Methods and compositions for the treatment and prevention of type 1 diabetes

Abstract

The present disclosure provides methods for preventing or treating type 1 diabetes, reducing risk factors associated with type 1 diabetes, and / or reducing the likelihood or severity of type 1 diabetes in a mammalian subject. Also provided is a method for attenuating an antigenic response to at least one type 1 diabetes-associated antigen in a mammal. The method includes sublingually administering an effective amount of an insulin-related peptide to the subject. TIFF2023505190000005.tif56128

Description

Technical Field

[0001] Sequence Listing This application is electronically submitted in ASCII format and includes a sequence listing that is incorporated herein by reference in its entirety. The ASCII copy created on November 23, 2020, is named 120177-0106_PCT_Sequence_Listing.txt and is 14,714 bytes in size.

Background Art

[0002] Background Type 1 diabetes (T1D, also known as "autoimmune diabetes" and previously known as "insulin-dependent diabetes" or "juvenile-onset diabetes") is a chronic disease caused by the autoimmune destruction of pancreatic β-cells and the accompanying loss of insulin production, clinically manifested as hyperglycemia, and accounting for 5-10% of all diabetes cases. The age of onset is usually childhood or adolescence; however, symptoms may also appear much later in life. The etiology of T1D is not fully understood, but T cell-mediated destruction of pancreatic β-cells is thought to be involved in pathogenesis. There is no known cure for T1D, and patients have to rely on daily insulin therapy to compensate for impaired β-cell function. Insulin treatment typically involves either frequent insulin injection therapy or continuous subcutaneous insulin infusion. Without insulin, these patients develop severe complications such as ketoacidosis, retinopathy, nephropathy, vascular disorders, and neuropathy. Subcutaneous delivery of insulin requires strict self-management, so compliance is often a serious problem. Furthermore, the act of parenteral administration of insulin can be traumatic for young people. Currently, no effective oral or sublingual insulin therapy is known. The need to develop effective treatments for the prevention and / or treatment of T1D is emphasized due to compliance concerns with severe disease states and the increasing global prevalence of T1D.

Summary of the Invention

[0003] overview This technology relates, as a whole, to compositions and methods for preventing, improving, or treating type 1 diabetes, and / or reducing the severity of one or more risk factors, signs, or symptoms associated with type 1 diabetes. In addition, this technology relates to administering an effective amount of an insulin-related peptide, such as Humulin® or its variants having one or more conservative amino acid substitutions, to subjects who have or are at risk of having type 1 diabetes.

[0004] This technology relates to a sublingual formulation of an insulin-related peptide that can significantly reduce the incidence and delay the onset of T1D in a technically recognized mouse model of T1D (non-obese diabetic (NOD) mice). Effective sublingual insulin treatment for T1D is a highly unmet need. Therefore, the disclosure of this technology provides a composition formulated for a preferred route of administration that is effective in methods for preventing and / or treating one or more signs or symptoms of T1D, including delaying the onset of hyperglycemia, delaying the onset of T1D, and / or reducing the likelihood of T1D in subjects, and that can improve patient compliance.

[0005] One embodiment relates to a method for delaying the onset of hyperglycemia in subjects requiring it. This method includes the step of sublingual administration of a therapeutically effective dose of an insulin-related peptide to the subject. Examples of suitable insulin-related peptides include a first amino acid sequence comprising the insulin β-chain 7-26 peptide sequence (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions; and a second amino acid sequence comprising the insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions.

[0006] Another aspect relates to a method for delaying the onset of type 1 diabetes in subjects requiring it. This method includes the step of sublingual administration of a therapeutically effective dose of an insulin-related peptide to the subject. Examples of suitable insulin-related peptides include a first amino acid sequence comprising the insulin β-chain 7-26 peptide sequence (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions; and a second amino acid sequence comprising the insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions.

[0007] Another aspect relates to a method for reducing the likelihood of type 1 diabetes in subjects requiring it. This method includes the step of sublingual administration of a therapeutically effective dose of an insulin-related peptide. Examples of suitable insulin-related peptides include a first amino acid sequence containing the insulin β-chain 7-26 peptide sequence (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions; and a second amino acid sequence containing the insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions.

[0008] Another aspect relates to a method for attenuating the antigenic response to at least one type 1 diabetes-related antigen in mammals. This method includes the step of sublingual administration of an effective amount of insulin-related peptide to a mammal. Examples of suitable insulin-related peptides include a first amino acid sequence containing the insulin β-chain 7-26 peptide sequence (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions; and a second amino acid sequence containing the insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions. Following sublingual administration of the insulin-related peptide, subjects may show a decrease in the levels of autoantibodies such as pancreatic islet cell antibodies (ICAs), glutamate decarboxylase-65 (GAD-65) antibodies, insulin autoantibodies (IAAs), insulinoma-related protein 2A (IA-2A) autoantibodies, insulinoma-related protein 2β (IA-2β) autoantibodies, and / or zinc transporter 8 (ZnT8) autoantibodies.

[0009] In another embodiment, a composition is provided comprising an insulin-related peptide; and an aqueous, pharmaceutically acceptable carrier comprising at least about 30 vol.% glycerin. This composition may be a pharmaceutical composition for attenuating the antigenic reaction to at least one type 1 diabetes-related antigen. The insulin-related peptide may be a peptide comprising a first amino acid sequence comprising the insulin β-chain 7-26 peptide sequence (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions; and a second amino acid sequence comprising the insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions. [Invention 1001] A method for delaying the onset of hyperglycemia, delaying the onset of type 1 diabetes, or reducing the likelihood of type 1 diabetes in a person who needs it, The step of sublingually administering a therapeutically effective dose of insulin-related peptide to the subject. The method, including the method described above. [Invention 1002] A method for delaying the onset of hyperglycemia in those who require it, The step of sublingually administering a therapeutically effective dose of insulin-related peptide to the subject. The method, including the method described above. [Invention 1003] A method for delaying the onset of type 1 diabetes in those who require it, The step of sublingually administering a therapeutically effective dose of insulin-related peptide to the subject. The method, including the method described above. [Invention 1004] A method to reduce the likelihood of type 1 diabetes in those who need it, The step of sublingually administering a therapeutically effective dose of insulin-related peptide to the subject. The method, including the method described above. [Invention 1005] A method for attenuating the antigenic response to at least one type 1 diabetes-related antigen in mammals, The step of sublingually administering an effective amount of insulin-related peptide to the mammal. The method, including the method described above. [Invention 1006] The method of the present invention 1005, wherein the at least one type 1 diabetes-related antigen comprises one or more of insulin, proinsulin, preproinsulin, and insulin β-chain peptides 9-23. [Invention 1007] The method of the present invention 1005, wherein the at least one type 1 diabetes-related antigen comprises one or more of human insulin, human proinsulin, human preproinsulin, and human insulin β-chain peptides 9-23. [Invention 1008] The method according to any one of the present invention 1005 to 1007, wherein the subject exhibits a decrease in the level of an autoantibody selected from pancreatic islet cell antibody (ICA), glutamate decarboxylase-65 (GAD-65) antibody, insulin autoantibody (IAA), insulinoma-associated protein 2A (IA-2A) autoantibody, insulinoma-associated protein 2β (IA-2β) autoantibody, or zinc transporter 8 (ZnT8) autoantibody. [Invention 1009] The method according to any one of items 1001 to 1008 of the present invention, wherein the insulin-related peptide is administered once a day for at least 5 days a week for at least 7 weeks. [Invention 1010] The method according to any one of items 1001 to 1008 of the present invention, wherein the insulin-related peptide is administered at least once daily for at least seven weeks. [Invention 1011] The method according to any one of the present invention 1001 to 1010, wherein the insulin-related peptide comprises a first amino acid sequence containing the insulin β-chain 7-26 peptide sequence (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions; and a second amino acid sequence containing the insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions. [Invention 1012] The method of the present invention 1011, wherein the first amino acid sequence corresponds to the insulin β-chain amino acid sequence; and / or the second amino acid sequence corresponds to the insulin α-chain amino acid sequence. [Invention 1013] The method according to any one of the present invention 1001 to 1012, wherein the insulin-related peptide comprises one or more of insulin, proinsulin, and preproinsulin. [Invention 1014] The method according to any one of the present invention 1001 to 1012, wherein the insulin-related peptide contains human insulin. [Invention 1015] The method of any of the present invention 1001 to 1012, wherein the insulin-related peptide comprises human proinsulin and / or human preproinsulin. [Invention 1016] The method of the present invention 1011, wherein the first amino acid sequence comprises the human insulin β-chain 6-26 peptide sequence (SEQ ID NO: 3). [Invention 1017] The method of the present invention 1011, wherein the first amino acid sequence corresponds to the human insulin β-chain amino acid sequence (SEQ ID NO: 1). [Invention 1018] The method of the present invention 1011, wherein the second amino acid sequence comprises the human insulin α chain 6-20 peptide sequence (SEQ ID NO: 4). [Invention 1019] The method of the present invention 1011, wherein the second amino acid sequence corresponds to the human insulin α-chain amino acid sequence (SEQ ID NO: 2). [Invention 1020] The method of the present invention 1011, wherein the second amino acid sequence comprises a variant of the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) comprising a substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly. [Invention 1021] The method of the present invention 1011, wherein the second amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) comprising a substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly. [Invention 1022] The method of the present invention 1011, wherein the second amino acid sequence comprises a variant of the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) comprising a substitution of α-chain 9-Ser by Thr, Ala, or Gly. [Invention 1023] The method of the present invention 1011, wherein the second amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) comprising a substitution of α-chain 9-Ser by Thr, Ala, or Gly. [Invention 1024] The method of the present invention 1011, wherein the first amino acid sequence comprises a variant of the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) comprising a substitution of α-chain 10-Ile by Val, Ala, or Gly. [Invention 1025] The method of the present invention 1001, wherein the second amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) comprising a substitution of α-chain 10-Ile by Val, Ala, or Gly. [Invention 1026] The method of the present invention 1011, wherein the first amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) comprising a substitution of α-chain 21-Asn with Ala or Gly. [Invention 1027] The aforementioned second amino acid sequence contains a variant of the human insulin α chain 6-20 peptide sequence (SEQ ID NO: 3), and the variant is Substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly; Substitution of α-chain 9-Ser by Thr, Ala, or Gly; and Substitution of α-chain 10-Ile by Leu, Val, Ala, or Gly The method of the present invention 1011, comprising one or more more selected amino acid substitutions. [Invention 1028] The method of the present invention 1011, wherein the second amino acid sequence corresponds to a variant of the human insulin α chain 1-20 peptide sequence (SEQ ID NO: 5). [Invention 1029] The method of the present invention 1011, wherein the first amino acid sequence corresponds to the human insulin β-chain amino acid sequence (SEQ ID NO: 1), and the second amino acid sequence corresponds to the human insulin α-chain amino acid sequence (SEQ ID NO: 2). [Invention 1030] The method of the present invention 1011, wherein the second amino acid sequence comprises a variant of the human insulin α-chain 4-20 peptide sequence (SEQ ID NO: 6) including an α-chain 4-Glu substitution by Asp. [Invention 1031] The insulin-related peptide comprises a first amino acid sequence containing the human insulin β-chain 3-26 peptide sequence (SEQ ID NO: 7) or a variant thereof; and a second amino acid sequence containing the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof; The variant of the human insulin β-chain 3-26 peptide sequence (SEQ ID NO: 7) has a substitution of β-chain 3-Asn by Lys and / or a substitution of β-chain 9-Ser by Pro. The method of the present invention 1011. [Invention 1032] The insulin-related peptide comprises a first amino acid sequence containing the human insulin β-chain 4-27 peptide sequence (SEQ ID NO: 8) or a variant thereof; and / or a second amino acid sequence containing the human insulin α-chain 1-20 peptide sequence or a variant thereof; A variant of the human insulin β-chain 4-27 peptide sequence (SEQ ID NO: 8) has a substitution of β-chain 9-Ser by Pro; and The variants of the human insulin α chain 1-20 peptide sequences are Substitution of α-chain 4-Glu by Asp; Substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly; Substitution of α-chain 9-Ser by Thr, Ala, or Gly; Substitution of α-chain 10-Ile by Leu, Val, Ala, or Gly; and Substitution of α-chain 18-Asn by His Having one or more more selected amino acid substitutions, The method of the present invention 1011. [Invention 1033] The insulin-related peptide comprises a first amino acid sequence corresponding to the human insulin β-chain amino acid sequence (SEQ ID NO: 1) or a variant thereof; and / or a second amino acid sequence corresponding to the human insulin α-chain amino acid sequence (SEQ ID NO: 2) or a variant thereof; The variant of the human insulin β-chain amino acid sequence (SEQ ID NO: 1) is Lys substitution of β-chain 3-Asn; Substitution of β-chain 9-Ser by Pro; Substitution of β-chain 29-Lys by Met; and Substitution of β-chain 30-Thr with Ser or Ala Having one or more more selected amino acid substitutions; and The variant of the human insulin α chain amino acid sequence is Substitution of α-chain 4-Glu by Asp; Substitution of α-chain 8-Thr by Ala; Substitution of α-chain 9-Ser by Gly; Substitution of α-chain 10-Ile by Val; Substitution of α-chain 18-Asn by His; and Substitution of α-chain 21-Asn by Gly Having one or more more selected amino acid substitutions, Method of Invention 1011 [Invention 1034] The method according to any one of the present invention 1001 to 1033, wherein the insulin-related peptide is a recombinant human insulin-related peptide. [Invention 1035] Effective amounts of insulin-related peptides; and A pharmaceutically acceptable aqueous carrier containing at least approximately 30 vol.% glycerin. A composition for attenuating the antigenic reaction to at least one type 1 diabetes-related antigen, comprising: [Invention 1036] The composition of the present invention 1035, wherein the insulin-related peptide comprises a first amino acid sequence having an insulin β-chain 7-26 peptide sequence or a variant thereof having one or more amino acid substitutions; and a second amino acid sequence having an insulin α-chain 6-20 peptide sequence or a variant thereof having one or more amino acid substitutions. [Invention 1037] Insulin-related peptides; and A pharmaceutically acceptable aqueous carrier containing at least approximately 30 vol.% glycerin. A composition containing the following: [Invention 1038] The composition of the present invention 1037, wherein the insulin-related peptide comprises a first amino acid sequence having an insulin β-chain 7-26 peptide sequence or a variant thereof having one or more amino acid substitutions; and a second amino acid sequence having an insulin α-chain 6-20 peptide sequence or a variant thereof having one or more amino acid substitutions. [Invention 1039] A composition according to any one of the present invention 1035 to 1038, wherein the aqueous pharmaceutically acceptable carrier further comprises a buffer. [Invention 1040] A composition according to Invention 1036 or Invention 1038, wherein the first amino acid sequence corresponds to the insulin β-chain amino acid sequence; and / or the second amino acid sequence corresponds to the insulin α-chain amino acid sequence. [Invention 1041] A composition according to any one of the present invention 1035 to 1039, wherein the insulin-related peptide is a recombinant human insulin-related peptide. [Invention 1042] A composition according to any one of the present invention 1035 to 1039, wherein the insulin-related peptide comprises one or more of insulin, proinsulin, and preproinsulin. [Invention 1043] A composition according to any one of the present invention 1035 to 1039, wherein the insulin-related peptide contains human insulin. [Invention 1044] A composition according to any one of the present invention 1035 to 1039, wherein the insulin-related peptide comprises human proinsulin and / or human preproinsulin. [Invention 1045] A composition according to Invention 1036 or Invention 1038, wherein the first amino acid sequence comprises the human insulin β-chain 6-26 peptide sequence (SEQ ID NO: 3). [Invention 1046] A composition according to Invention 1036 or Invention 1038, wherein the first amino acid sequence corresponds to the human insulin β-chain amino acid sequence (SEQ ID NO: 1). [Invention 1047] A composition according to any one of the present invention 1034 to 1036, wherein the second amino acid sequence comprises the human insulin α chain 6-20 peptide sequence (SEQ ID NO: 4). [Invention 1048] A composition according to Invention 1036 or Invention 1038, wherein the second amino acid sequence corresponds to the human insulin α-chain amino acid sequence (SEQ ID NO: 2). [Invention 1049] A composition according to Invention 1036 or Invention 1038, comprising a variant of the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) in which the second amino acid sequence includes a substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly. [Invention 1050] A composition according to Invention 1036 or Invention 1038, wherein the second amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) comprising a substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly. [Invention 1051] A composition according to Invention 1036 or Invention 1038, wherein the second amino acid sequence comprises a variant of the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) including a substitution of α-chain 9-Ser by Thr, Ala, or Gly. [Invention 1052] A composition according to Invention 1036 or Invention 1038, wherein the second amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) comprising a substitution of α-chain 9-Ser by Thr, Ala, or Gly. [Invention 1053] A composition according to Invention 1036 or Invention 1038, comprising a variant of the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) in which the first amino acid sequence includes a substitution of α-chain 10-Ile by Val, Ala, or Gly. [Invention 1054] A composition according to Invention 1036 or Invention 1038, wherein the second amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) comprising a substitution of α-chain 10-Ile by Val, Ala, or Gly. [Invention 1055] A composition according to Invention 1036 or Invention 1038, wherein the first amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) comprising a substitution of α-chain 21-Asn with Ala or Gly. [Invention 1056] The aforementioned second amino acid sequence contains a variant of the human insulin α chain 6-20 peptide sequence (SEQ ID NO: 4), and the variant is Substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly; Substitution of α-chain 9-Ser by Thr, Ala, or Gly; and Substitution of α-chain 10-Ile by Leu, Val, Ala, or Gly A composition according to Invention 1036 or Invention 1038, comprising one or more more selected amino acid substitutions. [Invention 1057] A composition according to Invention 1036 or Invention 1038, wherein the second amino acid sequence corresponds to a variant of the human insulin α chain peptide sequence 1-20 (SEQ ID NO: 5). [Invention 1058] A composition according to Invention 1036 or Invention 1038, wherein the first amino acid sequence corresponds to the human insulin β-chain amino acid sequence (SEQ ID NO: 1), and the second amino acid sequence corresponds to the human insulin α-chain amino acid sequence (SEQ ID NO: 2). [Invention 1059] A composition of the present invention 1036 or 1038, wherein the second amino acid sequence comprises a variant of the human insulin α-chain 6-20 peptide sequence, wherein the second amino acid sequence includes a substitution of α-chain 4-Glu by Asp. [Invention 1060] The insulin-related peptide comprises a first amino acid sequence containing the human insulin β-chain 3-26 peptide sequence (SEQ ID NO: 7) or a variant thereof; and a second amino acid sequence containing the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof; The variant of the human insulin β-chain 3-26 peptide sequence (SEQ ID NO: 7) has a substitution of β-chain 3-Asn by Lys and / or a substitution of β-chain 9-Ser by Pro. A composition according to any of the present invention 1035 to 1039. [Invention 1061] The insulin-related peptide comprises a first amino acid sequence containing the human insulin β-chain 4-27 peptide sequence (SEQ ID NO: 8) or a variant thereof; and / or a second amino acid sequence containing the human insulin α-chain 1-20 peptide sequence (SEQ ID NO: 5) or a variant thereof; The variant of the human insulin β-chain 4-27 peptide sequence (SEQ ID NO: 8) has a substitution of β-chain 9-Ser by Pro; and The variant of the human insulin α chain peptide sequence 1-20 (SEQ ID NO: 5) is Substitution of α-chain 4-Glu by Asp; Substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly; Substitution of α-chain 9-Ser by Thr, Ala, or Gly; Substitution of α-chain 10-Ile by Leu, Val, Ala, or Gly; and Substitution of α-chain 18-Asn by His Having one or more more selected amino acid substitutions, A composition according to any of the present invention 1035 to 1039. [Invention 1062] The insulin-related peptide comprises a first amino acid sequence corresponding to the human insulin β-chain amino acid sequence (SEQ ID NO: 1) or a variant thereof; and / or a second amino acid sequence corresponding to the human insulin α-chain amino acid sequence (SEQ ID NO: 2) or a variant thereof; The variant of the human insulin β-chain amino acid sequence (SEQ ID NO: 1) is Lys substitution of β-chain 3-Asn; Substitution of β-chain 9-Ser by Pro; Substitution of β-chain 29-Lys by Met; and Substitution of β-chain 30-Thr with Ser or Ala Having one or more more selected amino acid substitutions; and The variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) is Substitution of α-chain 4-Glu by Asp; Substitution of α-chain 8-Thr by Ala; Substitution of α-chain 9-Ser by Gly; Substitution of α-chain 10-Ile by Val; Substitution of α-chain 18-Asn by His; and Substitution of α-chain 21-Asn by Gly Having one or more more selected amino acid substitutions, A composition according to any of the present invention 1035 to 1039. [Invention 1063] A method of any of the present inventions 1001 to 1010, comprising the step of sublingually administering an effective amount of any of the compositions of the present inventions 1035 to 1062. [Invention 1064] The method of the present invention 1005, comprising reducing the antigenic reaction to lower the level of one or more type 1 diabetes-related autoantibodies in the subject. [Invention 1065] The method of the present invention 1064, wherein the one or more type 1 diabetes-related autoantibodies include insulin autoantibodies (IAAs). [Invention 1066] The method of the present invention 1005, wherein the subject exhibits a reduction in the generation of insulin autoantibodies (IAA). [Invention 1067] The method of the present invention 1005, comprising reducing the antigenic reaction to at least one type 1 diabetes-related antigen, thereby reducing the development of insulin autoantibodies (IAAs) after sublingual administration of an insulin-related peptide. [Invention 1068] The method of the present invention 1005, wherein the at least one type 1 diabetes-related antigen comprises insulin. [Invention 1069] The method according to any one of the invention 1064 to 1068, wherein the insulin-related peptide comprises a first amino acid sequence containing the insulin β-chain 7-26 peptide sequence (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions; and a second amino acid sequence containing the insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions. [Invention 1070] The method of the present invention 1069, wherein the insulin-related peptide contains insulin. [Invention 1071] The insulin-related peptide is substituted as follows: Substitution of Asn with Lys, His, or Gly; Glu replacement by Asp; Substitution of Ile with Ala, Gly, Leu, or Val; Substitution of Lys with Met; Substitution of Ser by Thr, Gly, Ala, or Pro; and Substitution of Thr by Ala, Ser, Gly, or Val The method of the present invention 1069, comprising a peptide comprising a variant of an insulin peptide sequence comprising one or more of the following. [Brief explanation of the drawing]

[0010] [Figure 1A] Figures 1A and 1B are charts showing the incidence and time to onset of type 1 diabetes in control NOD mice, and in NOD mice that were treated with Humulin® at 6 weeks of age (Figure 1A) and 10 weeks of age (Figure 1B). [Figure 1B] Refer to the explanation in Figure 1A. [Modes for carrying out the invention]

[0011] Detailed explanation I. Definition To provide a substantial understanding of this technology, it should be recognized that specific aspects, styles, embodiments, variations, and features of this technology are described below at various detailed levels. Definitions of specific terms used herein are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this technology belongs.

[0012] As used herein and in the appended claims, the singular forms “one (a),” “one (an),” and “it” include both singular and plural referents unless the context clearly indicates otherwise. For example, a reference to “one cell” includes a combination of two or more cells, and so on.

[0013] As used herein, “administration” of a drug, substance, or peptide to a subject means sublingual administration of the composition of this technology to the subject.

[0014] As used herein, “conservative amino acid substitution” means that it does not substantially alter the structural and functional properties of the parent sequence (for example, the substituted amino acid should not disrupt the helices present in the parent sequence or disrupt other types of secondary structures that characterize the parent sequence or are necessary for its functionality).

[0015] As used herein, the term “effective dose” means an amount sufficient to achieve the desired therapeutic and / or preventive effect, for example, an amount that results in partial or complete improvement of one or more symptoms of type 1 diabetes. In relation to therapeutic or preventive application, in some embodiments, the amount of composition administered to a subject depends on the type, degree, and severity of the disease, as well as the individual’s characteristics, such as overall health, age, sex, weight, and tolerance to the drug. Those skilled in the art can determine an appropriate dose in accordance with these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. For example, the insulin-related peptides of this technology, such as Humulin® or its variants having one or more conservative amino acid substitutions, may be administered to subjects having one or more signs, symptoms, or risk factors of type 1 diabetes, including but not limited to hyperglycemia, hypoinsulinemia, decreased serum C peptide levels, elevated A1C levels, presence of T1D-related autoantibodies, excessive urinary excretion (polyuria), thirst (polydipsia), persistent hunger (polyphagia), weight loss, visual impairment, fatigue, mental confusion, nausea, vomiting, ketoacidosis, retinopathy, nephropathy, vascular disorders, and neuropathy. The insulin-related peptides may also be administered to disease-free subjects who are genetically predisposed to developing T1D (e.g., first-degree relatives of a type 1 diabetes patient who have been determined to be genetically predisposed to developing type 1 diabetes). For example, a “therapeutic dose” of insulin-related peptide includes a level that at least improves the presence, frequency, or severity of one or more signs, symptoms, or risk factors of type 1 diabetes. A therapeutic dose may reduce or improve the physiological effects of type 1 diabetes, and / or risk factors for type 1 diabetes, and / or the likelihood of developing type 1 diabetes. A therapeutic dose may be administered in one or more doses.

[0016] As used herein, the term “insulin-related peptide” refers to a peptide comprising a first amino acid sequence comprising an insulin β-chain (B-chain) or a bioactive fragment thereof, or any variant thereof having one or more amino acid substitutions, and / or an insulin α-chain (A-chain) or a bioactive fragment thereof, or a second amino acid sequence comprising any variant thereof having one or more amino acid substitutions. In some embodiments, the insulin-related peptide comprises an amino acid sequence comprising an insulin β-chain peptide sequence 7-26 (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions. In some embodiments, the insulin-related peptide comprises an amino acid sequence comprising an insulin α-chain peptide sequence 6-20 (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions. In some embodiments, the insulin-related peptide comprises a first amino acid sequence containing the insulin β-chain 7-26 peptide sequence (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions, and a second amino acid sequence containing the insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions. In some embodiments, the insulin-related peptide comprises a first amino acid sequence containing the human insulin β-chain 7-26 peptide sequence (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions, and a second amino acid sequence containing the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions. In some embodiments, the "insulin-related peptide" comprises larger fragments of the insulin β-chain and insulin α-chain.For example, in some embodiments, the insulin-related peptide may include the insulin β-chain 6-26 peptide sequence (SEQ ID NO: 3), the insulin β-chain 3-26 peptide sequence (SEQ ID NO: 7), the insulin β-chain 4-27 peptide sequence (SEQ ID NO: 8), the insulin β-chain sequence (SEQ ID NO: 1), the insulin α-chain 1-20 peptide sequence (SEQ ID NO: 5), the insulin α-chain 4-20 peptide sequence (SEQ ID NO: 6), or the insulin α-chain sequence (SEQ ID NO: 2), or variants thereof. The insulin-related peptide may be of human origin or of any mammalian species. In some embodiments, the insulin-related peptide is a recombinant human insulin-related peptide such as Humun® or a variant thereof having one or more conserved amino acid substitutions. In some embodiments, the insulin-related peptide includes one or more of insulin, proinsulin, and preproinsulin.

[0017] As used herein, the terms “Type 1 diabetes” or “T1D” refer to a disorder characterized by insulin deficiency resulting from the loss of pancreatic β-cells and leading 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) testing (HbA1C value ≥ 6.5%), (2) oral glucose tolerance test (OGTT; post-load plasma glucose value ≥ 200 mg / dL), (3) random blood glucose testing (glucose value ≥ 200 mg / dL at any time point, combined with symptoms of diabetes), (4) fasting plasma glucose (FPG) testing (fasting blood glucose ≥ 126 mg / dL), and (5) C-peptide values ​​less than 0.2 nmol / L.

[0018] As used herein, “to treat” or “treatment” encompasses the treatment of type 1 diabetes and / or its signs or symptoms in subjects such as humans, including (i) inhibiting type 1 diabetes, i.e., preventing its onset; (ii) alleviating type 1 diabetes, i.e., causing regression of the disorder; (iii) slowing the progression of type 1 diabetes; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more signs or symptoms of type 1 diabetes, including but not limited to hyperglycemia, hypoinsulinemia, decreased serum C-peptide levels, elevated A1C levels, presence of T1D-related autoantibodies, polyuria, polydipsia, polyphagia, weight loss, visual disturbances, fatigue, confusion, nausea, vomiting, and ketoacidosis.

[0019] As used herein, “preventing” a disorder or condition means reducing the occurrence or likelihood of the disorder or condition in a treated sample compared to an untreated control sample, or delaying the onset of one or more signs or symptoms of the disorder or condition, including but not limited to hyperglycemia, hypoinsulinemia, decreased serum C-peptide levels, elevated A1C levels, presence of T1D-related autoantibodies, polyuria, polydipsia, polyphagia, weight loss, visual disturbances, fatigue, confusion, nausea, vomiting, and ketoacidosis, compared to an untreated control sample. As used herein, preventing type 1 diabetes means preventing or delaying the onset of type 1 diabetes. As used herein, preventing type 1 diabetes also includes preventing the recurrence of one or more signs or symptoms of type 1 diabetes.

[0020] It should also be recognized that the various forms of treatment or prevention of medical conditions described herein include not only complete treatment or prevention but also incomplete treatment or prevention, and that "substantial" means that some biological or medically relevant outcome is achieved. Treatment may be continuous, long-term treatment for a chronic disease, or a single or small number of doses for the treatment of an acute condition.

[0021] As used herein, the terms “subject” and “patient” are interchangeable.

[0022] II. Overview This technology relates to the remarkable discovery of a sublingual formulation of an insulin-related peptide that can significantly reduce the incidence and delay the onset of T1D in a technically recognized mouse model of T1D (non-obese diabetic (NOD) mice). Effective sublingual insulin treatment for T1D is a highly unmet need. Therefore, the methods and compositions of this technology provide a desirable route of administration that is effective as a treatment for T1D and can improve patient compliance.

[0023] III. Insulin-related peptides Insulin is a 51-amino acid protein secreted by the pancreatic β-cells of the islets of Langerhans. Insulin is initially synthesized as preproinsulin in the rough endoplasmic reticulum of pancreatic β-cells. After the signal peptide in the preprohormone is removed by proteolysis, a proinsulin molecule is produced, consisting of a 21-amino acid α-chain (or A-chain) peptide, a 30-amino acid β-chain (or B-chain) peptide, and an intervening C-chain peptide (C-peptide). Subsequently, proinsulin is processed in the Golgi complex, where the C-peptide is removed, and the α-chain and β-chain are linked via two disulfide bonds at cysteine ​​residues, producing bioactive insulin. A third disulfide bond connects two cysteine ​​residues within the α-chain. Insulin and C-peptide are secreted simultaneously in equimolar amounts in response to various stimuli, such as glucose.

[0024] As shown in Table 1, the amino acid sequences of the A and B chains of insulin are highly conserved among vertebrates. In addition, the positions of the three disulfide bonds are also the same in most species. Due to these highly conserved features, the three-dimensional higher-order structure of insulin is very similar across species. For this reason, insulin from one species is often bioactive and has similar physiological effects in other species. Table 1 discloses SEQ ID NO: 10-24 in order of appearance.

[0025] (Table 1) Insulin sequences from specific species TIFF0007879594000001.tif246170

[0026] The insulin-related peptide of this technology, formulated for sublingual administration, comprises a peptide containing an amino acid sequence that constitutes an insulin β-chain (B-chain) or its bioactive fragment, or any variant thereof having one or more amino acid substitutions, and may contain one or more of the B-chain sequences shown in Table 1. In some embodiments, the insulin-related peptide of this technology, formulated for sublingual administration, comprises a peptide containing an amino acid sequence that constitutes an insulin α-chain (A-chain) or its bioactive fragment, or any variant thereof having one or more amino acid substitutions, and may contain one or more of the A-chain sequences shown in Table 1. In some embodiments, the insulin-related peptide of this technology, formulated for sublingual administration, comprises a peptide containing a first amino acid sequence that constitutes an insulin β-chain (B-chain) or its bioactive fragment, or any variant thereof having one or more amino acid substitutions, and a second amino acid sequence that constitutes an insulin α-chain (A-chain) or its bioactive fragment, or any variant thereof having one or more amino acid substitutions, and may contain one or more of the B-chain and A-chain sequences shown in Table 1. In some embodiments, the insulin-related peptide of the present technology comprises a second amino acid sequence including the insulin β-chain peptide sequence 7-26 (SEQ ID NO: 9) or a variant thereof having one or more amino acid substitutions, and the insulin α-chain peptide sequence 6-20 (SEQ ID NO: 4) or a variant thereof having one or more amino acid substitutions. In some embodiments, the insulin-related peptide of the present technology comprises larger fragments of the insulin β-chain and insulin α-chain.For example, in some embodiments, the insulin-related peptide may include the insulin β-chain peptide sequences 6-26 (SEQ ID NO: 3), insulin β-chain peptide sequences 3-26 (SEQ ID NO: 7), insulin β-chain peptide sequences 4-27 (SEQ ID NO: 8), insulin β-chain sequence (SEQ ID NO: 1), insulin α-chain peptide sequences 1-20 (SEQ ID NO: 5), insulin α-chain peptide sequences 4-20 (SEQ ID NO: 6), or insulin α-chain sequence (SEQ ID NO: 2). The insulin-related peptide may be of human origin or of any mammalian species. For example, the insulin-related peptide of this disclosure may include one or more of the insulin A-chain or B-chain shown in Table 1. In some embodiments, the insulin-related peptide is a recombinant human insulin-related peptide such as Humulin® or a variant thereof having one or more conserved amino acid substitutions. In some embodiments, the insulin-related peptide includes one or more of insulin, proinsulin, and preproinsulin. In some embodiments, insulin-related peptides are rapid-acting, medium-acting, or long-acting insulin analogs.

[0027] In addition to the insulin-related peptide sequences provided in Table 1, exemplary and non-limiting insulin-related peptides of this technology are also provided in Table 2.

[0028] (Table 2) Exemplary insulin-related peptides TIFF0007879594000002.tif65157

[0029] Appropriate substitutional variants of peptides listed herein include conserved amino acid substitutions. Amino acids can be grouped according to their physicochemical properties as follows: (a) Nonpolar amino acids: Ala(A), Ser(S), Thr(T), Pro(P), Gly(G), Cys(C); (b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q); (c) Basic amino acids: His(H) Arg(R) Lys(K); (d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and (e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W).

[0030] Substitution of an amino acid within a peptide by another amino acid within the same group is called a conservative substitution and can maintain the physicochemical properties of the original peptide. In other embodiments, the peptide variants described herein include the following substitutions: Substitution of Asn with Lys, His, or Gly Glu replacement by Asp Substitution of Ile with Ala, Gly, Leu, or Val Lys replacement using Met Substitution of Ser by Thr, Gly, Ala, or Pro Substitution of Thr by Ala, Ser, Gly, or Val It may include one or more of the following.

[0031] Peptides can be synthesized by any of the methods well known in the art. Suitable methods for the chemical synthesis of proteins 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).

[0032] IV. Type 1 diabetes Type 1 diabetes (T1D), also known as "autoimmune diabetes" (formerly known as "insulin-dependent diabetes" or "juvenile-onset diabetes"), is a chronic disease characterized by hyperglycemia due to insulin deficiency resulting from the loss of pancreatic beta cells. The age of symptom onset is usually childhood or adolescence; however, symptoms can sometimes appear much later. The etiology of T1D is not fully understood, but T-cell-mediated destruction of beta cells is thought to be involved in the pathogenesis of the disease. There is no cure, and patients depend on insulin injections for life. Intensive glycemic control has reduced the incidence of microvascular and macrovascular complications, but the majority of T1D patients still develop these complications.

[0033] A. Clinical findings Clinical signs and symptoms of T1D include hyperglycemia, hypoinsulinemia, decreased serum C-peptide levels, elevated A1C levels, presence of T1D-related autoantibodies, excessive urinary excretion (polyuria), thirst (polydipsia), persistent hunger (polyphagia), weight loss, vision changes, fatigue, confusion, nausea, vomiting, and ketoacidosis. Chronic symptoms of T1D include retinopathy, nephropathy, vascular disorders, and neuropathy.

[0034] B. Diagnosis In humans, T1D is diagnosed by a combination of symptoms and the results of specific blood tests. Fasting plasma glucose (FPG) testing diagnoses diabetes if the fasting blood glucose level is 126 mg / dL or higher. Oral glucose tolerance test (OGTT) diagnoses diabetes if the plasma glucose level 2 hours after loading is 200 mg / dL or higher. Random blood glucose testing is sufficient to make a diagnosis if the blood glucose level is 200 mg / dL or higher at any point in time, combined with symptoms of diabetes. Hemoglobin A1C (HbA1C; glycated hemoglobin) testing, which measures the average glucose level over the past 2-3 months, diagnoses diabetes if the HbA1C level is 6.5% or higher. If elevated levels are detected in asymptomatic individuals, retesting, preferably with the same test, as soon as possible at a later date is recommended to confirm the diagnosis. Endogenous insulin production can be assessed by measuring serum C-peptide in a fasting state or after stimulation, most commonly after intravenous glucagon administration. C-peptide can also be measured in urine. The normal range for fasting serum C-peptide levels in humans is 0.26–1.27 nmol / L. C-peptide levels below 0.2 nmol / L are associated with the diagnosis of T1D in humans.

[0035] Progression to T1D is typically preceded by prodromal symptoms of anti-islet autoantibody development. Biomarkers of T1D-related autoimmunity that can be found several months to several years before symptom onset include many T1D-related autoantibodies such as insulin autoantibody (IAA), islet cell antibody (ICA), 65 kDa glutamate 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 of β-cells. In predisposed but disease-free individuals, the detection of multiple islet cell autoantibodies is a strong predictor of subsequent T1D development.

[0036] C. Prognostic Indicators Methods for evaluating the signs, symptoms, or complications of T1D are known in the art. Once diabetes is diagnosed, a key treatment goal is to maintain mean glucose as close to the normal range as possible without causing unacceptable levels of hypoglycemia. For most T1D patients, the goal is to maintain an HbA1c level <7.0% (estimated mean glucose <154 mg / dL). In addition to HbA1c testing, other exemplary methods for assaying the signs, symptoms, or complications of T1D include, but are not limited to, fasting plasma glucose (FPG) testing, oral glucose tolerance tests (OGTT), random blood glucose testing, C-peptide testing, and tests to monitor levels of T1D-related autoantibodies.

[0037] D. Methods of prevention and treatment The following discussion is presented for illustrative purposes only and is not intended to be limiting.

[0038] One aspect of this technology is to provide a method for preventing or delaying the onset or symptoms of T1D in individuals who are prone to developing T1D or are at risk of developing T1D (for example, first-degree relatives of T1D patients who have been determined to be genetically predisposed to developing T1D).

[0039] Subjects at risk of T1D can be identified, for example, by one or a combination of diagnostic or prognostic assays known in the art. In prophylactic applications, the insulin-related peptide of the art is administered to subjects who are susceptible to or at risk of T1D in amounts sufficient to eliminate or reduce the risk, or to delay the onset of the disease, including the biochemical and / or behavioral symptoms of the disease, its complications, and the intermediate pathological phenotypes exhibited during the onset of the disease. Prophylactic administration of the insulin-related peptide can be performed before the appearance of symptoms characteristic of the disease, so that the disease is prevented or, alternatively, its progression is delayed.

[0040] Individuals at risk of T1D or hyperglycemia include, but are not limited to, those identified as being related (usually first-degree relatives) to a diabetic individual or having a high-risk HLA genotype (e.g., DR3 / 4-DQ2 / 8 genotype). Screening for serum markers, including insulin autoantibodies (IAAs) and islet β-cell-associated serum autoantibodies (ICAs): IA-2A, IA-2β, IAA, GAD-65, and ZnT8, can also identify individuals at high risk of developing T1D. Assessment of C-peptide levels is a widely used measure of pancreatic β-cell function and can also be used to assess the risk of individuals developing T1D.

[0041] Another aspect of this technology includes methods for treating T1D in subjects diagnosed with, suspected of having, or at risk of having T1D. In therapeutic applications, compositions comprising insulin-related peptides of this technology are administered to subjects suspected of having the disease or already suffering from the disease (e.g., subjects exhibiting hyperglycemia, elevated serum autoantibodies associated with T1D, or decreased C-peptide levels) in amounts sufficient to cure or at least partially suppress the symptoms of the disease, including its complications.

[0042] In certain embodiments, T1D subjects treated with a sublingual formulation of the insulin-related peptide of the Technology exhibit normalization of blood glucose levels, T1D-related autoantibodies, and / or C-peptide levels by at least 5%, at least 10%, at least 50%, at least 75%, or at least 90% compared to untreated T1D subjects. In certain embodiments, T1D subjects treated with a sublingual formulation of the insulin-related peptide of the Technology exhibit blood glucose levels, T1D-related autoantibodies, and / or C-peptide levels similar to those observed in normal control subjects.

[0043] E. Mode of administration, pharmaceutical composition, and effective dosage In vivo methods typically involve the administration of drugs, such as those described herein, to mammals, including humans. When used in vivo for therapeutic purposes, the drugs of this technology are administered to the mammal in an amount effective to achieve the desired outcome or to treat the mammal. The dosage and administration plan depend on the severity of the disease in the subject, the characteristics of the specific insulin-related peptide used (e.g., its therapeutic index, duration of action, etc.), the subject, and the subject's medical history.

[0044] The effective dose of the insulin-related peptide in this technology can be determined during preclinical and clinical trials by methods well known to physicians and clinicians. The effective dose of the insulin-related peptide useful in this method can be administered to the mammal in need by many well-known methods for administering pharmaceutical compounds. In certain embodiments, the insulin-related peptide in this technology is formulated for sublingual administration.

[0045] The insulin-related peptides described herein may be incorporated alone or in combination into pharmaceutical compositions for administration to a subject for the treatment or prophylaxis of T1D. Such compositions may comprise insulin-related peptides and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes buffers, glycerin, saline, solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, and similar materials that are compatible with the pharmaceutical administration. Auxiliary active compounds may also be incorporated into the composition.

[0046] For the convenience of the patient or the treating physician, the administered preparation may be provided in a kit containing all the necessary equipment for the treatment process (e.g., vials of the drug, vials of diluents, etc.).

[0047] Sublingual compositions generally contain an inert diluent or an edible carrier. For sublingual therapeutic administration, insulin-related peptides can be incorporated with an aqueous, pharmaceutically acceptable carrier or excipient (e.g., glycerin) and used in the form of tablets, lozenges, or capsules. In some embodiments, the aqueous pharmaceutically acceptable carrier contains 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, and at least about 47 The aqueous pharmaceutically acceptable carrier comprises 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 comprises a buffer. The pH can be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide.A pharmaceutically compatible binder and / or auxiliary substance may be included as part of the composition.

[0048] Dosage, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (lethal dose for 50% of the population). The dose-to-therapeutic ratio is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a high therapeutic index are advantageous.

[0049] Data obtained from cell culture assays and animal studies can be used to determine the range of doses for use in humans. Doses of such compounds may be within the range of circulating concentrations, including the ED50, which has little toxicity. Doses may vary within this range depending on the dosage form and route of administration used. For any compound used in the methods of this technique, the therapeutically effective dose can first be estimated from cell culture assays and / or animal studies. Using such information, a useful dose in humans can be accurately determined. Plasma levels can be measured, for example, by high-performance liquid chromatography. Dosage and administration plans depend on the severity of the disease in the subject, the characteristics of the specific insulin-related peptide used (e.g., its therapeutic index, duration of action, etc.), the subject, and the subject's medical history.

[0050] Typically, the effective dose of insulin-related peptide sufficient to achieve a therapeutic or preventive effect is in the range of approximately 0.000001 mg / kg / day to approximately 10,000 mg / kg / day. Appropriately, the dose range is approximately 0.0001 mg / kg / day to approximately 100 mg / kg / day. For example, the dose may be 1 mg / kg or 10 mg / kg daily, every two or three days, or in the range of 1 to 10 mg / kg weekly, every two or three weeks. In one embodiment, the single dose of the peptide is in the range of 0.001 to 10,000 micrograms / kg. In one embodiment, the concentration of insulin-related peptide in the carrier is in the range of 0.2 to 2000 micrograms / ml / delivery. In some embodiments, the effective dose of insulin-related peptide sufficient to achieve a therapeutic or preventive effect is measured in units of insulin. For example, the dosage may range from 0.5 to 1 unit of insulin per kg of body weight per day. An exemplary treatment plan requires sublingual administration of an insulin-related peptide once daily for at least 5 days a week for at least 7 weeks. In some embodiments, treatment requires sublingual administration at least once daily for at least 7 weeks. In therapeutic applications, relatively high doses at relatively short intervals may be required until disease progression is reduced or terminated, or until the subject shows partial or complete improvement of disease symptoms. A preventive plan can then be implemented for the patient.

[0051] Those skilled in the art will recognize that certain factors, including but not limited to the severity of the disease, previous treatments, the subject's overall health status and / or age, and other pre-existing conditions, may influence the dosage and timing required to effectively treat the subject. [Examples]

[0052] The present technology will be further illustrated by the following embodiments, but these embodiments should not be construed as limiting.

[0053] material and method Sublingual formulation. A commercially available high-dose Humulin® solution (Humulin®® U-500) containing 500 units of insulin per 1 mL is mixed with an additional equal volume (1:1 (vol:vol)) of 100% glycerin. Each dose contains 10 μL of Humulin®-glycerin solution containing 2.5 units (approximately 87 micrograms) of insulin in a solution with a final concentration of 52 vol.% glycerin.

[0054] NOD mice. Non-obese diabetic (NOD) mice, described by Makino (Adv. Immunol. 51:285-322 (1992)), will be used in the studies described herein. NOD mice provide an animal model for the spontaneous development of type 1 diabetes. NOD mice develop insular inflammation as a result of leukocyte infiltration into the pancreatic islets, which then leads to destruction of the pancreatic islets and the type 1 diabetic phenotype.

[0055] Serum C-peptide assay. Mouse C-peptide ELISA (ALPCO) is used to quantify the C-peptide protein product of mouse I and mouse II proinsulin genes. Briefly, serum is collected by inserting a needle into the submandibular vein and drawing approximately 0.2 mL of blood. The blood is centrifuged in a refrigerated centrifuge at 17,000 rpm for 30 minutes to collect the serum, which is then stored at -80°C.

[0056] Autoantibody titer assay. A commercially available ELISA assay is used to detect the presence of anti-insulin antibodies in serum samples collected from subjects.

[0057] Example 1: Use of insulin-related peptides to delay the onset of hyperglycemia in a mouse model of type 1 diabetes. This embodiment demonstrates the use of a sublingual formulation of the insulin-related peptide of this technology in a method for delaying the onset of hyperglycemia in a mouse model of type 1 diabetes.

[0058] method Five-week-old female NOD mice were randomly assigned to one of three groups: (1) a control group (1:1 glycerin / phosphate-buffered saline (PBS)); (2) insulin-related peptide treatment (Humulin®) initiated at six weeks of age; or (3) insulin-related peptide treatment (Humulin®) initiated at ten weeks of age. Mice in treatment groups (2) and (3) received 87 μg of Humulin® (10 μL solution) sublingually once daily until 30 weeks of age. Blood glucose was measured once a week until 20 weeks of age, and then twice a week thereafter. Mice were classified as having diabetes after three consecutive blood glucose readings exceeding 300 mg / dL (hyperglycemia).

[0059] result As shown in Figure 1A, compared to the control group, sublingual Humulin® treatment significantly reduced both the incidence of type 1 diabetes (T1D %) and the time to onset of type 1 diabetes (weeks) in treatment group 2 (i.e., mice treated with Humulin® at 6 weeks of age) compared to the control group. Figure 1B shows the results for treatment group 3 (i.e., mice treated with Humulin® at 10 weeks of age) compared to the control group. Table 3 provides statistics related to the survival curves shown in Figures 1A and 1B.

[0060] (Table 3) Comparison of survival curves TIFF0007879594000003.tif106137

[0061] These results demonstrate that sublingual formulations of insulin-related peptides of this technology, such as Humulin®, are useful in a method of treating type 1 diabetes, in which the treatment includes delaying the onset of hyperglycemia or reducing the likelihood of developing type 1 diabetes in those who require it.

[0062] Example 2: Use of insulin-related peptides in maintaining serum C-peptide levels This embodiment demonstrates the use of a sublingual formulation of the insulin-related peptide of this technology in a method for maintaining serum C-peptide levels in a mouse model of type 1 diabetes. C-peptide is a component of proinsulin that links α-insulin and β-insulin chains, and after cleavage and removal, it is co-secreted with insulin by pancreatic β-cells. The 31-amino acid C-peptide is produced in equimolar amounts with endogenous insulin and is widely used as a measure of insulin secretion (or pancreatic β-cell function) rather than being a product of therapeutically administered exogenous insulin.

[0063] method Five-week-old female NOD mice were randomly assigned to one of the three groups described in Example 1: (1) control group (oil only); (2) insulin-related peptide treatment (Humulin®) initiated at six weeks of age; or (3) insulin-related peptide treatment (Humulin®) initiated at ten weeks of age. Mice in treatment groups (2) and (3) were administered 87 μg of Humulin® sublingually once daily until 30 weeks of age. Baseline C peptide was measured from serum collected at six weeks of age. Subsequently, C peptide was measured from serum collected every two weeks, starting at 20 weeks of age and continuing until 30 weeks of age.

[0064] result NOD mice treated with insulin-related peptides such as Humulin® or its variants having one or more conservative amino acid substitutions are predicted to maintain or enhance serum C-peptide levels compared to untreated control NOD mice that develop clinical symptoms of type 1 diabetes. These results suggest that a sublingual formulation of the insulin-related peptide of this technology is useful in the treatment of type 1 diabetes in subjects with biological markers or a history of predisposition to developing type 1 diabetes.

[0065] Example 3: Use of insulin-related peptides to attenuate antigenic reactions This embodiment demonstrates the use of a sublingual formulation of the insulin-related peptide of this technology in a method for attenuating antigenic reactions in subjects at risk of or diagnosed with type 1 diabetes. The development of type 1 diabetes is preceded and accompanied by the appearance of numerous autoantibodies against various islet cell antigens. In genetically predisposed but disease-free individuals (e.g., first-degree relatives of type 1 diabetes patients), the detection of multiple islet cell autoantibodies is a strong predictor of subsequent development of type 1 diabetes. These autoantibodies include, but are not limited to, islet cell antibodies (ICA, against β-cell cytoplasmic proteins), antibodies against glutamate decarboxylase (GAD-65), insulin autoantibodies (IAA), and autoantibodies against tyrosine phosphatases IA-2A and IA-2β, as well as ZnT8.

[0066] method Five-week-old female NOD mice were randomly assigned to one of the three groups described in Example 1: (1) control group (oil only); (2) insulin-related peptide treatment (Humulin®) initiated at six weeks of age; or (3) insulin-related peptide treatment (Humulin®) initiated at ten weeks of age. Mice in treatment groups (2) and (3) were administered 87 μg of humulin sublingually once daily until 30 weeks of age. At six weeks, the subjects were evaluated for baseline levels of autoantibodies. Thereafter, autoantibody titers were measured once every week, starting at 15 weeks of age and continuing until 30 weeks of age.

[0067] result NOD mice treated with insulin-related peptides such as Humulin® or its variants having one or more conservative amino acid substitutions are predicted to show attenuated antigenic responses (e.g., pancreatic islet cell autoantibody levels) compared to untreated controls. These results suggest that sublingual formulations of the insulin-related peptides of this technology, such as Humulin® or its variants having one or more conservative amino acid substitutions, are useful in the treatment of type 1 diabetes in patients who require them.

[0068] Example 4: Use of insulin-related peptides to delay the onset of hyperglycemia in humans This embodiment demonstrates the use of a sublingual formulation of the insulin-related peptide of this technology in a method for treating type 1 diabetes in disease-free individuals who are predisposed to developing type 1 diabetes (for example, first-degree relatives of type 1 diabetes patients who have been determined to be genetically predisposed to developing type 1 diabetes).

[0069] method Subjects identified as being at risk of developing type 1 diabetes receive daily sublingual administration of the insulin-related peptide of this technology. The dosage ranges from 0.1 mg / kg to 50 mg / kg. Subjects are evaluated weekly for the presence and / or severity of signs and symptoms associated with type 1 diabetes, including but not limited to hyperglycemia, hypoinsulinemia, serum C-peptide levels, A1C levels, or the presence of autoantibodies. Treatment may be continued indefinitely or until one or more signs or symptoms of type 1 diabetes develop.

[0070] result Subjects susceptible to developing type 1 diabetes who receive a therapeutically effective sublingual dose of the insulin-related peptide of this technology are expected to show a delay and / or reduction in the severity of signs or symptoms associated with the development of type 1 diabetes, or their elimination. These results indicate that sublingual formulations of the insulin-related peptide of this technology, such as Humulin® or its bioactive fragment, or any variant thereof having one or more amino acid substitutions, are useful in the treatment of type 1 diabetes in subjects in need, and in particular in delaying the onset of hyperglycemia and / or reducing the likelihood of developing type 1 diabetes in such subjects.

[0071] Equal portions The Art should not be limited in terms of the specific embodiments described herein, which are intended merely as examples of individual aspects of the Art. As will be obvious to those skilled in the art, many modifications and variations of the Art can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and apparatus within the scope of the Art will be obvious to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the appended claims. The Art should be limited only by the conditions of the appended claims, along with the entire scope of equivalents to which such claims are granted. The Art is not limited to specific methods, reagents, compounds, compositions, or biological systems, which are naturally subject to change. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit them.

[0072] In addition, if any feature or aspect of the disclosure is described in terms of the Markush group, a person skilled in the art will recognize that the disclosure is also described in terms of any individual member or subgroup of any member of that Markush group.

[0073] As will be understood by those skilled in the art, for all purposes, and especially in terms of providing written explanations, all scopes disclosed herein also encompass all possible sub-scopes and combinations of sub-scopes. Any enumerated scope can be readily described and made possible to fully explain that the same scope may be divided into at least half, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-restrictive example, each scope discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. Similarly, as will be understood by those skilled in the art, all language such as “up to,” “at least,” “greater than,” “less than,” and similar terms refers to a scope that includes the enumerated numbers and may subsequently be divided into such sub-scopes as described above. Finally, as will be understood by those skilled in the art, a scope includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, the group having 1 to 5 cells refers to the group having 1, 2, 3, 4, or 5 cells, and so on.

[0074] The foregoing explanation is provided to aid the reader's understanding. Neither the information provided nor the cited references are considered prior art to the compositions and methods disclosed herein.

[0075] All patents, patent applications, provisional patent applications, and publications referenced or cited herein, including all drawings and tables, are incorporated by reference in their entirety to the extent that they do not contradict the express teachings herein.

[0076] Other embodiments are described in the following claims.

Claims

1. A composition for delaying the onset of type 1 diabetes or reducing the likelihood of type 1 diabetes in a subject that requires it, wherein the composition comprises a therapeutically effective amount of insulin-related peptide and is intended for sublingual administration to the subject.

2. The composition according to claim 1, wherein the composition is administered once a day for at least 5 days a week for at least 7 weeks.

3. The composition according to claim 1, wherein the composition is administered at least once daily for at least seven weeks.

4. The composition according to claim 1, wherein the insulin-related peptide comprises a first amino acid sequence comprising the insulin β-chain 7-26 peptide sequence (SEQ ID NO: 9) or a variant thereof having one or more conservative amino acid substitutions; and a second amino acid sequence comprising the insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof having one or more conservative amino acid substitutions.

5. The composition according to claim 4, wherein the first amino acid sequence corresponds to the insulin β-chain amino acid sequence; and / or the second amino acid sequence corresponds to the insulin α-chain amino acid sequence.

6. (a) The insulin-related peptide comprises one or more of insulin, proinsulin, and preproinsulin, or (b) The insulin-related peptide contains human insulin, or (c) The insulin-related peptide comprises human proinsulin and / or human preproinsulin, The composition according to any one of claims 1 to 5.

7. (a) The first amino acid sequence includes the human insulin β-chain 6-26 peptide sequence (SEQ ID NO: 3), or (b) The first amino acid sequence corresponds to the human insulin β-chain amino acid sequence (SEQ ID NO: 1), or (c) The second amino acid sequence contains the human insulin α chain 6-20 peptide sequence (SEQ ID NO: 4), or (d) The second amino acid sequence corresponds to the human insulin α-chain amino acid sequence (SEQ ID NO: 2), or (e) The second amino acid sequence includes a variant of the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) which includes a substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly, or (f) The second amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) which includes a substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly, or (g) The second amino acid sequence contains a variant of the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) which includes a substitution of α-chain 9-Ser by Thr, Ala, or Gly, or (h) The second amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) which includes a substitution of α-chain 9-Ser by Thr, Ala, or Gly, or (i) The second amino acid sequence includes a variant of the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) which includes a substitution of α-chain 10-Ile by Val, Ala, or Gly, or (j) The second amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) which includes a substitution of α-chain 10-Ile by Val, Ala, or Gly, or (k) The second amino acid sequence corresponds to a variant of the human insulin α-chain amino acid sequence (SEQ ID NO: 2) which includes a substitution of α-chain 21-Asn by Ala or Gly, or (l) The second amino acid sequence contains a variant of the human insulin α chain 6-20 peptide sequence (SEQ ID NO: 4), and the variant is Substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly; Substitution of α-chain 9-Ser by Thr, Ala, or Gly; and Substitution of α-chain 10-Ile by Leu, Val, Ala, or Gly Includes one or more more selected amino acid substitutions, or (m) The second amino acid sequence corresponds to a variant of the human insulin α chain peptide sequence 1-20 (SEQ ID NO: 5), and the variant is Substitution of α-chain 4-Glu by Asp; Substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly; Substitution of α-chain 9-Ser by Thr, Ala, or Gly; Substitution of α-chain 10-Ile by Leu, Val, Ala, or Gly; and Substitution of α-chain 18-Asn by His Includes one or more more selected amino acid substitutions, or (n) The first amino acid sequence corresponds to the human insulin β-chain amino acid sequence (SEQ ID NO: 1); and the second amino acid sequence corresponds to the human insulin α-chain amino acid sequence (SEQ ID NO: 2), or (o) The second amino acid sequence contains a variant of the human insulin α-chain 4-20 peptide sequence (SEQ ID NO: 6) which includes an α-chain 4-Glu substitution by Asp, or (p) The insulin-related peptide comprises a first amino acid sequence containing the human insulin β-chain 3-26 peptide sequence (SEQ ID NO: 7) or a variant thereof; and a second amino acid sequence containing the human insulin α-chain 6-20 peptide sequence (SEQ ID NO: 4) or a variant thereof; The variant of the human insulin β-chain peptide sequence 3-26 (SEQ ID NO: 7) has a substitution of β-chain 3-Asn by Lys and / or a substitution of β-chain 9-Ser by Pro, and the variant of the human insulin α-chain peptide sequence 6-20 (SEQ ID NO: 4) is Substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly; Substitution of α-chain 9-Ser by Thr, Ala, or Gly; Substitution of α-chain 10-Ile by Leu, Val, Ala, or Gly; and Substitution of α-chain 18-Asn by His Includes one or more more selected amino acid substitutions, or (q) The insulin-related peptide comprises a first amino acid sequence containing the human insulin β-chain peptide sequence 4-27 (SEQ ID NO: 8) or a variant thereof; and / or a second amino acid sequence containing the human insulin α-chain peptide sequence 1-20 or a variant thereof; The variant of the human insulin β-chain 4-27 peptide sequence (SEQ ID NO: 8) has a substitution of β-chain 9-Ser by Pro; and The variants of the human insulin α chain 1-20 peptide sequences are Substitution of α-chain 4-Glu by Asp; Substitution of α-chain 8-Thr by Ser, Val, Ala, or Gly; Substitution of α-chain 9-Ser by Thr, Ala, or Gly; Substitution of α-chain 10-Ile by Leu, Val, Ala, or Gly; and Substitution of α-chain 18-Asn by His Having one or more more selected amino acid substitutions, or (r) The insulin-related peptide comprises a first amino acid sequence corresponding to the human insulin β-chain amino acid sequence (SEQ ID NO: 1) or a variant thereof; and / or a second amino acid sequence corresponding to the human insulin α-chain amino acid sequence (SEQ ID NO: 2) or a variant thereof; The variant of the human insulin β-chain amino acid sequence (SEQ ID NO: 1) is Lys substitution of β-chain 3-Asn; Substitution of β-chain 9-Ser by Pro; Substitution of β-chain 29-Lys by Met; and Substitution of β-chain 30-Thr with Ser or Ala Having one or more more selected amino acid substitutions; and The variant of the human insulin α chain amino acid sequence is Substitution of α-chain 4-Glu by Asp; Substitution of α-chain 8-Thr by Ala; Substitution of α-chain 9-Ser by Gly; Substitution of α-chain 10-Ile by Val; Substitution of α-chain 18-Asn by His; and Substitution of α-chain 21-Asn by Gly Having one or more more selected amino acid substitutions, The composition according to claim 4.

8. The composition according to any one of claims 1 to 7, wherein the insulin-related peptide is a recombinant human insulin-related peptide.

9. The composition according to any one of claims 1 to 8, wherein the therapeutically effective amount of insulin-related peptide is formulated as a composition further comprising an aqueous, pharmaceutically acceptable carrier containing at least about 30 vol.% glycerin.

10. The composition according to claim 9, wherein the aqueous pharmaceutically acceptable carrier further comprises a buffer.

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