Insulin analogues for the treatment of metabolic disorders and diseases in humans

Abstract

The disclosure described herein provides compositions and uses of insulin analogs for the stable and effective treatment of metabolic diseases or disorders in a subject. In some embodiments, the insulin analog is a derivatized native insulin peptide (comprising an A chain and a B chain) that includes a difference of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from native insulin. In typical embodiments, the insulin analogs described herein include a substitution at the N-terminus, the C-terminus, or both, comprising a hydrophobic moiety. The insulin analogs described herein exhibit potent receptor binding and extended plasma half-life. Pharmaceutical compositions of the insulin analogs described herein are described for the treatment of metabolic diseases or disorders, such as diabetes, in a subject.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit from international application PCT / CN2023 / 131773, filed on November 15, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic sequence list (AMBO_002_001WO_SeqList_ST26.xml; size: 25,113 bytes; and creation date: November 5, 2024) are incorporated herein by reference in their entirety. Background Technology

[0005] Insulin is an important peptide hormone associated with a range of diseases and disorders. It demonstrates an unparalleled ability to lower glucose in almost all forms of diabetes. Insulin is a hormone secreted by the pancreas that regulates blood glucose levels and plays a role in maintaining normal blood glucose levels. However, in people with diabetes, insulin cannot function properly due to a lack of insulin, insulin resistance, and loss of beta cell function. Consequently, blood glucose cannot be used as an energy source, leading to elevated blood glucose levels and hyperglycemia. Therefore, insulin therapy is crucial for patients with abnormal insulin secretion (type I) or insulin resistance (type II), and insulin administration is essential for regulating blood glucose levels. However, like other proteins and peptide hormones, insulin has a very short half-life in the body, thus requiring repeated administration.

[0006] Furthermore, insulin therapy can produce an overreaction, which can lead to life-threatening hypoglycemia. Hypoglycemia is a common complication of insulin replacement therapy in diabetic patients and can cause serious morbidity, including altered mental status, loss of consciousness, seizures, and death. In fact, the fear of such complications constitutes a major obstacle to strict control of blood glucose levels, and for patients with long-term type 2 diabetes, these efforts (“strict control”) may lead to increased mortality.

[0007] Therefore, there is an urgent need for new diabetes treatment technologies that can reduce the risk of hypoglycemia while preventing blood glucose levels from rising above the normal range, and at the same time exhibit an ideal half-life to limit the overall injection frequency for patients. Summary of the Invention

[0008] This document describes compositions and uses of insulin analogs for the stable and effective treatment of metabolic diseases or disorders in subjects. In some embodiments, the insulin analog is a derived natural insulin peptide (comprising an A chain and a B chain) comprising differences of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from natural insulin. In typical embodiments, the insulin analogs described herein comprise substitutions of hydrophobic portions at the N-terminus, C-terminus, or both ends. The insulin analogs described herein exhibit potent insulin receptor binding and prolonged plasma half-life. This document describes pharmaceutical compositions of insulin analogs for the treatment of metabolic diseases or disorders, such as diabetes, in subjects. In some embodiments, the insulin analog is administered to human subjects once weekly. Attached Figure Description

[0009] Figures 1A to 1M The structure of the insulin analogue of this disclosure is shown. Figure 1A The structure of insulin analog 1 is shown. Figure 1B The structure of insulin analog 2 is shown. Figure 1C The structure of insulin analog 3 is shown. Figure 1D The structure of insulin analog 4 is shown. Figure 1E The structure of insulin degludec insulin, an insulin analogue, is shown. Figure 1F The structure of insulin analog APi3543 is shown. Figure 1G The structure of insulin analog APi3544 is shown. Figure 1H The structure of insulin analog APi3545 is shown. Figure 1I The structure of insulin analog APi3546 is shown. Figure 1J The structure of the insulin analog APi3547 is shown. Figure 1K The structure of the insulin analog APi3548 is shown. Figure 1L The structure of insulin analog APi3549 is shown. Figure 1M The structure of the insulin analog APi3550 is shown.

[0010] Figure 2 Glucose curves are shown for subcutaneous administration of different doses of insulin analog #1 in a diabetic mouse model.

[0011] Figures 3A to 3B The safety profile of diabetic mice treated with a single subcutaneous injection of different doses of insulin analog #1 is demonstrated, as evidenced by mean body weight (monitored over a three-day period). Figure 3A The security situation is shown in a line graph. Figure 3B The safety status is shown in a bar chart.

[0012] Figure 4The calculated IU / mg (biological potency) for eight insulin analogs is shown.

[0013] Figure 5 The blood glucose levels in ICR mice after administration are shown.

[0014] Figure 6 We summarized the calculated and experimentally measured biopotency of insulin analogs relative to insulin reference standards in ICR mice.

[0015] Figure 7 The biopotency (IU / mg) of insulin analogs as measured relative to an insulin reference standard is shown in ICR mice.

[0016] Figures 8A to 8C The glucose-lowering effects of insulin analogs 2, 3, and 4 compared to insulin degludec were shown. Figure 8A The results show the glucose levels (nmol / L) of rats injected with insulin analogs over a 120-hour period compared to rats injected with degludec insulin. Figure 8B Normalized glucose levels (%) were shown in rats injected with insulin analogs over a 120-hour period compared to rats injected with degludec insulin. Figure 8C The glucose levels δ (nmol / L) of rats injected with insulin analogs over a 120-hour period are shown compared with those injected with degludec insulin.

[0017] Figure 9 The area under the glucose curve (AUC) values ​​from 0 to 96 hours are shown for rats injected with insulin analogs 2, 3, and 4 compared to rats injected with degludec insulin.

[0018] Figure 10 Normalized glucose levels (%) are shown in rats injected with insulin analogs 2, 3, and 4 over a 120-hour period compared with those injected with insulin degludec and insulin icotin.

[0019] Figures 11A to 11C The effects of insulin analogs 2, 3, and 4 on C-peptide levels are shown. Figure 11A The c-peptide levels (ng / mL) of rats injected with insulin analogs over a 120-hour period are shown compared with those injected with degludec insulin. Figure 11B Normalized C-peptide levels (%) were shown in rats injected with insulin analogs over a 120-hour period compared to rats injected with degludec insulin. Figure 11C Normalized C-peptide levels (%) were shown in rats injected with insulin analogs over 120 hours compared with those injected with degludec insulin and ico insulin.

[0020] Figure 12The HbA1C levels of rats injected with insulin analogs 2, 3, and 4 are shown compared with those injected with insulin degludec at 96 hours post-injection.

[0021] Figure 13 Normalized body weight (%) of rats injected with insulin analogs 2, 3, and 4 over a 120-hour period is shown compared to rats injected with degludec insulin.

[0022] Figures 14A to 14C The glucose-lowering effect of insulin analogs compared to insulin degludec was demonstrated. Figure 14A The glucose levels (nmol / L) of rats injected with insulin analogs 1, 2, 3, 4, APi3545, APi3546, APi3547, and APi3549 over a 120-hour period are shown compared with those injected with insulin degludec. Figure 14B Normalized glucose levels (%) were shown in rats injected with insulin analogs 1, 2, 3, 4, APi3545, APi3546, APi3547, and APi3549 over a 120-hour period compared with those injected with insulin degludec. Figure 14C Normalized glucose levels (%) in rats injected with insulin analogs 1, 2, 3, and 4 over a 120-hour period compared to rats injected with insulin degludec.

[0023] Figure 15 The area under the glucose curve (AUC) values ​​from 0 to 72 hours are shown for rats injected with insulin analogs 2, 3, 4, APi3545, APi3546, APi3547, and APi3549 compared with rats injected with degludec insulin.

[0024] Figure 16 Normalized body weight (%) of rats injected with insulin analogs 2, 3, 4, APi3545, APi3546, APi3547, and APi3549 over a 120-hour period is shown compared with that of rats injected with degludec insulin.

[0025] Figures 17A to 17B The effect of insulin analogs on C-peptide levels compared to degludec insulin is shown. Figure 17A Normalized C-peptide levels (%) are shown in rats injected with insulin analogs 2, 3, 4, APi3545, APi3546, APi3547, and APi3549 over a 120-hour period compared to rats injected with degludec insulin. Figure 17B Normalized C-peptide levels (%) were shown in rats injected with insulin analogs 2, 3, and 4 over a 120-hour period compared to rats injected with degludec insulin.

[0026] Figure 18The HbA1C levels of rats injected with insulin analogs 2, 3, 4, APi3545, APi3546, APi3547, and APi3549 are shown compared with those of rats injected with insulin degludec at 96 hours post-injection.

[0027] Figure 19 The study showed that insulin analogs 1, 2, 3, and 4 lacked IGF-1R activation compared to IGF-1, insulin, and degludec insulin. Detailed Implementation

[0028] A simple system is used to describe fragments and analogues of insulin. Specifically, insulin consists of a peptide A chain and a peptide B chain. Accordingly, A1 corresponds to the first N-terminal amino acid in the A chain, numbered sequentially from the N-terminus to the C-terminus. For example, the A chain containing A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 is understood as containing amino acid A1 at the N-terminus of the peptide chain and amino acid A21 at the C-terminus of the peptide chain. Similarly, B1 corresponds to the first N-terminal amino acid in the B chain, numbered sequentially from the N-terminus to the C-terminus. For example, a B chain containing B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 is understood to contain amino acid B1 located at the N-terminus of the peptide chain and amino acid B29 located at the C-terminus of the peptide chain.

[0029] The term "analyte" is defined herein as a peptide in which one or more amino acid residues of a parental (or wild-type) insulin peptide (comprising A and B chains) are replaced by additional amino acid residues. In some embodiments, the replaced amino acid is a non-natural amino acid. In some embodiments, the replaced amino acid has a D-configuration.

[0030] In some embodiments, the insulin analog comprises at least one γ-carboxy-L-glutamic acid (Gla). In some embodiments, the insulin analog comprises at least one N-3-methyl-L-histidine (His(3-Me)). In some embodiments, the insulin analog comprises at least one 3-(3-pyridyl)-L-alanine (Pal). In some embodiments, the insulin analog comprises at least one trans-4-hydroxy-L-proline (Hyp). In some embodiments, the insulin analog comprises at least one (s)-piperidine-2-carboxylic acid (Pip). In some embodiments, the insulin analog comprises at least one 2-furanyl-L-alanine (2Fal). In some embodiments, the insulin analog comprises at least one L-α-aminoadipic acid (Aad). In some embodiments, the insulin analog comprises at least one 5-iodo-L-histidine (His(5-I)). In some embodiments, the insulin analog comprises at least one 2-thienyl-L-alanine (Thi). In some embodiments, the insulin analog comprises at least one 4-phosphono-L-phenylalanine (Ppa). In some embodiments, the insulin analog comprises at least one 4-thiazolyl-L-alanine (Ala(4-thiazolyl)). In some embodiments, the insulin analog comprises at least one 3-pyridyl-L-alanine (3-Pal).

[0031] In some embodiments, the analogs described herein contain a non-natural or unnatural amino acid mutation at any of positions A14, B16, and B25. In some embodiments, the insulin analog also contains one or more fatty acid substituents, such as one or more C20 (20-carbon) diacids. In some embodiments, the incorporation of a non-natural amino acid mutation at positions A14, B16, and B25 reduces the enzymatic degradation of pepsin, chymotrypsin, or carboxypeptidase A analogs, and increases their solubility and slows receptor-mediated clearance.

[0032] In a typical embodiment, the insulin analog is a peptide analog. In a typical embodiment, the insulin analog is chemically synthesized. In a typical embodiment, the insulin analog described herein is not produced by recombinant technology.

[0033] In some embodiments, the analogues described herein exhibit an extended half-life compared to wild-type insulin or first-generation insulin products (animal insulin), or second-generation insulin products (i.e., biosynthetic insulin, such as Humulin or Novolin) or third-generation insulin products (biosynthetically prepared insulin, such as Humalog or NovoRapid).

[0034] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 1); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Gla, or Aad, or Ppa, A15 is Gln, A16 is Leu, A17 is Glu, and A18 is As n, A19 is Tyr, A20 is Cys, A21 is Asn; and the B chain from the N-terminus to the C-terminus contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 2); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is His(3-Me), or Pal, or His(5-I), and B17 is L eu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is His(3-Me), or Pal, or 2Fal, or Thi, or Ala(4-thiazolyl), or 3-Pal, B26 is Tyr, B27 is Thr, B28 is Pro, or Hyp, or Pip, and B29 is Lys.

[0035] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 3); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Gla, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly ... Gly, A11 is Ile, A12 is Cys, A13 is Leu, A14 is Gla, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly, A10 is Gly, A11 is Gly, A12 is Gly, A13 is Gly, A14 is Gly, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly, A19 is Gly, A10 is Gly, A11 is Gly, A12 is Gly, A13 is Gly, A14 is Gly, A15 is Gly, A16 is Gly, A1 It is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N end to the C end contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 4); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is His(3-Me), B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is His(3-Me), B26 is Tyr, B27 is Thr, B28 is Pro, and B29 is Lys.

[0036] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 3); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Gla, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly ... Gly, A11 is Ile, A12 is Cys, A13 is Leu, A14 is Gla, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly, A10 is Gly, A11 is Gly, A12 is Gly, A13 is Gly, A14 is Gly, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly, A19 is Gly, A10 is Gly, A11 is Gly, A12 is Gly, A13 is Gly, A14 is Gly, A15 is Gly, A16 is Gly, A1 It is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N end to the C end contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 6); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is Pal, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is Pal, B26 is Tyr, B27 is Thr, B28 is Pro, and B29 is Lys.

[0037] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 3); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Gla, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly ... Gly, A11 is Ile, A12 is Cys, A13 is Leu, A14 is Gla, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly, A10 is Gly, A11 is Gly, A12 is Gly, A13 is Gly, A14 is Gly, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly, A19 is Gly, A10 is Gly, A11 is Gly, A12 is Gly, A13 is Gly, A14 is Gly, A15 is Gly, A16 is Gly, A1 It is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N end to the C end contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 8); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is Pal, B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is Pal, B26 is Tyr, B27 is Thr, B28 is Hyp, and B29 is Lys.

[0038] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 3); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Gla, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly ... Gly, A11 is Ile, A12 is Cys, A13 is Leu, A14 is Gla, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly, A10 is Gly, A11 is Gly, A12 is Gly, A13 is Gly, A14 is Gly, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A19 is Gly, A19 is Gly, A10 is Gly, A11 is Gly, A12 is Gly, A13 is Gly, A14 is Gly, A15 is Gly, A16 is Gly, A1 It is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N end to the C end contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 10); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is Pal, B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is Pal, B26 is Tyr, B27 is Thr, B28 is Pip, and B29 is Lys.

[0039] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 11); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Aad, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A1 9 is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N-terminus to the C-terminus contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 12); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is His(5-I), B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is 2Fal, B26 is Tyr, B27 is Thr, B28 is Pro, and B29 is Lys.

[0040] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 13); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Ppa, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A1 9 is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N-terminus to the C-terminus contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 14); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is His(5-I), B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is Thi, B26 is Tyr, B27 is Thr, B28 is Pro, and B29 is Lys.

[0041] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 11); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Aad, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A1 9 is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N-terminus to the C-terminus contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 16); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is His(5-I), B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is Ala(4-thiazolyl), B26 is Tyr, B27 is Thr, B28 is Pro, and B29 is Lys.

[0042] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 11); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Aad, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A1 9 is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N-terminus to the C-terminus contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 18); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is His(5-I), B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is 3-Pal, B26 is Tyr, B27 is Thr, B28 is Pro, and B29 is Lys.

[0043] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 11); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Aad, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A1 9 is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N-terminus to the C-terminus contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 12); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is His(5-I), B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is 2Fal, B26 is Tyr, B27 is Thr, B28 is Pro, and B29 is Lys.

[0044] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 13); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Ppa, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A1 9 is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N-terminus to the C-terminus contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 14); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is His(5-I), B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is Thi, B26 is Tyr, B27 is Thr, B28 is Pro, and B29 is Lys.

[0045] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 11); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Aad, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A1 9 is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N-terminus to the C-terminus contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 16); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is His(5-I), B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is Ala(4-thiazolyl), B26 is Tyr, B27 is Thr, B28 is Pro, and B29 is Lys.

[0046] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21 (SEQ ID NO: 11); wherein A1 is Gly, A2 is Ile, A3 is Val, A4 is Glu, A5 is Gln, A6 is Cys, A7 is Cys, A8 is Thr, A9 is Ser, A10 is Ile, A11 is Cys, A12 is Ser, A13 is Leu, A14 is Aad, A15 is Gln, A16 is Leu, A17 is Glu, A18 is Asn, A1 9 is Tyr, A20 is Cys, and A21 is Asn; and the B chain from the N-terminus to the C-terminus contains B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29 (SEQ ID NO: 18); where B1 is Phe, B2 is Val, B3 is Asn, B4 is Gln, B5 is His, B6 is Leu, B7 is Cys, B8 is Gly, B9 is Ser, B10 is His, B11 is Leu, B12 is Val, B13 is Glu, B14 is Ala, B15 is Leu, B16 is His(5-I), B17 is Leu, B18 is Val, B19 is Cys, B20 is Gly, B21 is Glu, B22 is Arg, B23 is Gly, B24 is Phe, B25 is 3-Pal, B26 is Tyr, B27 is Thr, B28 is Pro, and B29 is Lys.

[0047] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the peptide A chain comprises SEQ ID NO:3 and the peptide B chain comprises SEQ ID NO:4. In some embodiments, the insulin analog comprising the peptide A chain and the peptide B chain is insulin analog 1, wherein the peptide A chain comprises SEQ ID NO:3 and the peptide B chain comprises SEQ ID NO:4.

[0048] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the peptide A chain comprises SEQ ID NO:3 and the peptide B chain comprises SEQ ID NO:6. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog 2, wherein the peptide A chain comprises SEQ ID NO:3 and the peptide B chain comprises SEQ ID NO:6.

[0049] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the peptide A chain comprises SEQ ID NO:3 and the peptide B chain comprises SEQ ID NO:8. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog 3, wherein the peptide A chain comprises SEQ ID NO:3 and the peptide B chain comprises SEQ ID NO:8.

[0050] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the peptide A chain comprises SEQ ID NO:3 and the peptide B chain comprises SEQ ID NO:10. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog 4, wherein the peptide A chain comprises SEQ ID NO:3 and the peptide B chain comprises SEQ ID NO:10.

[0051] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the peptide A chain comprises SEQ ID NO:11 and the peptide B chain comprises SEQ ID NO:12. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog APi3543, wherein the peptide A chain comprises SEQ ID NO:11 and the peptide B chain comprises SEQ ID NO:12. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog APi3547, wherein the peptide A chain comprises SEQ ID NO:11 and the peptide B chain comprises SEQ ID NO:12.

[0052] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the peptide A chain comprises SEQ ID NO:13 and the peptide B chain comprises SEQ ID NO:14. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog APi3544, wherein the peptide A chain comprises SEQ ID NO:13 and the peptide B chain comprises SEQ ID NO:14. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog APi3548, wherein the peptide A chain comprises SEQ ID NO:13 and the peptide B chain comprises SEQ ID NO:14.

[0053] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the peptide A chain comprises SEQ ID NO:11 and the peptide B chain comprises SEQ ID NO:16. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog APi3545, wherein the peptide A chain comprises SEQ ID NO:11 and the peptide B chain comprises SEQ ID NO:16. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog APi3549, wherein the peptide A chain comprises SEQ ID NO:11 and the peptide B chain comprises SEQ ID NO:16.

[0054] In some embodiments, the insulin analog comprises a peptide A chain and a peptide B chain, wherein the peptide A chain comprises SEQ ID NO:11 and the peptide B chain comprises SEQ ID NO:18. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog APi3546, wherein the peptide A chain comprises SEQ ID NO:11 and the peptide B chain comprises SEQ ID NO:18. In some embodiments, the insulin analog comprising a peptide A chain and a peptide B chain is insulin analog APi3550, wherein the peptide A chain comprises SEQ ID NO:11 and the peptide B chain comprises SEQ ID NO:18.

[0055] This disclosure also provides substituent-substituted insulin analogs. In some embodiments, the N-terminus of the B chain, or the C-terminus of the B chain, or both the N-terminus and C-terminus, is substituted. In some embodiments, the B chain contains a substituent linked to a lysine (Lys) residue in the B chain. In some embodiments, at least one lysine ε-amino group of the insulin analog is substituted with a lipophilic substituent. In some embodiments, the Lys ε-amino group of the insulin analog is substituted with a lipophilic substituent via a spacer group.

[0056] In some embodiments, the N-terminus of the B chain (i.e., via the N-terminal nitrogen group on the B peptide chain) or the C-terminus of the B chain (i.e., at the C-terminal lysine residue), or both the N-terminus and C-terminus, is covered by a C20 diacid or AEEA-AEEA-γ-Glu-(CH2). m -OH (AEEA = 2-[2-(2-aminoethoxy)ethoxy]acetic acid) is substituted, where m is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some embodiments, the N-terminus of the B chain, or the C-terminus of the B chain, or both the N-terminus and C-terminus, is replaced by AEEA-AEEA-γ-Glu-(CH2). m -OH (AEEA = 2-[2-(2-aminoethoxy)ethoxy]acetic acid) substitution, where m is 20.

[0057] In typical embodiments, the insulin analogs described herein comprise at least one linker. In some embodiments, chain A comprises an intramolecular linker. In some embodiments, chain A comprises an intermolecular linker. In some embodiments, chain B comprises an intramolecular linker. In some embodiments, chain B comprises an intermolecular linker. In some embodiments, A6 and A11 are linked via an intramolecular linker. In some embodiments, A7 and B7 are linked via an intermolecular linker. In some embodiments, A20 and B19 are linked via an intermolecular linker. In some embodiments, the linker is cleavable or non-cleavable. In some embodiments, the linker is cleavable or readily cleavable. In some embodiments, the linker is cleaved by pH-mediated cleavage. In some embodiments, the linker is cleaved by an enzymatic process. In some embodiments, the linker is cleaved by chemical reduction. In typical embodiments, the linker comprises at least a disulfide bond or a bridge.

[0058] In some embodiments, the insulin analogs of this disclosure exhibit binding affinity (ECG) to the insulin receptor or GLP-1 receptor in the nanomolar range. 50 In some embodiments, the binding affinity (ECG) of insulin analogs to insulin receptors or GLP-1 receptors is... 50 <1000 nM, <900 nM, <800 nM, <700 nM, <600 nM, <500 nM, <400 nM, <300 nM, <200 nM. In some embodiments, the binding affinity (ECG) of the insulin analog to the insulin receptor or GLP-1 receptor is... 50 The value is about 100 nM or less, for example, about 75 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM or less, or about 1 nM or less. In some or any of the embodiments, the insulin analogs of this disclosure exhibit EC50 in the picomol range. 50 In an exemplary embodiment, the binding affinity (ECG) of the insulin analog to the insulin receptor or GLP-1 receptor is [not specified]. 50 <1000 pM, <900 pM, <800 pM, <700 pM, <600 pM, <500 pM, <400 pM, <300 pM, <200 pM. In some embodiments, the binding affinity (ECG) of the insulin analog to the insulin receptor or GLP-1 receptor is... 50 The value is approximately 100 pM or less, for example, approximately 75 pM or less, approximately 50 pM or less, approximately 25 pM or less, approximately 10 pM or less, approximately 5 pM or less, or approximately 1 pM or less.

[0059] In some embodiments, the binding affinity (ECG) of insulin analogs to insulin receptors or GLP-1 receptors is [not specified]. 50 The binding affinity of insulin analogs to insulin receptors is less than 10 nM. In some embodiments, the binding affinity (ECG) of insulin analogs to insulin receptors is... 50 The binding affinity of the insulin analog to the insulin receptor is less than 9 nM, or 8 nM, or 7 nM, or 6 nM. In some embodiments, the binding affinity (ECG) of the insulin analog to the insulin receptor is... 50 The molecular weight is less than 5 nM. In some embodiments, the binding affinity (ECG) of the insulin analog to the insulin receptor is [missing value]. 50 The concentrations are below 4 nM, or 3.5 nM, or 3 nM, or 2.5 nM, or 2 nM, or 1.5 nM. In some embodiments, the binding affinity (ECG) of the insulin analog to the insulin receptor is [not specified]. 50 Less than 1 nM. In some embodiments, the binding affinity (ECG) of the insulin analog to the insulin receptor is [missing value]. 50 The binding affinity of the insulin analog to the insulin receptor is less than 0.9 nM, or 0.8 nM, or 0.7 nM, or 0.6 nM, or 0.5 nM, or 0.4 nM, or 0.3 nM, or 0.2 nM, or 0.1 nM. In some embodiments, the binding affinity (ECG) of the insulin analog to the insulin receptor is... 50 (Below 0.09 nM, or 0.08 nM, or 0.07 nM, or 0.06 nm, or 0.05 nM, or 0.04 nM, or 0.03 nM, or 0.02 nM, or 0.01 nM.)

[0060] This disclosure relates to insulin analogs that retain the high activity of natural insulin at the insulin receptor, wherein the insulin analogs exhibit 70%, 80%, 90%, 95%, 100% or higher activity at the insulin receptor relative to natural insulin.

[0061] In some embodiments, the insulin analogues of the present invention include activity at both the insulin receptor and the GLP-1 receptor. In some embodiments, the insulin analogues of the present invention include an extended duration of action at each of these receptors.

[0062] In some embodiments, the binding affinity of insulin analogs is measured by surface plasmon resonance or other similar mass-dependent spectroscopic methods. In some embodiments, the binding affinity of insulin analogs is measured by gel transfer assay. In some embodiments, the binding affinity of insulin analogs is measured by competition assay. In some embodiments, the binding affinity of insulin analogs is measured by ELISA assay.

[0063] In some embodiments, binding to insulin analogs is determined by imaging. In some embodiments, binding to insulin analogs is determined by fluorescence imaging. In some embodiments, binding to insulin analogs is determined by radioligand binding assays.

[0064] In some embodiments, the binding affinity of insulin analogs is measured using recombinant cell lines. In some embodiments, the binding affinity of insulin analogs is measured using mammalian recombinant cell lines. In some embodiments, the binding affinity of insulin analogs is measured using human recombinant cell lines. In some embodiments, the binding affinity of insulin analogs is measured using non-human recombinant cell lines. In some embodiments, the recombinant cell lines express metabolic or hormone receptors.

[0065] In some embodiments, the binding affinity (ECG) of insulin analogs to insulin receptors is [not specified]. 50 The activity of luciferase is determined by measuring luciferase activity using a recombinant cell reporter gene assay, system, or kit. In some embodiments, the binding affinity of the insulin analog to the insulin receptor (ECG) is also considered. 50 The activity of luciferase was determined by measuring luciferase activity using a recombinant cell reporter gene assay, in which luciferase expression is controlled by cAMP response elements (CREs) and in which recombinant cells constitutively express insulin receptors.

[0066] Compositions and Formulations

[0067] In some embodiments, insulin analogs exhibit a prolonged plasma elimination half-life of at least 5, 10, 20, 30, 40, 50, or 60 minutes. In some embodiments, insulin analogs exhibit a prolonged plasma elimination half-life of at least 1.5, 2.0, 2.5, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 hours. In some embodiments, insulin analogs exhibit a prolonged plasma elimination half-life of at least 10, 15, or 20 hours. In some embodiments, insulin analogs exhibit a prolonged plasma elimination half-life of at least 24 hours. In some embodiments, insulin analogs exhibit a prolonged plasma elimination half-life of at least one week. In some embodiments, insulin analogs exhibit a prolonged plasma elimination half-life of at least 24 hours. In some embodiments, the insulin analog exhibits a prolonged plasma elimination half-life of at least 168 hours. In some embodiments, the insulin analog exhibits a prolonged plasma elimination half-life of at least 24 hours. In some embodiments, the insulin analog exhibits a prolonged plasma elimination half-life of at least one week.

[0068] In some embodiments, the compositions of the present invention may include liquid, solid, or semi-solid forms. Non-limiting examples of composition forms include liquid solutions, injectable solutions, infusionable solutions, dispersions, suspensions, tablets, pills, powders, liposomes, suppositories, gels, or hydrogels.

[0069] In some embodiments, insulin analogs of a given concentration remain stable after being stored at a given temperature for days, weeks, months, or years. In some embodiments, insulin analogs remain stable at a certain temperature for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, or 5 years, wherein the temperature includes… Including, but not limited to, at least -200℃, -100℃, -80℃, -70℃, -60℃, -50℃, -40℃, -30℃, -20℃, -10℃, -5℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, or 25℃.

[0070] In some embodiments, the insulin analogs of the present invention include about 0.01 mg / kg to 0.05 mg / kg, 0.05 mg / kg to 0.10 mg / kg, 0.10 mg / kg to 0.15 mg / kg, 0.15 mg / kg to 0.20 mg / kg, 0.20 mg / kg to 0.25 mg / kg, 0.25 mg / kg to 0.30 mg / kg, 0.30 mg / kg to 0.35 mg / kg, 0.35 mg / kg to 0.40 mg / kg, 0.40 mg / kg to 0.45 mg / kg, 0.45 mg / kg to 0.50 mg / kg, 0.50 mg / kg to 0.55 mg / kg, 0.55 mg / kg to 0.60 mg / kg, 0.60 mg / kg to 0.65 mg / kg, 0.65 mg / kg to 0.70 mg / kg, and 0.70 mg / kg to 0.75 mg / kg. Concentrations of 0.75 mg / kg to 0.80 mg / kg, 0.80 mg / kg to 0.85 mg / kg, 0.85 mg / kg to 0.90 mg / kg, 0.90 mg / kg to 0.95 mg / kg, and 0.95 mg / kg to 1.00 mg / kg.

[0071] In some embodiments, the insulin analogs of the present invention include about 50 nmol / kg to 75 nmol / kg, 75 nmol / kg to 100 nmol / kg, 100 nmol / kg to 125 nmol / kg, 125 nmol / kg to 150 nmol / kg, 150 nmol / kg to 175 nmol / kg, 175 nmol / kg to 200 nmol / kg, 200 nmol / kg to 225 nmol / kg, 225 nmol / kg to 250 nmol / kg, 250 nmol / kg to 275 nmol / kg, 275 nmol / kg to 300 nmol / kg, 300 nmol / kg to 325 nmol / kg, 325 nmol / kg to 350 nmol / kg, 350 nmol / kg to 375 nmol / kg, 375 nmol / kg to 400 nmol / kg, and 400 nmol / kg to 425 nmol / kg. nmol / kg, 425 nmol / kg to 450 nmol / kg, 450 nmol / kg to 475 nmol / kg, 475 nmol / kg to 500 nmol / kg, 500 nmol / kg to 525 nmol / kg, 525 nmol / kg to 550 nmol / kg, 550 nmol / kg to 575 nmol / kg, 575 nmol / kg to 600 nmol / kg, 600 nmol / kg to 625 nmol / kg, 625 nmol / kg to 650 nmol / kg, 650 nmol / kg to 675 nmol / kg, 675 nmol / kg to 700 nmol / kg, 700 nmol / kg to 725 nmol / kg, 725 nmol / kg to 750 nmol / kg, 750 nmol / kg to 775 nmol / kg, 775 nmol / kg to 800 nmol / kg nmol / kg, 800 nmol / kg to 825 nmol / kg, 825 nmol / kg to 850 nmol / kg, 850 nmol / kg to 875 nmol / kg, 875 nmol / kg to 900 nmol / kg, 900 nmol / kg to 925 nmol / kg, 925 nmol / kg to 950 nmol / kg, 950 nmol / kg to 975 nmol / kg, 975 nmol / kg to 1000 nmol / kg, 1000 nmol / kg to 1025 nmol / kg, 1025 nmol / kg to 1050 nmol / kg, 1050 nmol / kg to 1075 nmol / kg, 1075nmol / kg to 1100 nmol / kg, 1100 nmol / kg to 1125 nmol / kg, 1125 nmol / kg to 1150 nmol / kg, 1150 nmol / kg to 1175 nmol / kg, 1175 nmol / kg to 1200 nmol / kg, 1200 nmol / kg to 1225 nmol / kg, 1225 nmol / kg to 1250 nmol / kg, 1250 nmol / kg to 1275 nmol / kg, 1275 nmol / kg to 1300 nmol / kg, 1300 nmol / kg to 1325 nmol / kg, 1325 nmol / kg to 1350 nmol / kg, 1350 nmol / kg to 1375 nmol / kg, 1375 nmol / kg to 1400 nmol / kg, 1400 nmol / kg to 1425 nmol / kg nmol / kg, 1425 nmol / kg to 1450 nmol / kg, 1450 nmol / kg to 1475 nmol / kg, 1475 nmol / kg to 1500 nmol / kg, 1500 nmol / kg to 1525 nmol / kg, 1525 nmol / kg to 1550 nmol / kg, 1550 nmol / kg to 1575 nmol / kg, 1575 nmol / kg to 1600 nmol / kg, 1600 nmol / kg to 1625 nmol / kg, 1625 nmol / kg to 1650 nmol / kg, 1650 nmol / kg to 1675 nmol / kg, 1675 nmol / kg to 1700 nmol / kg, 1700 nmol / kg to 1725 nmol / kg, 1725 nmol / kg to 1750 nmol / kg, 1750 Concentrations ranging from nmol / kg to 1775 nmol / kg, 1775 nmol / kg to 1800 nmol / kg, 1800 nmol / kg to 1825 nmol / kg, 1825 nmol / kg to 1850 nmol / kg, 1850 nmol / kg to 1875 nmol / kg, 1875 nmol / kg to 1900 nmol / kg, 1900 nmol / kg to 1925 nmol / kg, and 1925 nmol / kg to 2000 nmol / kg.

[0072] In some embodiments, the insulin analogs of the present invention include about 1 IU / mg to 3 IU / mg, 3 IU / mg to 5 IU / mg, 5 IU / mg to 7 IU / mg, 7 IU / mg to 9 IU / mg, 9 IU / mg to 11 IU / mg, 11 IU / mg to 13 IU / mg, 13 IU / mg to 15 IU / mg, 15 IU / mg to 17 IU / mg, 17 IU / mg to 19 IU / mg, 19 IU / mg to 21 IU / mg, 21 IU / mg to 23 IU / mg, 23 IU / mg to 25 IU / mg, 25 IU / mg to 27 IU / mg, 27 IU / mg to 29 IU / mg, 29 IU / mg to 31 IU / mg, 31 IU / mg to 33 IU / mg, 33 IU / mg to 35 IU / mg, 35 IU / mg to 37 IU / mg. IU / mg, 37 IU / mg to 39 IU / mg, 39 IU / mg to 41 IU / mg, 41 IU / mg to 43 IU / mg, 43 IU / mg to 45 IU / mg, 45 IU / mg to 47 IU / mg, 47 IU / mg to 49 IU / mg, 49 IU / mg to 51 IU / mg, 51 IU / mg to 53 IU / mg, 53 IU / mg to 55 IU / mg, 55 IU / mg to 57 IU / mg, 57 IU / mg to 59 IU / mg, 59 IU / mg to 61 IU / mg, 61 IU / mg to 63 IU / mg, 63 IU / mg to 65 IU / mg, 65 IU / mg to 67 IU / mg, 67 IU / mg to 69 IU / mg, 69 IU / mg to 71 IU / mg, 71 Concentrations ranging from 73 IU / mg to 75 IU / mg, 75 IU / mg to 77 IU / mg, 77 IU / mg to 79 IU / mg, 79 IU / mg to 81 IU / mg, 81 IU / mg to 83 IU / mg, 83 IU / mg to 85 IU / mg, 85 IU / mg to 87 IU / mg, 87 IU / mg to 89 IU / mg, 89 IU / mg to 91 IU / mg, 91 IU / mg to 93 IU / mg, 93 IU / mg to 95 IU / mg, 95 IU / mg to 97 IU / mg, 97 IU / mg to 99 IU / mg, and 99 IU / mg to 100 IU / mg.

[0073] In some embodiments, the insulin analogues of the present invention comprise concentrations at human effective doses.

[0074] Pharmaceutical Composition

[0075] The disclosure herein also describes pharmaceutical compositions comprising insulin analogs. The compositions of the present invention are prepared by conventional processes involving the dissolution and mixing of ingredients to give a desired composition.

[0076] In typical embodiments, the pharmaceutical composition comprises an insulin analog and a pharmaceutically acceptable medium or carrier. In some embodiments, the pharmaceutically acceptable carrier is a microsphere, micelle, or nanoparticle. In some embodiments, the pharmaceutically acceptable carrier is a protein, such as a receptor or albumin.

[0077] In some embodiments, the pharmaceutical composition comprises any one of an isotonic agent, an excipient, a preservative, or a buffer. In some embodiments, the isotonic agent is one or more of propylene glycol, sodium chloride, potassium chloride, mannitol, sorbitol, trehalose, lactitol, xylitol, glycerol, sucrose, glycine, lactose, glucose, maltose, lysine, isoleucine, aspartic acid, L-glycine, L-histidine, arginine, inositol, or polyethylene glycol. In some embodiments, the preservative comprises any one of phenol, m-cresol, o-cresol, thimerosal, methylparaben, propylparaben, butylparaben, chlorobutanol, and phenoxyethanol. In some embodiments, the buffer comprises any one of dipotassium hydrogen phosphate, sodium bicarbonate, and disodium hydrogen phosphate. In some embodiments, the pharmaceutical composition comprises zinc acetate.

[0078] In some embodiments, the pharmaceutical composition comprises a surfactant. In some embodiments, the surfactant is at least one of the following: polysorbate, poloxamer, ethylene / polypropylene block polymer, lecithin, alcohol, sodium lauryl sulfate, bile acids and their salts, polymeric surfactant, long-chain fatty acids, phospholipids, ethoxylated castor oil, polyethylene glycol-modified glycerol, acetylated monoglycerides, dehydrated sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, monoglycerides, diglycerides, glycerol, glycerophospholipids, glyceryl polyesters, sphingomyelin, glycosphingolipids, sodium docusate, calcium docusate, and potassium docusate.

[0079] In some embodiments, the pharmaceutical composition comprises a chelating agent. Suitable examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) or its salts and mixtures thereof.

[0080] In typical embodiments, the pharmaceutical composition remains stable after multiple free-thaw cycles. In typical embodiments, the pharmaceutical composition remains stable after exposure to high temperatures. Stability can be measured by any means, including visually inspecting the composition against a dark background in sunlight for signs of turbidity, changes in color or transparency, or any other visible precipitation.

[0081] In some embodiments, the pH range of the pharmaceutical composition is from about 7.0 to about 11.0. In some embodiments, the pH is adjusted to about one of the following values: 7.0, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0.

[0082] In some embodiments, the pH of the pharmaceutical composition is adjusted using a pH adjuster selected from the group consisting of sodium hydroxide, potassium hydroxide, hydrochloric acid, and N-methylglucosamine.

[0083] In some embodiments, the pharmaceutical composition comprises an antidiabetic agent or an antiobesity agent.

[0084] How to use

[0085] The disclosure of this document also provides methods for treating human diseases or disorders using therapeutically effective amounts of insulin analogs. In a typical embodiment, the human disease or disorder is diabetes and / or obesity.

[0086] In a typical embodiment, a method of treating diabetes includes administering a therapeutically effective amount of an insulin analog to a patient.

[0087] In some embodiments, the treatment results in a decrease in HbA1C measured in the patient. In some embodiments, the clinician measures the patient's glucose levels before administering the insulin analogue.

[0088] In some embodiments, the treatment results in a reduction in food intake, weight loss, appetite suppression, or induction of satiety in the patient.

[0089] In some embodiments, administration of the insulin analogue of the present invention provides a more sustained glucose-lowering effect compared to administration of insulin degludec. In some embodiments, the glucose-lowering effect of the insulin analogue of the present invention lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0090] In some embodiments, administration of the insulin analog 1 of the present invention provides a more sustained glucose-lowering effect compared to administration of insulin degludec. In some embodiments, the glucose-lowering effect of the insulin analog 1 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0091] In some embodiments, administration of the insulin analog 2 of the present invention provides a more sustained glucose-lowering effect compared to administration of insulin degludec. In some embodiments, the glucose-lowering effect of insulin analog 2 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0092] In some embodiments, administration of the insulin analog 3 of the present invention provides a more sustained glucose-lowering effect compared to administration of insulin degludec. In some embodiments, the glucose-lowering effect of the insulin analog 3 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0093] In some embodiments, administration of the insulin analog 4 of the present invention provides a more sustained glucose-lowering effect compared to administration of insulin degludec. In some embodiments, the glucose-lowering effect of insulin analog 4 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the glucose-lowering effect of insulin analog 4 lasts for 48 hours.

[0094] In some embodiments, administration of the insulin analog APi3545 of the present invention provides a more sustained glucose-lowering effect compared to administration of insulin degludec. In some embodiments, the glucose-lowering effect of the insulin analog APi3545 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the glucose-lowering effect of the insulin analog APi3545 lasts for 48 hours.

[0095] In some embodiments, administration of the insulin analog APi3546 of the present invention provides a more sustained glucose-lowering effect compared to administration of insulin degludec. In some embodiments, the glucose-lowering effect of the insulin analog APi3546 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0096] In some embodiments, administration of the insulin analog APi3547 of the present invention provides a more sustained glucose-lowering effect compared to administration of insulin degludec. In some embodiments, the glucose-lowering effect of the insulin analog APi3547 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0097] In some embodiments, administration of the insulin analog APi3549 of the present invention provides a more sustained glucose-lowering effect compared to administration of insulin degludec. In some embodiments, the glucose-lowering effect of the insulin analog APi3549 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0098] Methods for detecting glucose levels are known in the art and include blood glucose meters, test strips, ELISA assays, etc.

[0099] In some embodiments, administration of the insulin analogue of the present invention provides a more durable C-peptide reduction effect compared to administration of insulin degludec. In some embodiments, the C-peptide reduction effect of the insulin analogue of the present invention lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0100] In some embodiments, administration of the insulin analog 1 of the present invention provides a more durable C-peptide reduction effect compared to administration of insulin degludec. In some embodiments, the C-peptide reduction effect of insulin analog 1 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0101] In some embodiments, administration of the insulin analog 2 of the present invention provides a more durable C-peptide reduction effect compared to administration of insulin degludec. In some embodiments, the C-peptide reduction effect of insulin analog 2 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0102] In some embodiments, administration of the insulin analog 3 of the present invention provides a more durable C-peptide reduction effect compared to administration of insulin degludec. In some embodiments, the C-peptide reduction effect of insulin analog 3 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0103] In some embodiments, administration of the insulin analog 4 of the present invention provides a more durable C-peptide-lowering effect compared to administration of insulin degludec. In some embodiments, the C-peptide-lowering effect of insulin analog 4 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0104] In some embodiments, administration of the insulin analog APi3545 of the present invention provides a more durable C-peptide reduction effect compared to administration of insulin degludec. In some embodiments, the C-peptide reduction effect of the insulin analog APi3545 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the C-peptide reduction effect of the insulin analog APi3545 lasts for 72 hours.

[0105] In some embodiments, administration of the insulin analog APi3546 of the present invention provides a more durable C-peptide reduction effect compared to administration of insulin degludec. In some embodiments, the C-peptide reduction effect of the insulin analog APi3546 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0106] In some embodiments, administration of the insulin analog APi3547 of the present invention provides a more durable C-peptide reduction effect compared to administration of insulin degludec. In some embodiments, the C-peptide reduction effect of the insulin analog APi3547 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0107] In some embodiments, administration of the insulin analog APi3549 of the present invention provides a more durable C-peptide reduction effect compared to administration of insulin degludec. In some embodiments, the C-peptide reduction effect of the insulin analog APi3549 lasts for 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0108] Methods for detecting C-peptide levels are known in the art and include test strips, ELISA assays, etc.

[0109] In some embodiments, administration of the insulin analogue of the present invention provides lower weight gain compared to administration of insulin degludec. In some embodiments, the lower weight gain of the insulin analogue of the present invention lasts for at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0110] In some embodiments, administration of the insulin analog 1 of the present invention provides lower weight gain compared to administration of insulin degludec. In some embodiments, the lower weight gain of insulin analog 1 lasts for at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0111] In some embodiments, administration of the insulin analog 2 of the present invention provides lower weight gain compared to administration of insulin degludec. In some embodiments, the lower weight gain of insulin analog 2 lasts for at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0112] In some embodiments, administration of the insulin analog 3 of the present invention provides lower weight gain compared to administration of insulin degludec. In some embodiments, the lower weight gain of insulin analog 3 lasts for at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0113] In some embodiments, administration of the insulin analog 4 of the present invention provides lower weight gain compared to administration of insulin degludec. In some embodiments, the lower weight gain of insulin analog 4 lasts for at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0114] In some embodiments, administration of the insulin analog APi3545 of the present invention provides lower weight gain compared to administration of insulin degludec. In some embodiments, the lower weight gain of insulin analog APi3545 lasts for at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0115] In some embodiments, administration of the insulin analog APi3546 of the present invention provides lower weight gain compared to administration of insulin degludec. In some embodiments, the lower weight gain of insulin analog APi3546 lasts for at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0116] In some embodiments, administration of the insulin analog APi3547 of the present invention provides lower weight gain compared to administration of insulin degludec. In some embodiments, the lower weight gain of insulin analog APi3547 lasts for at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0117] In some embodiments, administration of the insulin analog APi3549 of the present invention provides lower weight gain compared to administration of insulin degludec. In some embodiments, the lower weight gain of insulin analog APi3549 lasts for at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0118] In some embodiments, the insulin analogs of the present invention do not provide insulin-like growth factor-1 receptor (IGF1R) activation. In some embodiments, insulin analog 1 does not provide IGF1R activation. In some embodiments, insulin analog 2 does not provide IGF1R activation. In some embodiments, insulin analog 3 does not provide IGF1R activation. In some embodiments, insulin analog 4 does not provide IGF1R activation.

[0119] In some embodiments, insulin analogs are administered orally, intravenously, subcutaneously, parenterally, transdermally, intramuscularly, rectally, vaginally, or topically. In some embodiments, insulin analogs are delivered via a transdermal patch. In some embodiments, insulin analogs are delivered via an implantable device or method.

[0120] In some embodiments, insulin analogs are administered by injection. In some embodiments, insulin analogs are administered by injection using a syringe. In some embodiments, insulin analogs are administered orally as tablets, pills, capsules, elixirs, syrups, extracts, or solutions. In some embodiments, insulin analogs are administered by injection at intervals of once daily, twice daily, once weekly, twice weekly, three times weekly, four times weekly, or five times weekly. In some embodiments, insulin analogs are administered by weekly injections. In some embodiments, insulin analogs are administered by injections every two or three weeks. In some embodiments, insulin analogs are administered by monthly injections.

[0121] In some embodiments, insulin analogs are administered by injection. In some embodiments, insulin analogs are administered by injection using a syringe. In some embodiments, insulin analogs are administered orally as tablets, pills, capsules, elixirs, syrups, extracts, or solutions. In some embodiments, insulin analogs are administered by injection at 12-hour intervals. In some embodiments, insulin analogs are administered by injection at 24-hour intervals. In some embodiments, insulin analogs are administered by injection at 48-hour intervals. In some embodiments, insulin analogs are administered by injection at 72-hour intervals. In some embodiments, insulin analogs are administered by injection at 96-hour intervals. In some embodiments, insulin analogs are administered by injection at 5-day intervals. In some embodiments, insulin analogs are administered by injection at 6-day intervals. In some embodiments, insulin analogs are administered by injection at 7-day intervals. In some embodiments, insulin analogs are administered by injection at 8-day intervals. In some embodiments, insulin analogs are administered by injection at 9-day intervals. In some embodiments, insulin analogs are administered by injection at 10-day intervals. In some embodiments, insulin analogs are administered by injection at intervals of 11 days. In some embodiments, insulin analogs are administered by injection at intervals of 12 days. In some embodiments, insulin analogs are administered by injection at intervals of 13 days. In some embodiments, insulin analogs are administered by injection at intervals of 14 days. In some embodiments, insulin analogs are administered by injection at intervals of 15 days. In some embodiments, insulin analogs are administered by injection at intervals of 16 days. In some embodiments, insulin analogs are administered by injection at intervals of 17 days. In some embodiments, insulin analogs are administered by injection at intervals of 18 days. In some embodiments, insulin analogs are administered by injection at intervals of 19 days. In some embodiments, insulin analogs are administered by injection at intervals of 20 days. In some embodiments, insulin analogs are administered by injection at intervals of 21 days. In some embodiments, insulin analogs are administered by injection at intervals of 22 days. In some embodiments, insulin analogs are administered by injection at intervals of 23 days. In some embodiments, insulin analogs are administered by injection at 24-day intervals. In some embodiments, insulin analogs are administered by injection at 25-day intervals. In some embodiments, insulin analogs are administered by injection at 26-day intervals. In some embodiments, insulin analogs are administered by injection at 27-day intervals.In some embodiments, insulin analogs are administered by injection at 28-day intervals. In some embodiments, insulin analogs are administered by injection at 29-day intervals. In some embodiments, insulin analogs are administered by injection at 30-day intervals. In some embodiments, insulin analogs are administered by injection at twice-weekly intervals. In some embodiments, insulin analogs are administered by injection at weekly intervals. In some embodiments, insulin analogs are administered by injection at twice-monthly intervals. In some embodiments, insulin analogs are administered by injection at monthly intervals. In some embodiments, insulin analogs are administered by injection at every-other-monthly intervals. In some embodiments, insulin analogs are administered by injection at twice-yearly intervals. In some embodiments, insulin analogs are administered by injection at once-yearly intervals.

[0122] In some embodiments, the compositions, formulations or pharmaceutical compositions disclosed herein may be used in the medical field for the treatment, prevention, relief or improvement of metabolic diseases and / or disorders.

[0123] This disclosure provides a method for administering the disclosed compositions, formulations, or pharmaceutical compositions to a subject in need, including a human subject, to slow, halt, or reverse disease progression. As a non-limiting example, disease progression can be measured using tests or diagnostic tools known to those skilled in the art. As another non-limiting example, disease progression can be measured by pathological changes in the pancreas, brain, or other tissues of the subject.

[0124] In some embodiments, a therapeutically effective amount of insulin analogue is administered to the subject. In some embodiments, the effective amount to be used for treatment will depend, for example, on the treatment environment and treatment goals. Those skilled in the art will understand that, according to some embodiments, the appropriate dose level for treatment will therefore vary in part depending on the molecule delivered, the indication for using the insulin analogue, the route of administration, and the subject's body type (weight, body surface or organ size) and / or condition (age and overall health status). In some embodiments, clinicians may titrate the dose and modify the route of administration to obtain optimal therapeutic effect.

[0125] In some embodiments, a therapeutically effective amount of the compositions, formulations, or pharmaceutical compositions disclosed herein inhibits and / or prevents symptoms of a specific disorder and / or disorder.

[0126] In some embodiments, insulin analogs and the compositions, formulations, or pharmaceutical compositions comprising insulin analogs disclosed herein are delivered systemically. For example, in some embodiments, insulin analogs are administered by intravenous injection. In some embodiments, insulin analogs are administered by subcutaneous injection. In some embodiments, insulin analogs are administered by intramuscular injection. In some embodiments, insulin analogs are administered by intravenous infusion. In some embodiments, systemically delivered insulin analogs are capable of crossing the blood-brain barrier.

[0127] Reagent kit and manufacturing method

[0128] In some embodiments, methods for preparing insulin analogs and compositions, formulations or pharmaceutical compositions disclosed herein are envisioned.

[0129] The insulin analogues and compositions, formulations or pharmaceutical compositions disclosed herein can be designed and / or synthesized using any suitable method known in the art.

[0130] In some embodiments, this disclosure provides a kit comprising an insulin analog and compositions, formulations, or pharmaceutical compositions containing insulin analogs disclosed herein. In some embodiments, the kit comprises an insulin analog and compositions, formulations, or pharmaceutical compositions containing insulin analogs disclosed herein, along with instructions for use. In some embodiments, the kit comprises, in a suitable container, an insulin analog and compositions, formulations, or pharmaceutical compositions containing insulin analogs disclosed herein, one or more reference standards, and various buffers, reagents, enzymes, and other standard components known in the art.

[0131] The container may include at least one vial, orifice, test tube, flask, bottle, syringe, or other container device in which insulin analogs and compositions, formulations, or pharmaceutical compositions containing insulin analogs disclosed herein may be placed, and in some cases, appropriately aliquoted therein. Where additional components are provided, the kit may contain additional containers for placing those components. The kit may also include means for containing insulin analogs and any other reagent containers in a tightly sealed manner for commercial sale. Such containers may include injection-molded or blow-molded plastic containers that retain the desired vials therein. The container and / or kit may include labels with instructions for use and / or warnings.

[0132] In some embodiments, the kit includes a container comprising an insulin analog and a pharmaceutically acceptable carrier or a pharmaceutical composition comprising an insulin analog, and instructions for use in treating a metabolic disease or slowing its progression in a subject in need. In some embodiments, the kit includes a container comprising an insulin analog and a pharmaceutically acceptable carrier or a pharmaceutical composition comprising an insulin analog, and instructions for administering the insulin analog, alone or in combination with another agent, to a subject in need to treat that subject's metabolic disease or slow its progression.

[0133] definition

[0134] In this disclosure, terms such as “comprises,” “comprising,” “containing,” and “having” may have the meanings given to them under U.S. Patent Law and may mean “includes,” “including,” etc.; “consisting essentially of” or “consistsessentially” also have the meanings given under U.S. Patent Law and these terms are open-ended, allowing for more than those listed, provided that the essential or novel features of the listed ones are not altered by the presence of more than those listed, but excluding prior art embodiments.

[0135] Unless explicitly stated or obvious from the context, the terms “a”, “an” and “the” as used herein are understood to be singular or plural.

[0136] Unless explicitly stated or obvious from the context, the term “or” as used herein shall be understood to be inclusive.

[0137] As used herein, unless otherwise stated, the term “about” means ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the stated value (e.g., amount, dose, temperature, time, percentage, etc.).

[0138] As used herein, the term "subject" means any mammal, including humans, livestock, and farm animals, as well as zoo, sports, and pet animals such as dogs, horses, and cats, and agricultural animals including cattle, sheep, pigs, and goats. Preferred mammals are humans, including adults, children, and the elderly. Subjects may also be pet animals, including dogs, cats, and horses. Preferred agricultural animals are pigs, cattle, and goats. "Patient" refers to a human subject.

[0139] As used herein, the phrases “therapeutic effective amount” and “effective amount” refer to the amount required to be administered to a patient or the patient’s cells, tissues, or organs to achieve a therapeutic effect (such as improvement or alternative cure). An effective amount is sufficient to elicit the biological or medical response in cells, tissues, systems, animals, or humans sought by researchers, veterinarians, physicians, or clinicians. The determination of an appropriate effective amount or therapeutically effective amount is within the realm of conventional technical skill.

[0140] As used herein, the terms “administering,” “administer,” “administration,” etc., refer to any mode of transferring, delivering, introducing, or transporting a therapeutic agent into a subject who requires treatment with such a drug. Such modes include, but are not limited to, intraocular, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

[0141] Exemplary embodiments

[0142] Exemplary Example 1. A human insulin analog comprising a peptide A chain and a peptide B chain, wherein the A chain comprises from the N-terminus to the C-terminus...

[0143] A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21;

[0144] The B chain contains, from the N-terminus to the C-terminus, […].

[0145] B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29;

[0146] in

[0147] A1 is Gly.

[0148] A2 is Ile.

[0149] A3 is Val.

[0150] A4 is Glu.

[0151] A5 is Gln.

[0152] A6 is Cys.

[0153] A7 is Cys.

[0154] A8 is Thr.

[0155] A9 is Ser.

[0156] A10 is Ile.

[0157] A11 is Cys.

[0158] A12 is Ser.

[0159] A13 is Leu.

[0160] A14 is Gla, or Aad, or Ppa.

[0161] A15 is Gln.

[0162] A16 is Leu.

[0163] A17 is Glu.

[0164] A18 is Asn.

[0165] A19 is Tyr.

[0166] A20 is Cys.

[0167] A21 is Asn; and

[0168] in

[0169] B1 is Phe.

[0170] B2 is Val.

[0171] B3 is Asn.

[0172] B4 is Gln.

[0173] B5 is His.

[0174] B6 is Leu.

[0175] B7 is Cys.

[0176] B8 is Gly.

[0177] B9 is Ser.

[0178] B10 is His.

[0179] B11 is Leu.

[0180] B12 is Val.

[0181] B13 is Glu.

[0182] B14 is Ala.

[0183] B15 is Leu.

[0184] B16 is His(3-Me), or Pal, or His(5-I).

[0185] B17 is Leu.

[0186] B18 is Val.

[0187] B19 is Cys.

[0188] B20 is Gly.

[0189] B21 is Glu.

[0190] B22 is Arg.

[0191] B23 is Gly.

[0192] B24 is Phe.

[0193] B25 is His(3-Me), or Pal, or 2Fal, or Thi, or Ala(4-thiazolyl), or 3-Pal.

[0194] B26 is Tyr.

[0195] B27 is Thr.

[0196] B28 is Pro, or Hyp, or Pip.

[0197] B29 is Lys.

[0198] Exemplary Example 2. The insulin analogue according to the foregoing exemplary embodiments, wherein the N-terminus of the B chain, or the C-terminus of the B chain, or both the N-terminus and the C-terminus, are substituted with substituents.

[0199] Exemplary Example 3. An insulin analog according to any one of the foregoing exemplary embodiments, wherein the substituent is AEEA-AEEA-γ-Glu-(CH2). m -OH, where m is 12, 14, 16, 18, 20 or 22.

[0200] Exemplary Example 4. An insulin analog according to any one of the foregoing exemplary embodiments, wherein A6 and A11 are connected by a connector.

[0201] Exemplary Example 5. An insulin analog according to any one of the foregoing exemplary embodiments, wherein A7 and B7 are connected by a connector.

[0202] Exemplary Example 6. An insulin analog according to any one of the foregoing exemplary embodiments, wherein A20 and B19 are connected via a connector.

[0203] Exemplary Example 7. An insulin analog according to any one of the foregoing exemplary embodiments, wherein the connector is cleavable or non-cleavable.

[0204] Exemplary Example 8. An insulin analog according to any one of the foregoing exemplary embodiments, wherein the linker comprises a disulfide bond.

[0205] Exemplary Example 9. An insulin analogue according to any one of the foregoing exemplary embodiments, wherein the insulin analogue has a binding affinity (ECG) to the insulin receptor. 50 (Below 10 nM)

[0206] Exemplary Example 10. An insulin analogue according to any one of the foregoing exemplary embodiments, wherein the insulin analogue has a binding affinity (IC50) to the insulin receptor. 50 (Below 5nM)

[0207] Exemplary Example 11. An insulin analogue according to any one of the foregoing exemplary embodiments, wherein the insulin analogue has a binding affinity (IC50) to the insulin receptor. 50 (Below 1 nM)

[0208] Exemplary Example 12. An insulin analog according to any one of the foregoing exemplary embodiments, wherein the binding affinity of the insulin analog is measured by surface plasmon resonance.

[0209] Exemplary Example 13. An insulin analog according to any one of the foregoing exemplary embodiments, wherein the binding affinity of the insulin analog is measured using a recombinant cell line.

[0210] Exemplary Example 14. An insulin analogue according to the foregoing exemplary embodiments, wherein a recombinant cell line expresses an insulin receptor.

[0211] Exemplary Example 15. The insulin analogue according to the foregoing exemplary embodiments, wherein the binding affinity (IC50) of the insulin analogue to the insulin receptor is... 50 The value is determined by measuring luciferase activity.

[0212] Exemplary Example 16. An insulin analogue according to any one of the foregoing exemplary embodiments, wherein the insulin analogue exhibits an in vivo plasma elimination half-life of at least 1 hour, or 2 hours, or 4 hours, or 6 hours, or 8 hours, or 10 hours, or 24 hours, or 48 hours, or 72 hours, or 168 hours in humans.

[0213] Exemplary Example 17. A pharmaceutical composition comprising an insulin analogue as described in any of the preceding exemplary embodiments and a pharmaceutically acceptable medium or carrier.

[0214] Exemplary Example 18. The pharmaceutical composition according to the foregoing exemplary embodiments further comprises any one of an isotonic agent, an excipient, a preservative, and a buffer.

[0215] Exemplary Example 19. The pharmaceutical composition according to any one of the foregoing exemplary embodiments further comprises a surfactant.

[0216] Exemplary Example 20. The pharmaceutical composition according to any one of the foregoing exemplary embodiments further comprises an antidiabetic agent.

[0217] Exemplary Example 21. The pharmaceutical composition according to any one of the foregoing exemplary embodiments, wherein the pharmaceutically acceptable carrier is a microsphere, micelle, or nanoparticle.

[0218] Exemplary Example 22. A method of treating diabetes, comprising administering to a patient a therapeutically effective amount of any of the foregoing exemplary embodiments of an insulin analogue.

[0219] Exemplary Example 23. The method according to the foregoing exemplary embodiments, wherein the treatment results in a decrease or stabilization of the average blood glucose level in the patient.

[0220] Exemplary Example 24. The method according to any one of the foregoing exemplary embodiments, wherein the insulin analog is administered orally, subcutaneously, intravenously, parenterally, transdermally, intramuscularly, rectally, vaginally, via pump, or topically.

[0221] Exemplary Example 25. The method according to any one of the foregoing exemplary embodiments, wherein the insulin analog is administered via subcutaneous, transdermal, intramuscular, or intravenous injection.

[0222] Exemplary Example 26. The method according to any one of the foregoing exemplary embodiments, wherein the insulin analog is administered once weekly.

[0223] Exemplary Example 27. The method according to any one of the foregoing exemplary embodiments, wherein administration of an insulin analog provides a more sustained glucose-lowering effect compared to administration of degludec insulin.

[0224] Exemplary Example 28. The method according to any one of the foregoing exemplary embodiments, wherein administration of an insulin analog provides a more durable C-peptide reduction effect compared to administration of degludec insulin.

[0225] Exemplary Example 29. The method according to any one of the foregoing exemplary embodiments, wherein administration of an insulin analog provides lower weight gain compared to administration of degludec insulin.

[0226] Exemplary Example 30. The method according to any one of the foregoing exemplary embodiments, wherein the administration of the insulin analog does not activate the insulin-like growth factor-1 receptor (IGF1R).

[0227] Example

[0228] The following examples are provided to provide a complete disclosure and description of how to make and use the invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor to represent that the following experiments are all or only the experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be accounted for. Unless otherwise stated, parts are parts by weight, molecular weights are weight-average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides; etc.

[0229] Unless otherwise stated, the practice of this invention will be carried out using conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the art.

[0230] Example 1: Synthesis of insulin analogs

[0231] Figures 1A to 1M The insulin analogues shown were synthesized via a combination of solid-phase and liquid-phase synthesis. Purification of the analogues was performed by HPLC using a specific buffer and a specific reversed-phase column, which comprised a combination of ion exchange and reversed-phase media packed within the column.

[0232] Example 2: Biopotency of 8 Insulin Analogs

[0233] It was determined that they were displayed separately. Figures 1A to 1D , Figures 1H to 1J as well as Figure 1L The biopotency of eight insulin analogs was determined. The calculated insulin concentration units (IU / mL) were assigned to the eight insulin analogs using a conversion factor of 1 μIU / L = 6 picomoles / L (J Diabetes Sci Technol. May 2019;13(3): 597–600, the contents of which are incorporated herein by reference in their entirety). Figure 4 The calculated IU / mg values ​​for eight insulin analogs are shown.

[0234] Bioequivalence of 8 insulin analogs according to Chinese Pharmacopoeia <1211> The concentration of insulin analogs and insulin reference standards was determined by comparison with the insulin reference standard. Specifically, insulin analogs and insulin reference standards were prepared using either diluent 1 or diluent 2. Diluent 1 contained 0.9% sodium chloride, pH 2.5 (adjusted with HCl), and 0.2% phenol. Diluent 2 contained 0.9% sodium chloride, pH 2.5 (adjusted with HCl). Insulin reference standards (27.2 IU / mg) and insulin analogs (calculated as 25 IU / mg) were prepared using diluent 1. Additionally, low-dose and high-dose solutions of the insulin reference standard and test analogs (0.05 IU / mL and 0.1 IU / mL, respectively) were prepared using diluent 2.

[0235] According to the Chinese Pharmacopoeia <1211> In this study, ICR mice (25-28g, 6 mice per group) were injected with 0.2-0.3 mL of low- and high-dose solutions of eight insulin analogs and insulin standards. The mice were then administered low- and high-dose insulin standards and insulin analogs according to a parallel design / protocol. Blood samples were collected from the orbital venous sinus 40 minutes after each administration. The glucose concentration of each blood sample was determined using the glucose oxidase-peroxidase method (Roche blood glucose test strips). Figure 5 The blood glucose levels after administration (i.e., 40 minutes later) are shown, and are correlated with high and low dose solutions of the analogue and reference standard, respectively. Figure 6 The relative biopotency of insulin analogs was summarized by comparing calculated and measured biopotency. Figure 7 This indicates that all eight insulin analogs tested had a glucose-lowering effect, with the three most active insulin analogs being insulin analog 2 (…). Figure 1B ), Api3545 ( Figure 1H ), Api3549 ( Figure 1L ).

[0236] Example 3: Subcutaneous administration of insulin analogs in the db / db mouse model showed controlled glucose curves over time.

[0237] The db / db mouse strain is a widely accepted model of type 2 diabetes, reproducing most aspects of human type 2 diabetes. Specifically, db / db mice carry a mutation in the leptin receptor, leading to obesity, decreased insulin receptor sensitivity, and subsequently elevated blood glucose levels, decreased β-cell function, and elevated HBA1c levels. As with human type 2 diabetes, the diabetic status in db / db mice is progressive. Animals at 6 weeks of age have near-normal or slightly elevated plasma glucose (PG) and relatively normal β-cell function. Fasting plasma glucose (FPG) levels gradually increase over several weeks, and β-cell function declines, resulting in very high FPG (>400 mg / dL) and complete β-cell degeneration by approximately 16 weeks of age. The db / db mouse model can be used for acute conditions but is more commonly used for chronic conditions to assess the cumulative effect of test compounds over several weeks of treatment. Variability is relatively low, so validating compounds with a group size of approximately 8 animals can yield good statistical significance.

[0238] Therefore, insulin analog 1 was tested. Figure 1A The ability to control glucose curves in a db / db type 2 diabetes model was assessed. Wild-type m / m mice were also included as a control in this study. Mice were male, aged 6–8 weeks, and the total group size was 8 mice. Insulin analogs were delivered subcutaneously in a single dose. The dose volume was 5 mL / kg (125 μL per 25 g mouse). Mice were injected with the following dose levels: insulin degludec 1.5 IU / kg (model) and insulin analog #1 ( Figure 1A The dosages were 0.04 mg / kg, 0.12 mg / kg, or 0.36 mg / kg. All insulin compounds (degludec insulin and analog #1) were diluted with 0.9% sodium chloride (mediator, physiological saline). The mediator control consisted of 0.9% sodium chloride.

[0239] Blood samples were collected at 0h (0-60 min before administration), 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 18h, 24h, and 30h after a single dose for measuring plasma glucose concentration. In addition, mouse body weight was measured daily for 3 days.

[0240] like Figure 2As shown, glucose curves at 2, 4, 6, and 8 hours were statistically significantly lower for all three study groups (1.5 IU / mg insulin degludec, 0.36 mg / kg, and 0.12 mg / kg analog 1) than for the db / db mediator control. Throughout the entire timeframe starting from the 2-hour time point, the glucose curve for 0.36 mg / kg analog 1 was statistically lower than that for the db / db mediator control. Furthermore, the response (reduction in glucose levels) to analog 1 at doses of 0.36 mg / kg, 0.12 mg / kg, and 0.04 mg / kg was dose-proportional.

[0241] Treatment with 1.5 IU / mg insulin degludec and analogue #1 at all dose levels had no significant effect on animal body weight, at least within the 1–3 day timeframe. Figures 3A to 3B This result indicates that insulin analog #1 can effectively regulate glucose curves while maintaining safety (by body weight).

[0242] Example 4: Efficacy of insulin analogs in non-fasting Spradolai rats

[0243] Male Spradolai (SD) rats aged 8–10 weeks were administered 175 nmol–200 nmol insulin analogs 1, 2, 3, 4, APi3545, APi3546, APi3547, and APi3549 via subcutaneous injection. Insulin degludec was used as a positive control. The mediator control consisted of glycerol (19.6 mg), m-cresol (1.72 mg), phenol (1.50 mg), zinc acetate (91.75 µg), and WFI (to 1 mL).

[0244] like Figures 8A to 8C , Figure 12 , Figures 14A to 14C and Figure 18 As shown, compared with rats injected with degludec insulin, rats injected with insulin analogs 1, 2, 3, 4, APi3545, APi3546, APi3547, and APi3549 exhibited a more sustained reduction in glucose levels. Insulin analogs 4 and APi3545 maintained low glucose levels for up to 48 hours. Insulin analog 3 showed the most thorough glucose-lowering effect.

[0245] like Figure 9 and Figure 15As shown, the area under the glucose curve (AUC) values ​​of insulin analogs 2, 3, and 4 followed their specific activity order. Furthermore, compared to rats injected with degludec insulin, rats injected with insulin analogs 1, 2, 3, 4, APi3545, APi3546, APi3547, and APi3549 exhibited a more sustained reduction in glucose levels. Insulin analogs 4 and APi3545 maintained low glucose levels for up to 48 hours. Insulin analog 3 showed the most thorough glucose-lowering effect.

[0246] like Figure 10 As shown, rats injected with insulin analog 2 exhibited a more sustained decrease in glucose levels compared to rats injected with degludec insulin.

[0247] like Figures 11A to 11C and Figures 17A to 17B As shown, compared with rats injected with degludec insulin, rats injected with insulin analogs 1, 2, 3, 4, APi3545, APi3546, APi3547, and APi3549 maintained C-peptide levels for a longer period. Insulin analog 2 showed the most thorough and longest-lasting C-peptide reduction effect. Insulin analog APi3545 maintained undetectable C-peptide levels for up to 72 hours.

[0248] like Figure 13 and Figure 16 As shown, rats injected with insulin analogs 1, 2, 4, APi3547, and APi3546 exhibited a smaller rate of weight gain compared to rats injected with degludec insulin.

[0249] Example 5: Structure-Property Relationship

[0250] Linked to B-Lys with fatty acids 29 Compared to its analogues, the fatty acid linkage to the N-terminus of the B chain (APi3547 and APi3549) essentially eliminates glucose-reducing activity.

[0251] B-28 bit Pip to Hyp replacement enhancement with connection to B-Lys 29 The glucose-lowering effect of insulin analogs of fatty acids.

[0252] In A-14, Gly is replaced with Aad; in B-16, His(3-Me) is replaced with Pal; and in B-25, His(3-Me) is replaced with Pal; and in B-25, His(3-Me) is replaced with 3-Pal. The fatty acid residues are linked to B-Lys. 29 It is almost equivalent to insulin analogs.

[0253] The substitutions in degludec insulin (Tyr→Aad in A14, Tyr→His(5-I) in B-16, and Phe→Ala(4-thiazolyl) in B-25) produced the longest-acting insulin analog tested (APi3545).

[0254] Compared to insulin degludec, all tested structural variations enhanced and prolonged the glucose-lowering effect of insulin analogs.

[0255] Example 6: Interaction between in vitro insulin analogs and IGF-1R receptors

[0256] Insulin-like growth factor (IGF-1R) is a tyrosine kinase receptor that regulates cell growth and proliferation and can be activated by IGF-1, IGF-2, and insulin.

[0257] The IGF-1R signaling pathway regulates multiple cell phenotypes related to tumor cell survival and growth, including cell cycle progression, apoptosis, and differentiation.

[0258] Epidemiological studies have reported a positive correlation between circulating IGF-1 levels and various primary cancers, such as breast cancer, colorectal cancer, and prostate cancer.

[0259] The agency may consider the activation of the IGFR-1R receptor by insulin analogs (investigative T1D and T2D drugs) as a potential carcinogenic risk.

[0260] IGF-1R (Luc) HEK293 reporter cells were exposed to 0.015–4 mg / mL IGF-1, insulin, and insulin analogs 1, 2, 3, and 4. Figure 19 As shown, insulin analogs 1, 2, 3 and 4 did not exhibit IGF-1R activation at concentrations up to 4 µg / mL (1.6 µM).

[0261] equivalent

[0262] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only conventional experiments. Such equivalents are intended to be covered by the following claims.

[0263] All references cited in this article are incorporated herein by reference in their entirety and for all purposes, to the extent that each individual publication or patent or patent application is specifically and individually cited in its entirety for all purposes.

[0264] This invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention will become apparent to those skilled in the art from the foregoing description and drawings, in addition to those described herein. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A human insulin analog comprising a peptide A chain and a peptide B chain, wherein the A chain comprises, from the N-terminus to the C-terminus, a peptide B chain. A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21; The B chain, from the N-terminus to the C-terminus, comprises B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-B29; in A1 is Gly. A2 is Ile. A3 is Val. A4 is Glu. A5 is Gln. A6 is Cys. A7 is Cys. A8 is Thr. A9 is Ser. A10 is Ile. A11 is Cys. A12 is Ser. A13 is Leu. A14 is Gla, or Aad, or Ppa. A15 is Gln. A16 is Leu. A17 is Glu. A18 is Asn. A19 is Tyr. A20 is Cys. A21 is Asn; and in B1 is Phe. B2 is Val. B3 is Asn. B4 is Gln. B5 is His. B6 is Leu. B7 is Cys. B8 is Gly. B9 is Ser. B10 is His. B11 is Leu. B12 is Val. B13 is Glu. B14 is Ala. B15 is Leu. B16 is His(3-Me), or Pal, or His(5-I). B17 is Leu. B18 is Val. B19 is Cys. B20 is Gly. B21 is Glu. B22 is Arg. B23 is Gly. B24 is Phe. B25 is His(3-Me), or Pal, or 2Fal, or Thi, or Ala(4-thiazolyl), or 3-Pal. B26 is Tyr. B27 is Thr. B28 is Pro, or Hyp, or Pip. B29 is Lys.

2. The insulin analog of claim 1, wherein the N-terminus of the B chain or the C-terminus of the B chain or both the N-terminus and the C-terminus are substituted with a substituent.

3. The insulin analog according to claim 1, wherein the substituent is AEEA-AEEA-γ-Glu-(CH2). m -OH, where m is 12, 14, 16, 18, 20 or 22.

4. The insulin analog of claim 1, wherein A6 and A11 are connected by a connector.

5. The insulin analog of claim 1, wherein A7 and B7 are connected by a connector.

6. The insulin analog of claim 1, wherein A20 and B19 are connected by a connector.

7. The insulin analog of claim 1, wherein the connector is cleavable or non-cleavable.

8. The insulin analog of claim 1, wherein the linker comprises a disulfide bond.

9. The insulin analogue of claim 1, wherein the insulin analogue has a binding affinity (ECG) to the insulin receptor. 50 (Below 10 nM) 10. The insulin analog of claim 1, wherein the insulin analog has a binding affinity (IC50) to the insulin receptor. 50 (Below 5 nM) 11. The insulin analogue of claim 1, wherein the insulin analogue has a binding affinity (IC50) to the insulin receptor. 50 (Below 1 nM) 12. The insulin analog of claim 1, wherein the binding affinity of the insulin analog is measured by surface plasmon resonance.

13. The insulin analog of claim 1, wherein the binding affinity of the insulin analog is measured using a recombinant cell line.

14. The insulin analog of claim 13, wherein the recombinant cell line expresses the insulin receptor.

15. The insulin analog of claim 14, wherein the binding affinity (IC50) of the insulin analog to the insulin receptor is... 50 The value is determined by measuring luciferase activity.

16. The insulin analog of claim 1, wherein the insulin analog exhibits an in vivo plasma elimination half-life of at least 1 hour, or 2 hours, or 4 hours, or 6 hours, or 8 hours, or 10 hours, or 24 hours, or 48 hours, or 72 hours, or 168 hours in humans.

17. A pharmaceutical composition comprising an insulin analogue according to claim 1 and a pharmaceutically acceptable medium or carrier.

18. The pharmaceutical composition according to claim 17, further comprising any one of an isotonic agent, an excipient, a preservative, and a buffer.

19. The pharmaceutical composition according to claim 17, further comprising a surfactant.

20. The pharmaceutical composition of claim 17, further comprising an antidiabetic agent.

21. The pharmaceutical composition of claim 17, wherein the pharmaceutically acceptable carrier is a microsphere, micelle, or nanoparticle.

22. A method of treating diabetes, comprising administering to a patient a therapeutically effective amount of the insulin analogue according to claim 17.

23. The method of claim 22, wherein the treatment reduces or stabilizes the average blood glucose level in the patient.

24. The method of claim 22, wherein the insulin analog is administered orally, subcutaneously, intravenously, parenterally, transdermally, intramuscularly, rectally, vaginally, via pump, or topically.

25. The method of claim 22, wherein the insulin analog is administered by subcutaneous, percutaneous, intramuscular, or intravenous injection.

26. The method of claim 22, wherein the insulin analog is administered once weekly.

27. The method of claim 22, wherein administration of the insulin analog provides a more sustained glucose-lowering effect compared to administration of degludec insulin.

28. The method of claim 22, wherein administration of the insulin analog provides a more durable C-peptide reduction effect compared to administration of degludec insulin.

29. The method of claim 22, wherein administration of the insulin analog provides lower weight gain compared to administration of degludec insulin.

30. The method of claim 22, wherein the administration of the insulin analog does not activate the insulin-like growth factor-1 receptor (IGF1R).

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