A method for inhibiting the development of or treating nonalcoholic fatty liver disease (NAFLD) in a human subject in need thereof, said method comprising the step of orally administering to the subject a water-free pharmaceutical composition comprising a therapeutic protein that inhibits the development of or treats the subject's NAFLD, a protease inhibitor, and a chelator of divalent cations, thereby inhibiting the development of or treating the subject's NAFLD, wherein the therapeutic protein consists of insulin.

2. The method of claim 1, where said pharmaceutical composition is administered for more than one month.

3. The method of claim 1, wherein said pharmaceutical composition is administered once daily or twice daily.

4. The method of claim 1, wherein said pharmaceutical composition comprises a liquid formulation, wherein said insulin, said protease inhibitor, and said chelator of divalent cations are in said liquid formulation.

5. The method of claim 4, wherein said liquid formulation is inside a capsule.

6. The method of claim 5, wherein said capsule is surrounded by a coating that resists degradation in the stomach.

7. The method of claim 4, wherein said liquid formulation is an oil-based liquid formulation.

8. The method of claim 1, wherein said pharmaceutical composition is a solid formulation.

9. The method of claim 1, wherein said insulin is present in said pharmaceutical composition in an amount between 8-32 mg inclusive, per dose for an adult patient or a corresponding amount per body weight for a pediatric patient.

10. The method of claim 1, wherein administering said composition inhibits the development of nonalcoholic fatty liver disease (NAFLD).

11. The method of claim 10, where said pharmaceutical composition is administered for more than one month.

12. The method of claim 10, wherein said pharmaceutical composition is administered once daily or twice daily.

13. The method of claim 10, wherein said insulin is present in said pharmaceutical composition in an amount between 6-14 mg inclusive, per dose for an adult patient or a corresponding amount per body weight for a pediatric patient.

14. The method of claim 1, wherein administering said composition treats the nonalcoholic fatty liver disease (NAFLD).

15. The method of claim 14, where said pharmaceutical composition is administered for more than one month.

16. The method of claim 14, wherein said pharmaceutical composition is administered once daily or twice daily.

17. The method of claim 14, wherein said insulin is present in said pharmaceutical composition in an amount between 6-14 mg inclusive, per dose for an adult patient or a corresponding amount per body weight for a pediatric patient.

18. A method for inhibiting the development of or treating nonalcoholic fatty liver disease (NAFLD) in a non-human subject in need thereof, said method comprising the step of orally administering to the non-human subject a water-free pharmaceutical composition comprising a therapeutic protein that inhibits the development of or treats the subject's NAFLD, a protease inhibitor, and a chelator of divalent cations, thereby inhibiting the development of or treating the subject's NAFLD, wherein the therapeutic protein consists of insulin.

19. The method of claim 18, where said pharmaceutical composition is administered for more than one month.

Description

EXPERIMENTAL DETAILS SECTION

(1) In all the animal experiments described herein, liquid dosage forms that are administered to a human in an enteric-coated capsule may, for example, be administered directly to the digestive system of the animal via a cannula. In general, liquid and solid dosage forms may be fed to animals via gavage.

Example 1: Testing of Oral GLP-1 Analogue and/or Insulin Formulations for Treating NAFLD

(2) Volunteers with NAFLD are administered one or more dosage forms having a pH-sensitive coating and/or capsule containing one or more protease inhibitors; EDTA; and a GLP-1 analogue, on an ongoing basis, for example for a time period of between 1-24 months. In other experiments, the dosage form contains insulin and a GLP-1 analogue. In still other experiments, subjects at risk of developing NAFLD are treated with the composition. The NAFLD status of the subjects is followed over the experimental period, to test the effectiveness of the compositions.

Example 2: Testing of Oral GLP-1 Analogue and/or Insulin Formulations for Other Metabolic Indications

(3) Volunteers with metabolic disorder (Grundy et al, 2004) are administered one or more dosage forms having a pH-sensitive coating and/or capsule and containing one or more protease inhibitors; EDTA; and a GLP-1 analogue, on an ongoing basis, for example for a time period of 1-24 months. In other experiments, the dosage form contains insulin and a GLP-1 analogue. In still other experiments, subjects at risk of developing metabolic disorder are treated with the composition. Subjects are followed over the experimental period for obesity (for example by measuring waist circumference), total cholesterol levels, hypertriglyceridemia, serum ApoB levels, total cholesterol/HDL ratios, ApoB/ApoA1 ratios, atherosclerosis, sub-clinical inflammation (as can be measured inter alia by measuring levels of C-reactive protein), the presence of a prothrombotic state (as can be measured inter alia by measuring levels of plasminogen activator inhibitor-1 [PAI-1]), the presence of platelet activation, the presence of endothelial dysfunction, the presence of a cardioembolic state, and/or insulin-induced enhancement of vasodilator responses (Sung et al, 2012; Chatrath et al 2012; Nseir et al, 2011).

Example 3: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Alzheimer's Disease in an Animal Model

(4) One or more dosage forms containing one or more protease inhibitors, EDTA, and a GLP-1 analogue is administered on an ongoing basis, for example for a time period of between 1-24 weeks, to experimental animals in the context of an animal model of Alzheimer's disease. In other experiments, the formulation contains insulin and a GLP-1 analogue as the active agents. In some experiments, the animal model is a streptozotocin (STZ)-induced rat model of AD. Intracerebral injection of STZ leads to hyper-phosphorylation of tau protein and causes a condition that mimics AD. Some experiments utilize sham-injected animals, whereby CSF is used instead of STZ, as a control group.

(5) After complete recovery from the procedure (typically several months after induction), animals are divided into groups and for treatment with oral GLP-1 or empty carrier (e.g. normal saline) for a period of several days, in some experiments for at least 30 days.

(6) Dose-dependent and time-course effects of oral GLP-1 on memory retention are measured during the course of treatment. Following treatment, the animals are sacrificed, and brain tissues are used to evaluate hippocampal and cortical GLP-1 levels, amyloid beta (Aβ) burden, tau phosphorylation, and inflammatory markers.

(7) In some experiments, the cognitive status of the subjects is followed over the experimental period, or neurodegenerative disease status is assessed according to Salcedo et al or a reference cited therein, to test the effectiveness of the compositions. Below are some representative protocols that can be used:

(8) Radial Arm Maze (RAM) Task

(9) Working memory in animals is tested with the RAM apparatus (Alamed) and using described test methods including training. The test is typically performed several months after STZ induction.

(10) Hole-Board (HB) Task

(11) Animals are tested for learning deficits in a food-motivated complex HB apparatus. The apparatus consists of an open field containing an array of holes surrounded by Plexiglass walls. Each hole contains a metal cup with a perforated bottom under which food pellets are placed. The test records the number of visits to food-baited holes (hits), the number of visits to unbaited holes (errors) and the time to complete the trial.

(12) Biochemical Studies

(13) Animals are euthanized at the completion of behavioral studies, and biochemical studies and histology are conducted on fixed brains. In some experiments, the hippocampus and frontal cortex from one hemisphere are dissected and used for biochemical studies, such as those listed below.

(14) Estimation of Active GLP-1 Levels

(15) Isolated hippocampal and cortex samples are homogenized with a 10-fold volume of chilled 50 mM phosphate-buffered saline (pH 7.8). The homogenate is divided into four equal portions and utilized for the estimation of GLP-1, Aβ42, tau and inflammatory markers. Active GLP-1 and Aβ42 levels can be tested using commercially available ELISA kits.

(16) Measurement of Phosphorylated Tau (p-tau)

(17) Hippocampal and cortical tissues are harvested and subjected to Western Blot analysis.

(18) Measurement of TNF-α and IL-1β Levels

(19) TNF-α and IL-4β levels in the hippocampal and cortex homogenate can be measured using available ELISA kits.

(20) Histology and Neuronal Count

(21) The hippocampus and cortex is sectioned and stained with cresyl violet (CV) acetate, and stained neurons are analyzed using an image analyzer. CV positive neurons numbers are compared with the sham control group, and average cell counts of the sections are obtained from each animal.

Example 4: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Stroke in an Animal Model

(22) The preventive and therapeutic effects of oral GLP-1 analogue are tested in an animal stroke model. One such model is a reperfusion injury model in rats, where rats undergo temporary occlusion of the middle cerebral artery for 90 min. Rats are administered GLP-1 analogue or carrier for several days prior and/or after reperfusion. Assessment of the neurological consequences, biochemical changes, and/or size of infarct is performed. Neurological function may be determined using a modified Bederson's test at one or several time points after occlusion, after which rats may be euthanized for histological investigation. In some experiments, peripheral blood is obtained for measurement of blood glucose level and evaluation of oxidative stress and/or brain tissues are collected to measure vascular endothelial growth factor (VEGF) levels (Sato et al).

Example 5: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Parkinson's Disease

(23) Patients with moderate Parkinson's disease (PD) are randomly assigned to receive either oral GLP-1 analogue or placebo for 12 months. PD progression is measured, in some cases after withdrawal of conventional PD medication. For example, the Movement Disorders Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) may be used, in some cases together with one or more non-motor tests, at several time points during treatment and in some cases after a further washout period (Aviles-Olmos et al).

Example 6: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Traumatic Brain Injury (TBI) in an Animal Model

(24) The ability of oral GLP-1 analogue formulations to protect against or facilitate recovery from TBI is evaluated. In some experiments, the GLP-1 analogue is administered for several days after injury. One model that may be used is an in vivo fluid percussion injury model (Eakin et al). Markers of cell death and measures of cognitive function may be utilized. Examples of the latter are the Morris Water Maze and other cognitive tests described herein.

Example 7: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Peripheral Nerve Injury in an Animal Model

(25) The ability of oral GLP-1 analogue formulations to protect against or facilitate recovery from peripheral nerve disease is evaluated. One model that may be used is sciatic nerve crush nerve injury. GLP-1 analogue or empty carrier is administered shortly after crush injury and continued for several subsequent days or weeks. Rats subjected to sciatic nerve crush may exhibit marked functional loss, electrophysiological dysfunction, and atrophy of the tibialis anterior muscle (TA). Recovery can be monitored by measuring neurological function, electrophysiological function, muscle atrophy, and/or morphological parameters several days or weeks after nerve crush (Yamamoto et al).

Example 8: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Cognition and Mood Disorders

(26) In other experiments, the ability of oral GLP-1 analogue formulations to treat bipolar disorder, major depressive disorder, schizophrenia, and/or schizoaffective disorder is evaluated. Individuals with these conditions or at risk of developing them are treated with GLP-1 analogue on an ongoing basis, for example for a time period of between 1-24 months, and clinical global improvement in psychiatric symptoms and/or manic, depressive, or schizophrenic symptoms is measured. In other experiments, cognitive function is determined, using tests known to those skilled in the art (McIntyre et al).

Example 9: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Amyotrophic Lateral Sclerosis (ALS) in an Animal Model

(27) In other experiments, the ability of oral GLP-1 analogue formulations to treat or prevent ALS is evaluated. GLP-1 analogue is administered on an ongoing basis, for example for between 1-18 weeks. In some experiments, an animal model such as the SOD1 G93A mutant mouse (Li et al) is utilized. Disease progression can be monitored by measuring activity level, e.g. running behavior, lumbar spinal cord structure, and, using brain tissue, neuron density and specific disease progression markers such as glial fribrillary acidic protein (GFAP), Caspase-3, Choline acetyl transferase (ChAT), and the neuronal cell neurofilament protein, SMI-32 (Li et al).

Example 10: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Huntington'S Disease in an Animal Model

(28) In other experiments, the ability of oral GLP-1 analogue formulations to treat or prevent Huntington's disease is evaluated. GLP-1 analogue is administered on an ongoing basis, for example for between 1-18 weeks. In some experiments, an animal model such as those described in Brooks and Dunnett and the references cited therein is utilized.

Example 11: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Diabetic Neuropathy in an Animal Model

(29) In other experiments, the ability of oral GLP-1 analogue formulations to treat or prevent diabetic neuropathy is evaluated. GLP-1 analogue is administered on an ongoing basis, for example for between 1-18 weeks. In some experiments, an animal model such as those described in Lai and Lo and the references cited therein is utilized.

Example 12: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Alzheimer'S Disease, Huntington'S Disease, Stroke, TBI, Peripheral Nerve Injury, AL, and Diabetic Neuropathy in Humans

(30) One or more dosage forms containing one or more protease inhibitors, EDTA, and a GLP-1 analogue is administered on an ongoing basis, for example for between 1-48 months, to subjects at risk of developing Alzheimer's disease, Huntington's disease stroke, TBI, peripheral nerve injury, ALS, or diabetic neuropathy. In other experiments, the formulation contains insulin and a GLP-1 analogue. To test the effectiveness of the compositions, the disease status of the subjects is followed over the experimental period, in some cases in combination with brain imaging. Disease status may be followed, for example, by performing physiological tests and/or determining cognitive status or neurological function, according to Salcedo et al or a reference cited therein, or other tests known to those skilled in the art.

Example 13: Testing of Oral GLP-1 Analogue Formulations for Treating and Preventing Diabetic Neuropathy in an Animal Model

(31) In other experiments, the ability of solid pharmaceutical formulations, containing insulin, a GLP-1 analogue, or a combination thereof, to inhibit or prevent the development of Type II Diabetes Mellitus (T2DM) in a subject with impaired glucose tolerance (IGT) is evaluated.

(32) The pharmaceutical composition is administered on an ongoing basis, for example for between 1-240 weeks, in some experiments daily at bedtime. Patients are followed by DM indicia known in the art, such as fasting plasma glucose (FPG) levels, frequently sampled intravenous glucose tolerance test (FSIVGTT), oral glucose tolerance test (OGTT), and HbA1C levels, to monitor the development of T2DM. Alternatively or in addition, insulin sensitivity and beta-cell function are measured.

(33) In the claims, the word “comprise”, and variations thereof such as “comprises”, “comprising”, and the like indicate that the components listed are included, but not generally to the exclusion of other components.

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