HK40078570B - Methods and compositions for the prevention of type 1 diabetes - Google Patents

Description

BACKGROUND

Type 1 diabetes (T1D; also known as "autoimmune diabetes," and formerly known as "insulin-dependent diabetes," or "juvenile-onset diabetes") is a chronic disease that results from an autoimmune-mediated destruction of pancreatic β-cells with consequent loss of insulin production, which manifests clinically as hyperglycemia, and accounts for 5-10% of all cases of diabetes. The age of symptomatic onset is usually during childhood or adolescence; however, symptoms can develop much later in life. Although the etiology of T1D is not completely understood, the pathogenesis is thought to involve T cell-mediated destruction of pancreatic β-cells. There is no known cure for T1D, and patients must rely on daily insulin therapy to compensate for impaired β-cell function. Insulin treatments typically involve either multiple daily insulin injection therapy or continuous subcutaneous insulin infusion. Without insulin, these patients develop serious complications such as ketoacidosis, retinopathy, nephropathy, vasculopathy, and neuropathy. Because subcutaneous delivery of insulin requires strict, self-regimentation, compliance is often a serious problem. Moreover, the act of parenteral insulin administration can be traumatic for juveniles. Presently, there are no known effective oral or sublingual insulin therapies. Compliance concerns coupled with serious morbidity and an increasing incidence of T1D worldwide, underscore the need to develop effective therapies for T1D prevention and/or treatment. Al-Waili, JPMA, 49(7), 199, p 167-169, describes a study to investigate the hypoglycemic effects of repeated sublingual doses of human insulin for the treatment of hypoglycemia in type I diabetes. Patil and Devarajan, Drug Deliv, 2016, 23(2), p 429-436, describes a study of insulin-loaded alginic acid nanoparticles for sublingual delivery. DE 200 21 079 U1 is concerned with compositions comprising a polypeptide and glycerin. Daniel et al., J. Exp. Med. Vol. 208(7), 2011, p1501-1510, describes a study said to be for prevention of type 1 diabetes in mice by tolerogenic vaccination with a strong agonist insulin mimetope. However, Bergman et al., J. Exp. Med. Vol. 214(7), 2017, p 2153-2156, describes a study said to show that tolerogenic insulin peptide therapy precipitates type 1 diabetes. Saporta, BioMed Research International, 2016, Article ID 9323804, is a review article on sublingual immunotherapy.

SUMMARY

The present invention provides a composition comprising human insulin and glycerin, for use in a method of delaying the onset of Type 1 diabetes or decreasing the likelihood of Type 1 diabetes in a mammalian subject in need thereof, the method comprising sublingually administering the composition to the subject. The present invention also provides the composition comprising human insulin and glycerin, for attenuating an antigenic response in a mammalian subject to at least one Type 1 diabetes-related antigen. Further exemplary embodiments of the present invention are set out in the appended set of claims.

The present technology relates to a sublingual formulation that is capable of significantly reducing the incidence and delaying the onset of T1D in an art-accepted mouse model of the disease (the non-obese diabetic (NOD) mouse). Effective sublingual insulin treatment for T1D is a highly unmet need. The disclosure of the present technology therefore provides a composition formulated for a desirable route of administration that is efficacious in methods for delaying the onset of T1D, and/or decreasing the likelihood of T1D in a subject, and which may improve patient compliance. Any references herein to methods of treatment of the human or animal body by therapy utilizing compounds and/or compositions are to be interpreted as references to the compounds and/or compositions for use in such methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A and 1B are charts showing the incidence and time to onset of Type 1 diabetes in control NOD mice and in NOD mice treated with Humulin^® beginning at 6 weeks of age ( FIG. 1A ) and at 10 weeks of age ( FIG. 1B ).

DETAILED DESCRIPTION I. Definitions

It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs.

As used in this specification and the appended claims, the singular forms "a", "an" and "the" include both singular and plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

As used herein, the "administration" of an agent, drug, or peptide to a subject refers to sublingual administration of the compositions of the present technology to the subject.

As used herein, the term "effective amount" refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in partial or full amelioration of one or more symptoms of Type 1 diabetes. In the context of therapeutic or prophylactic applications, in some embodiments, the amount of a composition administered to the subject will depend on the type, degree, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight, and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. For example, in the methods described herein, compositions of the present technology may be administered to a subject having one or more signs, symptoms, or risk factors of Type 1 diabetes, including, but not limited to, hyperglycemia, hypoinsulinemia, reduced serum C-peptide levels, elevated A1C levels, presence of T1D-associated autoantibodies, excessive excretion of urine (polyuria), thirst (polydipsia), constant hunger (polyphagia), weight loss, vision changes, fatigue, mental confusion, nausea, vomiting, ketoacidosis, retinopathy, nephropathy, vasculopathy, and neuropathy. The compositions may also be administered to a disease-free subjects genetically predisposed to the development of T1D (e.g., first-degree relatives of patients with Type 1 diabetes, where the relatives have been determined to be genetically predisposed to the development of Type 1 diabetes). For example, a "therapeutically effective amount" of the compositions includes levels at which the presence, frequency, or severity of one or more signs, symptoms, or risk factors of Type 1 diabetes are, at a minimum, ameliorated. A therapeutically effective amount may reduce or ameliorate the physiological effects of Type 1 diabetes, and/or the risk factors of Type 1 diabetes, and/or the likelihood of developing Type 1 diabetes. A therapeutically effective amount can be given in one or more administrations.

As used herein, the term "Type 1 diabetes" or "T1D," refers to a disorder characterized by insulin deficiency due to pancreatic β-cell loss that leads to hyperglycemia. T1D can be diagnosed using a variety of diagnostic tests as described below. These include, but are not limited to, (1) glycated hemoglobin A1C (HbA1C) test (HbA1C level ≥ 6.5%), (2) oral glucose tolerance test (OGTT; post-load plasma glucose level ≥ 200 mg/dL), (3) random blood glucose test (glucose level ≥ 200 mg/dL at any time of day combined with symptoms of diabetes), (4) fasting plasma glucose (FPG) test (fasting blood sugar ≥126 mg/dL), (5) C-peptide level of less than 0.2 nmol/L.

"Treating" or "treatment" as used herein covers the treatment of Type 1 diabetes and/or its signs or symptoms in a subject, such as a human, and includes: (i) inhibiting Type 1 diabetes, i.e., arresting its development; (ii) relieving Type 1 diabetes, i.e., causing regression of the disorder; (iii) slowing the progression of Type 1 diabetes; and/or (iv) inhibiting, relieving, or slowing progression of one or more signs or symptoms of Type 1 diabetes, including, but not limited to, hyperglycemia, hypoinsulinemia, reduced serum C-peptide levels, elevated A1C levels, presence of T1D-associated autoantibodies, polyuria, polydipsia, polyphagia, weight loss, vision changes, fatigue, mental confusion, nausea, vomiting, and ketoacidosis.

As used herein, "preventing" or "prevention" of a disorder or condition refers to a compound that reduces the occurrence or likelihood of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more signs or symptoms of the disorder or condition relative to the untreated control sample, including, but not limited to, hyperglycemia, hypoinsulinemia, reduced serum C-peptide levels, elevated A1C levels, presence of T1D-associated autoantibodies, polyuria, polydipsia, polyphagia, weight loss, vision changes, fatigue, mental confusion, nausea, vomiting, and ketoacidosis. As used herein, preventing Type 1 diabetes refers to preventing or delaying the onset of Type 1 diabetes. As used herein, prevention of Type 1 diabetes also includes preventing a recurrence of one or more signs or symptoms of Type 1 diabetes.

It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described herein are intended to mean "substantial," which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

As used herein, the terms "subject" and "patient" are used interchangeably.

II. General

The present technology relates to the surprising discovery of a sublingual formulation that is capable of significantly reducing the incidence and delaying the onset of T1D in an art-accepted mouse model of the disease (the non-obese diabetic (NOD) mouse). Effective sublingual insulin treatment for T1D is a highly unmet need. The present technology therefore provide a desirable route of administration that is efficacious as a T1D therapeutic and may improve patient compliance.

III. Insulin

Insulin hormone is a 51-amino acid protein that is secreted by pancreatic β-cells in the Islets of Langerhans. Insulin is first synthesized as preproinsulin in the rough endoplasmic reticulum of the pancreatic β-cells. After the signal peptide in the preprohormone is removed by proteolytic cleavage, a proinsulin molecule composed of an alpha chain (or A-chain) peptide with 21 amino acids, a beta chain (or B-chain) peptide with 30 amino acids, and an intervening C chain peptide (C-peptide) is produced. Subsequent processing of proinsulin in the Golgi complex produces biologically active insulin by removing the C-peptide and linking the alpha and beta chains through two disulfide bonds at cysteine residues. A third disulfide bond connects two cysteine residues within the alpha chain. Insulin and C-peptide are secreted simultaneously in equimolar amounts in response to various stimuli, such as glucose.

In some embodiments, the human insulin is a recombinant human insulin, such as Humulin^®.

The peptides may be synthesized by any of the methods well known in the art. Suitable methods for chemically synthesizing the protein include, for example, those described by Stuart and Young in Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Company (1984), and in Methods Enzymol., 289, Academic Press, Inc., New York (1997).

IV. Type 1 Diabetes

Type 1 diabetes (T1D), also known as "autoimmune diabetes," (previously known as "insulin-dependent diabetes," or "juvenile-onset diabetes") is a chronic disease characterized by insulin deficiency due to pancreatic β-cell loss that leads to hyperglycemia. The age of symptomatic onset is usually during childhood or adolescence; however, symptoms can sometimes develop much later. Although the etiology of T1D is not completely understood, the pathogenesis of the disease is thought to involve T cell-mediated destruction of β-cells. A cure is not available, and patients depend on lifelong insulin injections. Although intensive glycemic control has reduced the incidence of microvascular and macrovascular complications, the majority of patients with T1D are still developing these complications.

A. Clinical Manifestations

The clinical signs and symptoms of T1D include hyperglycemia, hypoinsulinemia, reduced serum C-peptide levels, elevated A1C levels, presence of T1D-associated autoantibodies, excessive excretion of urine (polyuria), thirst (polydipsia), constant hunger (polyphagia), weight loss, vision changes, fatigue, mental confusion, nausea, vomiting, and ketoacidosis. Chronic symptoms of T1D include retinopathy, nephropathy, vasculopathy, and neuropathy.

B. Diagnosis

T1D in humans is diagnosed by a combination of symptoms and the results of certain blood tests. In a fasting plasma glucose (FPG) test, diabetes is diagnosed if a fasting blood sugar level is 126 mg/dL or higher. In an oral glucose tolerance test (OGTT), diabetes is diagnosed if the 2-hour post-load plasma glucose level is 200 mg/dL or higher. In a random blood glucose test, a blood glucose level of 200 mg/dL or greater at any time of day combined with symptoms of diabetes is sufficient to make the diagnosis. In a hemoglobin A1C (HbA1C; glycohemoglobin) test, which measures the average glucose level over the prior two to three months, diabetes is diagnosed if the HbA1C level is 6.5% or higher. If elevated values are detected in asymptomatic people, repeat testing, preferably with the same test, is recommended as soon as practicable on a subsequent day to confirm the diagnosis. Endogenous insulin production can be assessed by measuring serum C-peptide either in the fasting state or after a stimulus, most commonly intravenously administered glucagon. C-peptide can also be measured in urine. The normal range for fasting serum C-peptide levels in humans is 0.26 to 1.27 nmol/L. A C-peptide level of less than 0.2 nmol/L is associated with a diagnosis of T1D in humans.

Progression to T1D is typically preceded by a prodrome of anti-islet autoantibody expression. Biomarkers of T1D-associated autoimmunity that may be found months to years before symptom onset include a number of T1D-associated autoantibodies such as insulin autoantibodies (IAA), islet cell antibodies (ICA), 65 kDa glutamic acid decarboxylase (GAD-65), insulinoma-associated protein 2A or 2β (IA-2A, IA-2β), and zinc transporter 8 (ZnT8), which are proteins associated with secretory granules in β-cells. In predisposed, but disease-free individuals, detection of multiple islet cell autoantibodies is a strong predictor for subsequent development of T1D.

C. Prognostic Indicators

Methods for assessing the signs, symptoms, or complications of T1D are known in the art. Once the diagnosis of diabetes is made, an important goal of therapy is to maintain the average glucose as near the normal range as possible without causing unacceptable amounts of hypoglycemia. The goal for most patients with T1D is to maintain an HbA1c level < 7.0% (estimated average glucose of < 154 mg/dL). In addition to the HbA1c test, other exemplary methods for assaying the signs, symptoms, or complications of T1D include, but are not limited to, the fasting plasma glucose (FPG) test, the oral glucose tolerance test (OGTT), the random blood glucose test, the C-peptide test, and tests to monitor the levels of T1D-associated autoantibodies.

D. Prophylactic and Therapeutic Methods

The following discussion is presented by way of example only, and is not intended to be limiting.

One aspect of the present technology provides for a method for preventing or delaying the onset of T1D or symptoms of T1D in a subject predisposed to the development of or at risk of having T1D (e.g., first-degree relatives of patients with T1D, where the relatives have been determined to be genetically predisposed to the development of T1D).

Subjects at risk for T1D can be identified by, e.g., any one or a combination of diagnostic or prognostic assays known in the art. In prophylactic applications, insulin is administered to a subject susceptible to, or otherwise at risk of T1D in an amount sufficient to eliminate or reduce the risk, or delay the onset of the disease, including biochemical and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. Administration of prophylactic insulin can occur prior to the manifestation of symptoms characteristic of the disease, such that the disease is prevented, or alternatively, delayed in its progression.

Subjects at risk for T1D include, but are not limited to, subjects who are related to a diabetic individual (usually a first-degree relative) or identified to have high-risk HLA genotypes (e.g., the DR3/4-DQ2/8 genotype). Screening for serologic markers including insulin autoantibodies (IAA) and serum autoantibodies associated with islet beta cells (ICA): IA-2A, IA-2β, IAA, GAD-65, and ZnT8 can also identify individuals at high risk for developing T1D. Assessing C-peptide levels is a widely-used measure of pancreatic β cell function and can also be used to assess an individual's risk for the development of T1D.

E. Modes of Administration, Pharmaceutical Compositions, and Effective Dosages

In vivo methods typically include the administration of an agent such as those described herein, to a mammal such as a human. When used in vivo for therapy, an agent of the present technology is administered to a mammal in an amount effective in obtaining the desired result or treating the mammal. The dose and dosage regimen will depend upon the degree of the disease in the subject, the subject, and the subject's history.

An effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount useful in the methods may be administered to a mammal in need thereof by any number of well-known methods for administering pharmaceutical compounds. In particular embodiments, the compositions of the present technology are formulated for sublingual administration.

The compositions include the human insulin and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes a buffer, glycerin, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. The compositions for use in the present invention comprise at least glycerin as a pharmaceutically acceptable carrier.

For the convenience of the patient or treating physician, the dosing formulations can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, etc.) for a treatment course.

Sublingual compositions generally include an inert diluent or an edible carrier. For the purpose of sublingual therapeutic administration, the insulin can be incorporated with an aqueous pharmaceutically acceptable carrier or excipient (which includes at least glycerin) and used in the form of tablets, troches, or capsules. In some embodiments, the aqueous pharmaceutically acceptable carrier comprises at least about 30 vol. % glycerin, at least about 31 vol. % glycerin, at least about 32 vol. % glycerin, at least about 33 vol. % glycerin, at least about 34 vol. % glycerin, at least about 35 vol. % glycerin, at least about 36 vol. % glycerin, at least about 37 vol. % glycerin, at least about 38 vol. % glycerin, at least about 39 vol. % glycerin, at least about 40 vol. % glycerin, at least about 41 vol. % glycerin, at least about 42 vol. % glycerin, at least about 43 vol. % glycerin, at least about 44 vol. % glycerin, at least about 45 vol. % glycerin, at least about 46 vol. % glycerin, at least about 47 vol. % glycerin, at least about 48 vol. % glycerin, at least about 49 vol. % glycerin, at least about 50 vol. % glycerin, at least about 51 vol. % glycerin, at least about 52 vol. % glycerin, at least about 53 vol. % glycerin, at least about 54 vol. % glycerin, at least about 55 vol. % glycerin, or at least about 60 vol. % glycerin. In some embodiments, the aqueous pharmaceutically acceptable carrier comprises at least about 30-70 vol. % glycerin, at least about 35-65 vol. %glycerin, at least about 40-60 vol. % glycerin, at least about 45-60 vol. % glycerin, at least about 50-60 vol. % glycerin, or at least about 50-55 vol. % glycerin. In some embodiments, the aqueous pharmaceutically acceptable carrier further comprise a buffer. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Pharmaceutically compatible binding agents and/or adjuvant materials can be included as part of the composition.

Dosage, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are advantageous.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the present technology, the therapeutically effective dose can be estimated initially from cell culture assays and/or animal studies. Such information can be used to determine useful doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography. The dose and dosage regimen will depend upon the degree of the disease in the subject, the subject, and the subject's history.

Typically, an effective amount of the insulin, sufficient for achieving a therapeutic or prophylactic effect, ranges from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of peptides ranges from 0.001-10,000 micrograms per kilogram body weight. In one embodiment, insulin concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. In some embodiments, an effective amount of insulin sufficient for achieving a therapeutic or prophylactic effect, is measured in units of insulin. For example, dosages can range from 0.5 to 1 unit of insulin/kg body weight/day. An exemplary treatment regimen entails sublingual administration of the insulin once a day, at least five days a week, for at least 7 weeks. In some embodiments, treatment entails sublingual administration at least once daily for at least 7 weeks. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regimen.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease, previous treatments, the general health and/or age of the subject, and other diseases present.

EXAMPLES

The present technology is further illustrated by the following examples, which should not be construed as limiting in any way.

Materials and Methods

Sublingual formulation. A commercial high dose Humulin^® solution (Humulin^® R U-500) containing 500 units of insulin per mL is mixed with an additional equal volume (1:1 (vol:vol)) 100% glycerin. Each dose contains 10 µL of the Humulin^®-glycerin solution, which contains 2.5 units (approximately 87 micrograms) of insulin in a solution having a final concentration of 52 vol. % glycerin.

NOD mice. Non-obese diabetic (NOD) mice, as described by Makino (Adv. Immunol. 51:285-322 (1992)), are used in the studies described herein. NOD mice provide an animal model for the spontaneous development of Type 1 diabetes. NOD mice develop insulitis as a result of leukocyte infiltration into the pancreatic islet, which in turn leads to the destruction of pancreatic islets and a Type 1 diabetic phenotype.

Serum C-peptide assay. The mouse C-peptide ELISA (ALPCO), which quantifies C-peptide protein products of mouse I and mouse II proinsulin genes, is used. Briefly, serum is collected by inserting a needle into the submandibular vein and collecting ~0.2 mL of blood. The blood is centrifuged for 30 minutes at 17,000 rpm in a refrigerated centrifuge, and serum is collected and stored -80°C.

Autoantibody titer assay. Commercially available ELISA assays are employed for detecting the presence of anti-insulin antibodies in serum samples collected from subjects.

Example 1: Use of Insulin in Delaying the Onset of Hyperglycemia in a Mouse Model of Type 1 Diabetes

This Example demonstrates the use of a sublingual formulation of insulin of the present technology in methods for delaying the onset of hyperglycemia in a mouse model of Type 1 diabetes.

Methods

Five-week old female NOD mice were randomly assigned to three groups: (1) control group (1:1 glycerin/phosphate buffered saline (PBS)); (2) insulin treatment (Humulin^®) started at six weeks of age; or (3) insulin treatment (Humulin^®) started at ten weeks of age. Mice in the treatment groups (2) and (3) were sublingually administered 87 µg of Humulin^® (10 µL of solution) once per day up to 30 weeks of age. Blood glucose measurements were taken once per week up to 20 weeks of age, and then twice per week thereafter. Mice were classified as diabetic after three consecutive blood glucose readings above 300 mg/dL (hyperglycemic).

Results

As shown in FIG. 1A , treatment with sublingual Humulin^® significantly reduced both the incidence (% T1D Onset) of Type 1 diabetes and onset time (weeks) of Type 1 diabetes in treatment group 2 (i.e., mice treated with Humulin^® starting at six (6) weeks of age) as compared to the control group. FIG. 1B , shows the results from treatment group 3 (i.e., mice treated with Humulin^® starting at ten (10) weeks of age) as compared to the control group. Table 3 provides the statistics associated with the survival curves shown in FIGs. 1A and 1B . Table 3. Comparison of Survival Curves

Chi square	3.98	0.812
Df	1	1
P value	0.0460	0.367
P value summary	*	ns
Are the survival curves significantly different?	Yes	No

Table 3. Comparison of Survival Curves

Chi square	5.45	0.725
Df	1	1
P value	0.020	0.389
P value summary	*	ns
Are the survival curves significantly different?	Yes	No

Table 3. Comparison of Survival Curves

Control	21 weeks	21 weeks
6 /10 wk Humulin initiation	27 weeks	24 weeks
Ratio (and its reciprocal)	0.778 (1.29)	0.8750 (1.14)
95% CI of ratio	0.396 to 1.53 (0.654 to 2.53)	0.453 to 1.69 (0.592 to 2.21)

These results demonstrate that the sublingual formulations of insulin of the present technology, such as Humulin^®, are useful in methods for treating Type 1 diabetes, where treatment includes delaying the onset of hyperglycemia or decreasing the likelihood of developing Type 1 diabetes in a subject in need thereof.

Example 2: Use of Insulin in Maintaining Serum C-Peptide Levels

This Example demonstrates the use of a sublingual formulation of insulin of the present technology in methods for maintaining serum C-peptide levels in a mouse model of Type 1 diabetes. C-peptide is the portion of proinsulin joining the alpha and beta insulin chains that is cleaved out prior to co-secretion with insulin from pancreatic beta cells. Produced in equimolar amounts to endogenous insulin, the 31-amino acid C-peptide is not a product of therapeutically administered exogenous insulin and has been widely used as a measure of insulin secretion (or pancreatic beta cell function).

Methods

Five-week old female NOD mice are randomly assigned to three groups as described in Example 1: (1) control group (oil only); (2) insulin treatment (Humulin^®) started at six weeks of age; or (3) insulin treatment (Humulin^®) started at ten weeks of age. Mice in the treatment groups (2) and (3) are sublingually administered 87 µg of Humulin^® once per day up to 30 weeks of age. Baseline C-peptide measurements are taken from serum drawn at week 6. Thereafter, C-peptide measurements are taken from serum drawn 1 time per 2 weeks beginning at week 20 up to 30 weeks of age.

Results

It is predicted that NOD mice receiving insulin peptide treatment, such as Humulin^® or a variant thereof having one or more conservative amino acid substitutions, will display maintenance or enhanced serum C-peptide levels as compared to untreated control NOD mice which develop clinical symptoms of Type 1 diabetes. These results will show that sublingual formulations of insulin of the present technology are useful in the treatment of Type 1 diabetes in a subject, particularly those with biological markers or history indicating a predisposition to the development of Type 1 diabetes.

Example 3: Use of Insulin in Attenuating an Antigenic Response

This Example demonstrates the use of a sublingual formulation of insulin of the present technology in methods for attenuating an antigenic response in subjects at risk for or having been diagnosed with Type 1 diabetes. The onset of Type 1 diabetes is preceded and accompanied by the appearance of a number of autoantibodies to a variety of pancreatic islet cell antigens. In genetically predisposed, but disease-free, individuals (e.g., first-degree relatives of patients with Type 1 diabetes), detection of multiple islet cell autoantibodies is a strong predictor for subsequent development of Type I diabetes. These autoantibodies include, but are not limited to, islet cell antibodies (ICA, against cytoplasmic proteins in the beta cell), antibodies to glutamic acid decarboxylase (GAD-65), insulin autoantibodies (IAA), and autoantibodies to tyrosine phosphatases IA-2A and IA-2β, and ZnT8.

Methods

Five-week old female NOD mice are randomly assigned to three groups as described in Example 1: (1) control group (oil only); (2) insulin treatment (Humulin^®) started at six weeks of age; or (3) insulin treatment (Humulin^®) started at ten weeks of age. Mice in the treatment groups (2) and (3) are sublingually administered 87 µg of humulin once per day up to 30 weeks of age. The subjects are assessed for baseline levels of autoantibodies at week 6. Thereafter, autoantibody titer measurements are taken once every other week beginning at week 15 up to 30 weeks of age.

Results

It is predicted that NOD mice receiving insulin treatment, such as Humulin^® or a variant thereof having one or more conservative amino acid substitutions, will display an attenuated antigenic response (e.g., of islet cell autoantibody levels) as compared to untreated controls. These results will demonstrate that sublingual formulations of insulin of the present technology, such as Humulin^® or a variant thereof having one or more conservative amino acid substitutions, are useful in the treatment of Type 1 diabetes in a subject in need thereof.

Example 4: Use of Insulin in Delaying the Onset of Hyperglycemia in Humans

This Example demonstrates the use of a sublingual formulation of insulin peptides of the present technology in methods for treating Type 1 diabetes in disease-free, individuals predisposed to the development of Type 1 diabetes (e.g., first-degree relatives of patients with Type 1 diabetes, where the relatives have been determined to be genetically predisposed to the development of Type 1 diabetes).

Methods

Subjects determined to be predisposed to the development of Type 1 diabetes receive daily, sublingual administrations of an insulin of the present technology. Dosages will range between 0.1 mg/kg to 50 mg/kg. Subjects will be evaluated weekly for the presence and/or severity of signs and symptoms associated with Type 1 diabetes, including, but not limited to, e.g., hyperglycemia, hypoinsulinemia, serum C-peptide levels, A1C levels, or presence of autoantibodies. Treatments may be maintained indefinitely or until such time as one or more signs or symptoms of Type 1 diabetes develop.

Results

It is predicted that subjects predisposed to the development of Type 1 diabetes receiving sublingually administered therapeutically effective amounts of insulin of the present technology will display delayed and/or reduced severity or elimination of the signs or symptoms associated with the development of Type 1 diabetes. These results will show that sublingual formulations of insulin of the present technology, such as Humulin^® or a biologically active fragment thereof or a variant of either of these having one or more conservative amino acid substitutions, are useful in the treatment of Type 1 diabetes in a subject in need thereof and in particular, in delaying the onset of hyperglycemia and/or decreasing the likelihood of developing Type 1 diabetes in the subject.

Where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

The foregoing description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the compositions and methods disclosed herein.

Claims (15)

1. A composition comprising human insulin and glycerin, for use in a method of delaying the onset of Type 1 diabetes or decreasing the likelihood of Type 1 diabetes in a mammalian subject in need thereof, the method comprising sublingually administering the composition to the subject.
2. The composition for use of claim 1, wherein the method is a method of delaying the onset of Type I diabetes in a subject in need thereof.
3. The composition for use of claim 1, wherein the method is a method of decreasing the likelihood of Type I diabetes in a subject in need thereof.
4. The composition for use of claim 1, for attenuating an antigenic response in a mammalian subject to at least one Type 1 diabetes-related antigen.
5. The composition for use of claim 4, wherein the subject displays reduced levels of autoantibodies selected from islet cell antibodies (ICA), glutamic acid decarboxylase-65 (GAD-65) antibodies, insulin autoantibodies (IAA), insulinoma-associated protein 2A (IA-2A) autoantibodies, insulinoma-associated protein 2β (IA-2β) autoantibodies, or zinc transporter 8 (ZnT8) autoantibodies.
6. The composition for use of any one of claims 1 to 5, wherein the method comprises administering the composition once a day, at least five days a week, for at least 7 weeks.
7. The composition for use of any one of claims 1 to 5, wherein the method comprises administering the composition at least once daily for at least 7 weeks.
8. The composition for use of any one of claims 1 to 7, wherein the composition comprises:

   the human insulin; and

   an aqueous pharmaceutically acceptable carrier, which comprises at least about 30 vol.% glycerin.
9. The composition for use of claim 8, wherein the aqueous pharmaceutically acceptable carrier further comprises a buffer.
10. The composition for use of claim 4, wherein attenuating the antigenic response comprises reducing the levels of one or more Type 1 diabetes-associated autoantibodies in the subject.
11. The composition for use of claim 10, wherein the one or more Type 1 diabetes-associated autoantibodies comprise insulin autoantibody (IAA).
12. The composition for use of claim 4, wherein the subject displays reduced development of insulin autoantibodies (IAA).
13. The composition for use of claim 4, wherein attenuating the antigenic response to at least one Type 1 diabetes related-antigen comprises decreasing development of insulin autoantibodies (IAA) after sublingual administration of the composition.
14. The composition for use of claim 4, wherein the at least one Type 1 diabetes related-antigen comprises insulin.
15. The composition for use of any one of the preceding claims, wherein the subject is human.