Treatment of diabetes

Abstract

The present disclosure provides methods and compositions for treating patients with diabetes and / or obesity.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Application No. 63 / 497,737, filed April 23, 2023; U.S. Application No. 63 / 509,743, filed June 22, 2023; and U.S. Application No. 63 / 590,388, filed October 13, 2023. The contents of these priority applications are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application contains a sequence list, which has been submitted electronically in XML format and is hereby incorporated in its entirety by reference. The XML copy created on April 23, 2024, is named 124921WO004.xml and has a size of 4,912 bytes. Background Technology

[0005] Diabetes mellitus (DM) (commonly referred to as diabetes) is a disease caused by inadequate control of blood sugar levels. It has two main phenotypes – type 1 (T1DM or T1D) and type 2 (T2DM or T2D). T1DM, formerly known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition. T1DM is thought to be caused by an autoimmune attack against the pancreatic beta cells, causing patients to lose the ability to produce any or enough insulin, a hormone that helps cells take up blood sugar. While genetic susceptibility plays a role, environmental triggers, such as viral infections, are often associated with the onset of T1DM. Although T1DM typically appears in childhood or adolescence, it can develop in adulthood.

[0006] Type 2 diabetes mellitus (T2DM) is caused by insulin's inability to properly control blood sugar. This insulin resistance can cause the pancreas to secrete more insulin, leading to hyperinsulinemia; however, blood sugar levels remain abnormally high. Over time, this condition can lead to beta cell depletion and insufficient insulin production, cardiovascular disease, and significantly increase the risk of heart attack, stroke, neuropathy, limb amputation, blindness, and kidney failure. Obesity is a major risk factor for T2DM.

[0007] Treatment for diabetes typically focuses on glycemic control, such as through the ingestion of exogenous insulin (e.g., via subcutaneous injection or powder inhalation). However, insulin therapy often leads to chronic hyperinsulinemia. In fact, basal insulin levels in diabetic patients can be several times higher than in healthy individuals (see, for example, Gregory et al., Diabetes (2019) 68(8):1565-76). Hyperinsulinemia, especially in the presence of excessive fat accumulation, induces insulin resistance, which can lead to even greater weight gain, peripheral fat accumulation, and obesity (see, for example, Van der Schueren et al., Lancet Diabetes Endocrinol (2021) 9(11):776-85; Schauer et al., Diabetes (2011) 60(1):306-14). This combined state of insulin resistance and insulin deficiency is often referred to as “double diabetes.” Furthermore, even patients with ideal glycemic control (HbA1c <7%) remain at high risk for cardiovascular disease and are prone to obesity, and these problems can be exacerbated by insulin therapy.

[0008] Another chronic disease affecting a large portion of the world's population is obesity. Obesity is associated with many complications, including cardiovascular disease and type 2 diabetes mellitus (T2DM), imposing a huge health and economic burden on patients and society.

[0009] Therefore, there remains an urgent need to develop safe, effective, and well-tolerated therapies for people with chronic conditions such as diabetes and obesity. Summary of the Invention

[0010] This disclosure provides methods for treating type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) or improving glycemic control in patients in need. In some embodiments, the patient is insulin-dependent, i.e., requires insulin intake or therapy (e.g., by injection). In further embodiments, the patient has T1DM or T2DM. In some embodiments, the patient may have insulin resistance (e.g., a patient with T2DM). A method for weight management in patients in need is also provided.

[0011] In one aspect, this disclosure provides an adjunctive therapy for insulin-dependent patients (e.g., patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM), the adjunctive therapy comprising administering (e.g., via subcutaneous injection) to a patient in need a dual agonist of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) and glucose-dependent insulinotropic peptide (GIP) receptor (GIPR), wherein the agonist comprises the following structures or pharmaceutically acceptable salts or esters thereof:

[0012]

[0013] (Formula I) (SEQ ID NO: 1),

[0014] R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and the agonist is administered at a body weight-independent dose of 1 mg to 20 mg (i.e., a steady or fixed dose).

[0015] In one aspect, this disclosure provides a method for treating a patient with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) or for improving glycemic control in a patient with T1DM or T2DM, the method comprising administering (e.g., by subcutaneous injection) a dual agonist of a glucagon-like peptide-1 (GLP-1) receptor and a glucose-dependent insulinotropic polypeptide (GIP) receptor to a patient in need, wherein the agonist comprises a structure of formula I or a pharmaceutical salt or ester thereof, wherein R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, the cycloalkyl or heterocyclic group optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and wherein the agonist is administered at a weight-independent dose of 1 mg to 20 mg (i.e., a steady dose or a fixed dose).

[0016] In one aspect, this disclosure provides a method for increasing insulin sensitivity or reducing insulin resistance in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM), the method comprising administering (e.g., by subcutaneous injection) to a patient in need a dual agonist of a glucagon-like peptide-1 (GLP-1) receptor and a glucose-dependent insulinotropic polypeptide (GIP) receptor, wherein the agonist comprises a structure of formula I or a pharmaceutical salt or ester thereof, wherein R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, the cycloalkyl or heterocyclic group optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and wherein the agonist is administered at a weight-independent dose of 1 mg to 20 mg (i.e., a steady dose or a fixed dose).

[0017] In one aspect, this disclosure provides a method for increasing insulin-independent glucose management in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM), the method comprising administering (e.g., by subcutaneous injection) to a patient in need a dual agonist of a glucagon-like peptide-1 (GLP-1) receptor and a glucose-dependent insulinotropic polypeptide (GIP) receptor, wherein the agonist comprises a structure of formula I or a pharmaceutical salt or ester thereof, wherein R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, the cycloalkyl or heterocyclic group optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and wherein the agonist is administered at a weight-independent dose of 1 mg to 20 mg (i.e., a steady dose or a fixed dose).

[0018] In one aspect, this disclosure provides a method for reducing the need for therapeutic insulin in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM), the method comprising administering (e.g., by subcutaneous injection) to a patient in need a dual agonist of a glucagon-like peptide-1 (GLP-1) receptor and a glucose-dependent insulinotropic polypeptide (GIP) receptor, wherein the agonist comprises a structure of formula I or a pharmaceutical salt or ester thereof, wherein R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, the cycloalkyl or heterocyclic group optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and wherein the agonist is administered at a weight-independent dose of 1 mg to 20 mg (i.e., a steady dose or a fixed dose).

[0019] In one aspect, this disclosure provides a method for relieving hypertension in patients in need, such as those with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM), comprising administering (e.g., by subcutaneous injection) a dual agonist of a glucagon-like peptide-1 (GLP-1) receptor and a glucose-dependent insulinotropic polypeptide (GIP) receptor to the patient in need, wherein the agonist comprises a structure of formula I or a pharmaceutical salt or ester thereof, wherein R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, the cycloalkyl or heterocyclic group optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and wherein the agonist is administered at a weight-independent dose of 1 mg to 20 mg (i.e., a steady dose or a fixed dose).

[0020] In one aspect, this disclosure provides a method for reducing atherogenic lipids (e.g., cholesterol, triglycerides, and / or low-density lipoprotein cholesterol) in patients in need (such as those with T1DM or T2DM), the method comprising administering (e.g., by subcutaneous injection) a dual agonist of a glucagon-like peptide-1 (GLP-1) receptor and a glucose-dependent insulinotropic polypeptide (GIP) receptor to the patient in need, wherein the agonist comprises a structure of formula I or a pharmaceutical salt or ester thereof, wherein R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, the cycloalkyl or heterocyclic group optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and wherein the agonist is administered at a weight-independent dose of 1 mg to 20 mg (i.e., a steady dose or a fixed dose).

[0021] In one aspect, this disclosure provides a method for weight management (weight loss) in patients in need, such as those with T1DM or T2DM, comprising administering (e.g., by subcutaneous injection) a dual agonist of a glucagon-like peptide-1 (GLP-1) receptor and a glucose-dependent insulinotropic polypeptide (GIP) receptor to the patient in need, wherein the agonist comprises a structure of formula I or a pharmaceutical salt or ester thereof, wherein R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, the cycloalkyl or heterocyclic group optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and wherein the agonist is administered at a weight-independent dose (i.e., a steady dose or a fixed dose) of 1 mg to 20 mg.

[0022] In some embodiments, a dual GLP-1R / GIPR agonist comprises the following structures or pharmaceutically acceptable salts or esters thereof:

[0023]

[0024] (Formula V) (SEQ ID NO:1).

[0025] In some embodiments, a dual GLP-1R / GIPR agonist comprises the following structures or pharmaceutically acceptable salts or esters thereof:

[0026]

[0027] (Formula VII) (SEQ ID NO:1).

[0028] In some embodiments, the application steps of the method are repeated at intervals of one to seven days. In a further embodiment, the application steps are repeated once a day.

[0029] In some embodiments, each administered dose is a dose that activates both the GLP-1 receptor and the GIP receptor. In some embodiments, the administered dose is about 1.5 mg, 1.8 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

[0030] In some embodiments, the patient has 25 kg / m 2 Or a higher BMI.

[0031] In some embodiments, the patient is an adult patient, an adolescent patient, or a pediatric patient.

[0032] In some embodiments, the patient receives another treatment for T1DM or T2DM, such as insulin therapy, diet therapy, exercise therapy, hypertension therapy, and / or lipid-lowering therapy.

[0033] This disclosure also provides the dual GLP-1R / GIPR agonist for use in the treatment methods described herein; and the use of the dual GLP-1R / GIPR agonist in the manufacture of a medicament for use in the treatment methods described herein.

[0034] In another aspect, this disclosure provides an article of manufacture (e.g., a kit, syringe (such as a pre-filled syringe), or syringe (such as a pre-filled syringe) for use in this treatment method. The article of manufacture includes one or more dose units to be administered (e.g., about 1 mg to 20 mg per dose, such as 1.8 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, or 15 mg). In some embodiments, the syringe or syringe includes a single dose unit and may be for single use.

[0035] A pharmaceutical composition is also provided comprising (ii) a dual GLP-1R / GIPR agonist or a pharmaceutically acceptable salt or ester thereof at a concentration of 15 mg / mL; (iii) a disodium hydrogen phosphate heptahydrate / sodium dihydrogen phosphate buffer, optionally at a concentration of 20 mM; (iii) propylene glycol; and (iv) phenol, further optionally, wherein the pharmaceutical composition is at pH 7.0.

[0036] Other features, objectives, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while the detailed description indicates embodiments and aspects of the invention, it is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the invention will be apparent to those skilled in the art based on the detailed description. Attached Figure Description

[0037] Figure 1A depicts the left plot comparing the cAMP production activities of GLP-1, liraglutide, and CT-859 (also referred to herein as "CT-9859"); and the right plot comparing the cAMP production activities of GLP-1, liraglutide, and CT-859. The X-axis shows the concentration in nmol, and the Y-axis shows the percentage of activity.

[0038] Figure 1B depicts the left graph comparing the cAMP production activities of GLP-1, liraglutide, exenatide-4 (Ex4), exenatide-Phe1 (Ex-Phe1), and CT-859; and the right graph comparing the cAMP production activities of GLP-1 and CT-859. The X-axis shows the logarithmic concentration in nM, and the Y-axis shows the percentage of activity.

[0039] Figure 2A depicts the left graph comparing the β-repressor protein-coupling activities of GLP-1, liraglutide, and CT-859; and the right graph comparing the β-repressor protein-coupling activities of GLP-1 and CT-859. The X-axis shows the concentration in nmol, and the Y-axis shows the percentage of activity.

[0040] Figure 2B depicts the left panel comparing the β-repressor protein-coupling activities of GLP-1, liraglutide, exenatide-4 (Ex4), exenatide-Phe1 (Ex-Phe1), and CT-859; and the right panel comparing the β-repressor protein-coupling activities of GLP-1 and CT-859. The X-axis shows the logarithmic concentration in nM, and the Y-axis shows the percentage of activity.

[0041] Figure 3 depicts normal mice (GLP1R) treated with the medium and CT-859 at doses of 20 nmol / kg or 200 nmol / kg. + / + ) and GLP-1R knockout mice (GLP1R - / - The graph shows the blood glucose level (mg / dL) (left), AUC blood glucose (min*mg / dL) (middle), and plasma insulin level (µIU / mL) (right) during the intraperitoneal glucose tolerance test.

[0042] Figure 4A depicts the blood glucose levels (mg / dL) in lean mice treated with the medium, 20 nmol / kg liraglutide (“lira”), or 20 nmol / kg CT-859 at 4 hours (left), 24 hours (middle), and 48 hours (right).

[0043] Figure 4B depicts AUC blood glucose (min*mg / dL) at 4 hours, 24 hours, and 48 hours after treatment with the medium, 20 nmol / kg liraglutide, or 20 nmol / kg CT-859.

[0044] Figure 5 depicts the plasma concentrations of liraglutide and CT-859 in lean mice at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 32 hours after treatment with 1 mg / kg CT-859 or 1 mg / kg liraglutide.

[0045] Figure 6 depicts fasting blood glucose levels (mg / dL) in diet-induced obese (DIO) mice administered the medium, 20 nmol / kg liraglutide, or 20 nmol / kg CT-859 for 20 days.

[0046] Figure 7 depicts fasting plasma insulin levels (µIU / mL) (left panel) and log(HOMA-IR) (a surrogate measure of insulin resistance) (right panel) in diet-induced obese (DIO) mice administered the medium, 20 nmol / kg liraglutide, or 20 nmol / kg CT-859 for 20 days.

[0047] Figure 8 depicts the weight loss (WL) in diet-induced obese (DIO) mice administered the medium, 20 nmol / kg liraglutide, or 20 nmol / kg CT-859 for 20 days.

[0048] Figure 9 depicts the cumulative food intake (FI) (g) in diet-induced obese (DIO) mice administered the medium, 20 nmol / kg liraglutide, or 20 nmol / kg CT-859 for 20 days.

[0049] Figure 10 depicts the plasma insulin levels (µIU / mL) (left panel) and log(HOMA-IR) (an alternative measure of insulin resistance) (right panel) in diet-induced obese (DIO) mice administered the medium, 200 nmol / kg liraglutide, or 200 nmol / kg CT-859 for 20 days.

[0050] Figure 11 depicts the weight loss in diet-induced obese (DIO) mice after 20 days of administration of a mediator, 200 nmol / kg liraglutide, or 200 nmol / kg CT-859.

[0051] Figures 12A and 12B depict food intake (g / 24h) in ob / ob mice that were administered the medium, 200 nmol / kg liraglutide, or 200 nmol / kg CT-868 for 1, 7, or 14 days (Figure 12A) or 8 days (Figure 12B).

[0052] Figure 13 depicts the changes in body weight in wild-type mice measured at baseline, 4, 24, 48, and 72 hours after administration of the mediator, 0.025 nmol / kg exenatide-4 (Ex-4), and 0.025 nmol / kg exenatide-Phe1 (Ex-Phe1). The X-axis shows time in hours (hrs), and the Y-axis shows the percentage change in body weight relative to baseline.

[0053] Figure 14 depicts a graph showing the cumulative food consumption in wild-type mice measured at baseline, 4 hours, 24 hours, 48 ​​hours, and 72 hours after administration of the medium, 0.025 nmol / kg exenatide-4 (Ex-4), and 0.025 nmol / kg exenatide-Phe1 (Ex-Phe1). The X-axis shows time in hours (hrs), and the Y-axis shows the cumulative food consumption in grams (g).

[0054] Figures 15A through 15J present data demonstrating that CT-859 is a biased dual GLP-1R / GIPR agonist. These figures show the mean (± SE) cAMP accumulation at GLP-1R (Figure 15A) and β-repressor protein coupling (Figure 15B), as well as GLP-1R internalization (Figure 15C), the time course of GLP-1R internalization after treatment with 100 nM GLP-1, liraglutide, and CT-859 (Figure 15D), and CT-859's inhibition of GLP-1-mediated β-repressor protein coupling at GLP-1R (Figure 15E). Figure 15F shows cAMP accumulation, and Figure 15G shows β-repressor protein coupling of GIPR, while Figure 15H shows GIPR internalization, and Figure 15I shows the time course of GIPR internalization after 100 nM GLP-1R and CT-859. Figure 15J shows that CT-859 inhibits GIP-mediated β-repressor protein coupling at GIPR.

[0055] Figure 16 shows the in vitro properties of GLP-1, liraglutide, and CT-859.

[0056] Figures 17A through 17D present data demonstrating that CT-859 is more potent than liraglutide in vivo. Figure 17A shows the mean (± SE) glucose response to IPGTT in lean C57BL / 6J mice four hours after administration of the mediator, CT-859 (0.01 nmol / kg), CT-859 (0.1 nmol / kg), CT-859 (1 nmol / kg), or CT-859 (10 nmol / kg), and Figure 17B shows the corresponding area under the glucose curve. Figure 17C shows the glucose response to IPGTT in lean C57BL / 6J mice four hours after administration of the mediator, liraglutide (1 nmol / kg), liraglutide (3 nmol / kg), liraglutide (10 nmol / kg), or liraglutide (30 nmol / kg), and Figure 17D shows the corresponding area under the glucose curve. Statistical differences were assessed using one-way ANOVA followed by Bonferroni post-hoc tests. * p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001.

[0057] Figures 18A to 18F present data demonstrating CT-859's binding to GIPR at higher doses. Figures 18A and 18B show GLP-1R four hours after administration of the medium, CT-859 (20 nmol / kg), and CT-859 (200 nmol / kg), respectively. + / + and GLP-1R - / - The mean (± SE) glucose response to IPGTT in mice, and Figure 18C shows the area under the glucose curve. Figures 18D and 18E show the GIPR at four hours after administration of the medium, CT-859 (20 nmol / kg), and CT-859 (200 nmol / kg), respectively. + / + and (E) GIPR - / - The glucose response to IPGTT in mice is shown in Figure 18F, and the area under the glucose curve is illustrated. Statistical differences were assessed using a two-way ANOVA followed by a Bonferroni post-hoc test. **** p < 0.0001.

[0058] Figures 19A to 19L present data showing that the biased GLP-1R agonist reduced glucose over a longer duration compared to the unbiased GLP-1R agonist. Figures 19A to 19C show the mean (± SE) glucose response to IPGTT in GIPR- / - mice at 4 hours (Figure 19A), 24 hours (Figure 19B), and 48 hours (Figure 19C) after administration of the mediator, liraglutide (20 nmol / kg), and CT-859 (20 nmol / kg), and Figure 19D shows the area under the glucose curve. Figures 19E to 19G show the glucose response to IPGTT in lean C57BL / 6J mice at 4 hours (Figure 19E), 24 hours (Figure 19F), and 48 hours (Figure 19G) after administration of the mediator, liraglutide (20 nmol / kg), and CT-859 (20 nmol / kg), and Figure 19H shows the area under the glucose curve. Figures 19I to 19K show the glucose response to IPPTT in C57BL / 6J-DIO mice at 4 hours (Figure 19I), 24 hours (Figure 19J), and 48 hours (Figure 19K) after administration of the mediator, liraglutide (20 nmol / kg), and CT-859 (20 nmol / kg), and Figure 19L shows the area under the glucose curve. Statistical differences were assessed using a two-way ANOVA followed by a Bonferroni post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0059] Figure 20 presents data demonstrating that CT-859 exposure is less than that of liraglutide and that it binds to GIPR only at high doses. Mean (± SE) plasma drug concentrations of liraglutide and CT-859 in CD1 mice are shown.

[0060] Figures 21A through 21H present data demonstrating the superiority of exenatide-Phe1 over exenatide-4 in improving glucose tolerance and reducing body weight. Figure 21A shows mean (± SE) cAMP accumulation, Figure 21B shows β-repressor protein coupling at GLP-1R, Figure 21C shows GLP-1R internalization, and Figure 21D shows the time course of GLP-1R internalization after treatment with 100 nM GLP-1, exenatide-4 (Ex-4), and exenatide-Phe1 (Ex-Phe1). Figure 21E shows glucose levels in C57BL / 6J-DIO mice during the IPGTT eight hours after administration of Ex-4 (2.4 nmol / kg) and Ex-Phe1 (2.4 nmol / kg), and Figure 21F shows the corresponding area under the glucose curve. Figure 21G shows the body weight in lean C57BL / 6J mice after a single ICV injection of Ex-4 (0.025 nmol) and Ex-Phe1 (0.025 nmol), and Figure 21H shows the cumulative food consumption in these mice. Statistical differences were assessed using a two-way ANOVA followed by a Bonferroni post-hoc test. * p < 0.05; **** p < 0.0001; for (G and H) * = p < 0.05: mediator vs. Ex-4 (0.025 nmol), # = p < 0.05: mediator vs. Ex-Phe1 (0.025 nmol), a = p < 0.05: Ex-4 (0.025 nmol) vs. Ex-Phe1 (0.025 nmol).

[0061] Figures 22A through 22D present data demonstrating that ICV administration of CT-859 is superior to liraglutide in reducing food consumption and body weight. Figure 22A shows the mean (± SE) body weight in lean C57BL / 6J mice after ICV administration of the mediator, liraglutide (0.025 nmol), and CT-859 (0.025 nmol), and Figure 22B shows the cumulative food consumption in these mice. Figure 22C shows the GLP-1R mice after ICV administration of the mediator and CT-859 (0.025 nmol). + / + and GLP-1R - / -Body weight in mice and cumulative food consumption in mice are shown in Figure 22D. Statistical differences were assessed using a two-way ANOVA followed by a Bonferroni post-hoc test. * = p < 0.05: mediator vs. liraglutide (0.025 nmol), # = p < 0.05: mediator vs. CT-859 (0.025 nmol), a = p < 0.05: liraglutide (0.025 nmol) vs. CT-859 (0.025 nmol).

[0062] Figures 23A to 23J present data demonstrating that CT-859 is superior to liraglutide in reducing body weight in DIO mice at both non-GIPR and GIPR combined doses. Figures 23A to 23E show the mean (± SE) body weight, food consumption, fasting blood glucose, fasting plasma insulin, and Log(HOMA-IR) of C57BL / 6J DIO mice treated for 19 days with the mediator, liraglutide (20 nmol / kg), and CT-859 (20 nmol / kg), respectively. Figures 23F to 23J show the body weight, food consumption, postprandial blood glucose, postprandial plasma insulin, and Log(HOMA-IR) of C57BL / 6J DIO mice treated for 19 days with the mediator, liraglutide (200 nmol / kg), and CT-859 (200 nmol / kg), respectively. Statistical differences were assessed by using one-way ANOVA or two-way ANOVA followed by a Bonferroni post-hoc test. * p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001; For (A) * = p < 0.05: mediator with liraglutide (20 nmol / kg), # = p < 0.05: mediator with CT-859 (20 nmol / kg), a = p < 0.05: liraglutide (20 nmol / kg) with CT-859 (20 nmol / kg); For (F) * = p < 0.05: mediator with liraglutide (200 nmol / kg), # = p < 0.05: mediator with CT-859 (200 nmol / kg), a = p < 0.05: liraglutide (200 nmol / kg) with CT-859 (200 nmol / kg).

[0063] Figures 24A to 24F present data demonstrating that CT-859 is superior to liraglutide in reducing adipose tissue weight in DIO mice. Figures 24A to 24C show the mean (± SE) inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and liver weight in C57BL / 6J DIO mice treated for 19 days with the medium, liraglutide (20 nmol / kg), and CT-859 (20 nmol / kg), respectively. Figures 24D to 24F show the iWAT, eWAT, and liver weight in C57BL / 6J DIO mice treated for 19 days with the medium, liraglutide (200 nmol / kg), and CT-859 (200 nmol / kg), respectively. Statistical differences were assessed using one-way ANOVA followed by a Bonferroni post-hoc test. * p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001.

[0064] Figure 25 depicts the changes in body weight in ob / ob mice after treatment with the medium (hollow white circles), 200 nmol / kg liraglutide, or 30 nmol / kg CT-868.

[0065] Figure 26 depicts GIPR knockout mice (GIPR knockout mice) administered with the mediator, 20 nmol / kg liraglutide, or 20 nmol / kg CT-859. - / - The graph shows the 4-hour blood glucose level (mg / dL) (far left), 24-hour blood glucose level (mg / dL) (second graph on the left), 48-hour blood glucose level (mg / dL) (third graph on the left) and the corresponding AUC blood glucose (min*mg / dL; far right) during the intraperitoneal glucose tolerance test.

[0066] Figure 27 depicts a pair of graphs showing the changes in body weight (%) and blood glucose levels (mg / dL) of Akita mice at baseline and 15 days after administration of the medium, 20 nmol / kg liraglutide, or 20 nmol / kg CT-868 for 8 and 15 days.

[0067] Figure 28 depicts a pair of graphs showing the 14-day changes in body weight (%); left graph; right graph after 7 and 14 days of administration of the medium + 0.5 LinBit insulin pills, 200 nmol / kg liraglutide + 0.5 LinBit insulin pills, and 200 nmol / kg CT-868 + 0.5 LinBit insulin pills in Akita mice at baseline and after administration of the medium + 0.5 LinBit insulin pills, 200 nmol / kg CT-868 + 0.5 LinBit insulin pills.

[0068] Figure 29 depicts a pair of graphs showing changes in body weight (%); left graph and blood glucose levels (mg / dL) in DIO-STZ mice after 12 days of administration of the medium + 1 LinBit insulin pill, 200 nmol / kg liraglutide + 1 LinBit insulin pill, or 200 nmol / kg CT-868 + 1 LinBit insulin pill.

[0069] Figure 30 depicts a pair of graphs showing changes in body weight (%; left panel) and blood glucose levels (mg / dL; left panel) in Akita mice after 14 days of administration of (mediator + 0.5 LinBit insulin pills), (mediator + 1.5 LinBit insulin pills), 300 nmol / kg CT-868 + 1 blank pill, or (300 nmol / kg CT-868 + 0.5 LinBit insulin pills).

[0070] Figure 31 depicts a pair of graphs showing 4-hour fasting blood glucose levels (mg / dL; left graph) and fasting plasma insulin levels (µIU / mL; right graph) in Akita mice after 14 days of administration of (mediator + 0.5 LinBit insulin pills) (hollow white bars and circles), (mediator + 1.5 LinBit insulin pills), 300 nmol / kg CT-868 + 1 blank pill, or (300 nmol / kg CT-868 + 0.5 LinBit insulin pills).

[0071] Figure 32 depicts the blood glucose levels (mg / dL; top left) and corresponding glucose AUC (percentage relative to the medium) in GLP-1R-KO mice during the intraperitoneal glucose tolerance test 1 hour after administration of the medium or 300 nmol / kg CT-868, as well as the plasma insulin levels (µIU / mL; bottom left) and corresponding insulin AUC (percentage relative to the medium; bottom right).

[0072] Figure 33 depicts the results of a pyruvate tolerance test used to assess endogenous glucose production; that is, blood glucose levels (mg / dL; left panel) and AUC blood glucose (min*mg / dL; right panel) in Akita mice after administration of the medium, 10 nmol / kg CT-868, 30 nmol / kg CT-868, or 100 nmol / kg CT-868 (each dose against the background of 3 U / kg insulin).

[0073] Figure 34 depicts the results of a pyruvate tolerance test used to assess endogenous glucose production; namely, blood glucose levels (mg / dL) in wild-type (left) and GLP-1R-KO (middle) mice after administration of the medium, 30 nmol / kg CT-868, or 100 nmol / kg CT-868, and the corresponding AUC blood glucose (mg / dL*min) in WT and GLP-1R-KO mice; right.

[0074] Figure 35 depicts three plots showing the percentage change in body weight relative to baseline (top), food consumption (g) (bottom left), and plasma glucose (mg / ml) in wild-type mice measured at baseline, 24 hours, 48 ​​hours, and 72 hours after administration of the medium, 0.025 nmol / kg CT-859, or 1 nmol / kg CT-859 (bottom right).

[0075] Figure 36 depicts three plots showing the percentage change in body weight relative to baseline (top), food consumption (g) (bottom left), and plasma glucose (mg / ml) in GLP-1R-KO (GLP-1R- / -) mice measured at baseline, 24 hours, 48 ​​hours, and 72 hours after administration of the medium, 0.025 nmol / kg CT-859, or 1 nmol / kg CT-859 (bottom right).

[0076] Figure 37A depicts the changes in body weight in wild-type mice measured at baseline, 4 hours after administration of the medium, 0.025 nmol / kg CT-859, and 0.025 nmol / kg liraglutide (Lira). The X-axis shows time in hours (hrs), and the Y-axis shows the percentage change in body weight relative to baseline.

[0077] Figure 37B depicts a graph showing food consumption in wild-type mice measured at baseline, 4 hours after administration of the medium, 0.025 nmol / kg CT-859, and 0.025 nmol / kg liraglutide (Lira). The X-axis shows time in hours (hrs), and the Y-axis shows food consumption in grams (g).

[0078] Figures 38A and 38B depict the crossover clinical study design described in Example 4. Pbo: placebo; 868: CT-868.

[0079] Figure 39 depicts the insulin secretion response of patients with type 2 diabetes mellitus (T2DM) to liraglutide and CT-868 compared to participants treated with placebo. Insulin secretion rate (ISR; pmol / kg / min) is plotted on the Y-axis, and glucose level (mmol / L) is plotted on the X-axis.

[0080] Figure 40 depicts a pair of graphs showing changes in glucose and insulin levels in overweight and obese adults with T2DM after administration of placebo, liraglutide, and CT-868.

[0081] Figure 41 depicts a graph showing plasma exposure to CT-868 in patients with type 2 diabetes mellitus (T2DM). Plasma concentrations of CT-868 (ng / mL) are plotted on the Y-axis, and time (hours) are plotted on the X-axis.

[0082] Figures 42A through 42H present data demonstrating that CT-868 is a cAMP-based dual agonist targeting the GLP-1 / GIP receptor with an imbalanced selectivity favorable to GLP-1R. Figures 42B and 42C show dose-response curves for cAMP assays in cells expressing human GLP-1R (Figure 42B) and human GIPR (Figure 42C). The curves depict responses to GLP-1, liraglutide, GIP, and / or CT-868. Figures 42D and 42E show dose-response curves for β-repressor protein 2 recruitment in cells expressing human GLP-1R (D) and human GIPR (E). The curves depict responses to GLP-1, liraglutide, GIP, and / or CT-868. Figures 24F to 24G show the time-course analysis of receptor internalization for human GLP-1R (F) and GIPR (G) in response to 100 nM GLP-1, liraglutide, GIP, and / or CT-868. Internalization was monitored at two-minute intervals for 120 minutes, and values ​​were normalized to mediator controls. Figures 24H to 24I show the receptor internalization analysis of human GLP-1R (H) and GIPR (I) in response to GLP-1, GIP, liraglutide, and / or CT-868 at 120 minutes (H) and 60 minutes (I) after treatment. Data for all figures represent the mean ± SEM of each treatment n = 6–9 replicates.

[0083] Figures 43A to 43I present data demonstrating the mechanism by which CT-868 enhances glucose homeostasis via GLP-1R and GIPR. Figures 43A to 43B show blood glucose and plasma insulin levels during IPGTT in lean C57BL / 6NJ mice treated with the medium or CT-868 at 3 nmol / kg, 10 nmol / kg, and 30 nmol / kg. Data are presented as mean ± SEM for n = 8 animals per group. Figures 43C to 43F show blood glucose and plasma insulin levels during IPGTT in wild-type and GLP-1 KO mice treated with the medium or CT-868 at 30 nmol / kg and 300 nmol / kg. Data are presented as mean ± SEM for n = 5–6 animals per group. Figure 43G shows the AUC analysis of blood glucose levels in wild-type and GLP-1 KO mice treated with the mediator or CT-868 at 30 nmol / kg and 300 nmol / kg during the ipPTT. Data are presented as mean ± SEM for n = 6 animals per group. Figures 43H to 43I show the MMTT in 23-week-old DIO mice 24 hours after administration of CT-868, liraglutide, or the mediator via subcutaneous injection (10 mL / kg). Mice underwent a 16-hour fasting period prior to the MMTT. Blood glucose and plasma insulin levels were monitored at 15, 30, 60, and 120 minutes postprandial. Data are presented as mean ± SEM for n = 8 animals per group. Statistical significance is expressed as follows: ** p < 0.01, *** p < 0.001, **** p < 0.0001, ap < 0.05 for the medium and 30 nmol / kg CT-868, and bp < 0.05 for 30 nmol / kg liraglutide and 30 nmol / kg CT-868. One-way ANOVA and Tukey's multiple comparison test were used. Abbreviations: IPGTT = Intraperitoneal glucose tolerance test; IVGTT = Intravenous glucose tolerance test; MMTT = Mixed diet tolerance test; DIO = Diet-induced obesity; ipPTT = Pyruvate tolerance test.

[0084] Figures 44A through 44D present data demonstrating the dose-dependent reduction in body weight of DIO and ob / ob mice with long-term CT-868 administration. Figure 44A shows the dose-dependent effect of CT-868 (3, 10, or 30 nmol / kg / day) on body weight in DIO mice over 14 days. Changes in body weight are expressed as a percentage of the initial body weight recorded on day 1. Data are presented as mean ± SEM for each treatment group n = 10 animals. Statistical significance was expressed as follows: * p < 0.05 for the medium versus 3 nmol / kg CT-868, # p < 0.05 for the medium versus 10 nmol / kg CT-868, ^ p < 0.05 for the medium versus 30 nmol / kg CT-868, ap < 0.05 for 3 nmol / kg CT-868 versus 10 nmol / kg CT-868, bp < 0.05 for 3 nmol / kg CT-868 versus 30 nmol / kg CT-868, and cp < 0.05 for 10 nmol / kg CT-868 versus 30 nmol / kg CT-868. Analysis was performed using two-way ANOVA and Tukey's multiple comparison test. Figure 44B shows the tissue weight analysis at the end of the study in DIO mice treated with CT-868. Figures show the dose-dependent effects of CT-868 (3, 10, or 30 nmol / kg / day) on iWAT, eWAT, and liver weight, respectively. Data are presented as mean ± SEM for each treatment group (n = 10 animals). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Analyses were performed using two-way ANOVA with Tukey's multiple comparison test. Figures 44C to 44D show comparative analyses of body weight change (44C) and 24-hour food consumption (44D) in ob / ob mice administered the medium, CT-868 (10 or 30 nmol / kg / day), or liraglutide (200 nmol / kg / day) daily over 24 days. Changes in body weight are expressed as a percentage of initial body weight recorded on day 1 (44C). Figure 44D presents food intake data for days 1, 8, and 15. Data represent mean ± SEM for each treatment group (n = 8 animals).The statistical significance of Figure 44C is expressed as follows: * p < 0.05 for the medium and 200 nmol / kg liraglutide, # p < 0.05 for the medium and 10 nmol / kg CT-868, ^ p < 0.05 for the medium and 30 nmol / kg CT-868, ap < 0.05 for 200 nmol / kg liraglutide and 10 nmol / kg CT-868, bp < 0.05 for 200 nmol / kg liraglutide and 30 nmol / kg CT-868, and cp < 0.05 for 10 nmol / kg CT-868 and 30 nmol / kg CT-868. The statistical significance of Figure F is expressed as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Analysis was performed using two-way ANOVA and Tukey's multiple comparison test. Abbreviations: DIO = diet-induced obesity; iWAT = inguinal white adipose tissue; eWAT = epididymal white adipose tissue; ob / ob mice = leptin-deficient mice.

[0085] Figures 45A and 45B present data demonstrating the positive effects of CT-868 on energy metabolism. Figure 45A shows the effect of CT-868 on RER (VCO2 / VO2 ratio) in DIO mice administered the medium or 20 nmol / kg / day of CT-868 daily for 12 days, and Figure 45B shows the dynamic activity. Data represent the inverse conversion adjusted mean + SEM for each treatment group n = 8 animals, and dark cycles (x pm to x am) are shown by gray shaded boxes. Significant differences from the medium: * p < 0.05, ** p < 0.01, *** p < 0.001. Analyses were performed using a generalized linear model, with treatment as a factor and body weight on day 1 and log(baseline) (mean from day -2 to day 0) as covariates. Abbreviations: VO2 = oxygen consumption; VCO2 = carbon dioxide production; RER = ventilation ratio.

[0086] Figures 46A through 46F present schematic diagrams and data related to the first-in-human single-escalation-dose and multiple-escalation-dose studies of CT-868. Figure 46A shows the study design for Part 1: Single-escalation-dose (SAD), and Figure 46B shows the study design for Part 2: Multiple-escalation-dose (MAD). In Part 1 (SAD phase), participants received a single dose of the study drug (CT-868 or a matched placebo) via subcutaneous injection. In Part 2 (MAD phase), participants were randomly assigned to receive either CT-868 or a placebo via subcutaneous injection (once daily from Day 1 to Day 14). All participants in Parts 1 and 2, except for the 'lean volunteers' (cohort S7), were overweight / obese individuals who were otherwise healthy. Figures 46C and 46D show the plasma concentration-time curves of CT-868 after single or multiple doses, respectively. This figure shows the mean CT-868 plasma concentrations by dose cohort after single or multiple doses of CT-868. Values ​​are plotted using a semi-logarithmic scale. Concentrations below the limit of quantitation (0.2 ng / mL) and with a zero mean are not plotted. Figure 46E shows the CT-868 AUC against glucose, insulin, and C-peptide after a dietary tolerance test. 0-last Relationship with baseline-adjusted AUC. Red data points indicate all doses up to 7.5 mg; blue data points indicate only the 11 mg dose. Data were analyzed with and without the 11 mg dose (cohort S9) because this MTT was performed 24 hours after administration, while the MTTs for placebo and cohorts S1 through S8 were performed 3 hours after administration. Daily administration of CT-868 for 14 consecutive days resulted in dose-dependent weight loss in participants with overweight / obesity (Figure 46F). Abbreviations: AUC = Area under the curve; BMI = Body Mass Index; BW = Body Weight; D = Day; MAD = Multiple escalation dose; MD = Multiple dose; SAD = Single escalation dose; T2DM = Type 2 diabetes.

[0087] Figures 47A and 47B illustrate schematic diagrams of the placebo-controlled and comparator-controlled crossover study (CT-868-003). Part 1 included 12 obese participants. In Group 1, 6 participants received a crossover treatment of CT-868 (Day 1: 5.0 mg, Day 2: 3.25 mg), matched placebo, and liraglutide (Day 1: 0.6 mg, Day 2: 1.2 mg), each for two days, with a 14-day washout period between treatments (see drug allocation diagram, STAR method). In Group 2, 6 participants received a crossover treatment of CT-868 and matched placebo, each for two days, with a 14-day washout period in between. Part 2 included 20 participants with T2DM, 13 in Group 1 (crossover between CT-868 [Day 1: 5.0 mg, Day 2: 3.25 mg, Day 3: 3.25 mg], matched placebo, and liraglutide [Day 1: 0.6 mg, Day 2: 1.2 mg, Day 3: 1.2 mg]), and seven in Group 2 (crossover between CT-868 and matched placebo). Abbreviations: GE, gastric emptying test; GGI = graded glucose infusion; T2DM = type 2 diabetes; w / o = washout.

[0088] Figures 48A and 48B present data demonstrating the potent pancreatic effect of CT-868 in participants with overweight / obesity and those with type 2 diabetes mellitus (T2DM). Figure 48A shows the relationship between the rate of insulin secretion (ISR) and ambient glucose during staggered glucose infusion in overweight / obesity participants 2 days after CT-868 administration, and Figure 48B shows the relationship between the rate of insulin secretion (ISR) and ambient glucose during staggered glucose infusion in participants with T2DM 2 days after CT-868 administration. Abbreviations: G = glucose; ISR = rate of insulin secretion; LS = least squares; T2DM = type 2 diabetes mellitus.

[0089] Figure 49 presents data showing the effects of CT-868 on plasma glucose, insulin, and glucagon during a mixed diet tolerance test in participants with T2DM. The figure illustrates the percentage change in plasma glucose, insulin, and glucagon relative to baseline during the mixed diet tolerance test performed 3 days after CT-868 administration.

[0090] Figure 50 presents data showing that CT-868 and liraglutide exhibited similar gastric emptying delays as placebo. Paracetamol plasma concentration-time curves for CT-868, placebo, and liraglutide in participants with T2DM during the paracetamol absorption assay. Abbreviation: AUC = Area under the curve; C max = Maximum plasma concentration; CV = Coefficient of variation; SD = Standard deviation, T max = Time to reach maximum plasma concentration.

[0091] Figure 51 illustrates the titration protocol for the Phase 2 clinical study described in Example 7.

[0092] Figure 52 depicts two graphs showing the percentage change in HbA1c relative to baseline up to week 26 in overweight or obese adults with type 2 diabetes (T2DM) after administration of placebo, 1.75 mg CT-868, 3.25 mg CT-868, or 4 mg CT-868 (Figure A; left), and the percentage of subjects who reached their target HbA1c at week 26 (Figure B; right).

[0093] Figure 53 depicts six plots showing changes in total cholesterol (top left), low-density lipoprotein (LDL; top center), high-density lipoprotein (HDL; top right), triglycerides (bottom left), very low-density lipoprotein (VLDL; bottom center), and apolipoprotein B (ApoB; bottom right) measured at baseline, 4 weeks after administration of placebo, 1.75 mg CT-868, 3.25 mg CT-868, or 4 mg CT-868, and at 4 weeks after administration of placebo.

[0094] Figure 54 depicts two graphs showing the mean changes in blood pressure (Figure A; left) and liver enzyme levels (Figure B; right) measured at week 26 after administration of placebo, 1.75 mg CT-868, 3.25 mg CT-868, or 4 mg CT-868 in overweight or obese adults with type 2 diabetes mellitus (T2DM). SBP: Systolic blood pressure; DBP: Diastolic blood pressure; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: Gamma-glutamyl transferase.

[0095] Figure 55 shows the overall trial design, including arms and doses, for the Phase 2 trial of CT-868 in overweight and obese adults with T1D.

[0096] Figure 56 shows the main study activities before and after randomization of the Phase 2 trial of CT-868 in overweight and obese adults with T1D, including 2-week CGM and insulin dose data collection and patient education on diet and exercise.

[0097] Figure 57 shows the main study activities before and after randomization in the Phase 2 trial of CT-868 in overweight and obese adults with T1D, including HbA1c, PRO, CRU visit, blood samples, study drug concentration (approximately 24 hours after administration), and extended subgroup PK sampling. Detailed Implementation

[0098] This disclosure provides a therapy (e.g., monotherapy or adjunctive therapy) for patients with insulin dependence or insulin resistance (e.g., patients with type 1 and type 2 diabetes) by using single-molecule agonists targeting both glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) and glucose-dependent insulinotropic peptide (GIP) receptor (GIPR). The therapies described herein can improve the outcomes of diabetes treatments such as insulin therapy in a variety of ways.

[0099] In some embodiments, the therapies described herein (e.g., monotherapy or adjunctive therapy) improve glycemic control in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). Despite advances in diabetes therapies such as insulin therapy, drug delivery, and glycemic monitoring, most patients with T1DM and T2DM still do not achieve current standard-of-care glycemic control targets. The therapies described herein can improve the effectiveness of existing diabetes therapies, including insulin therapy. Furthermore, the therapies described herein can improve insulin-independent glucose disposal, partly due to GIP-mediated effects. In specific embodiments, the therapies described herein (e.g., monotherapy or adjunctive therapy) improve glycemic control in patients with T1DM.

[0100] In some embodiments, the therapies described herein (e.g., monotherapy or adjunctive therapy) reduce the insulin dose required to maintain glucose control while improving glycated hemoglobin A1c (HbA1c) levels without exacerbating the risk of hypoglycemia and diabetic ketoacidosis (DKA). In specific embodiments, the therapies described herein (e.g., monotherapy or adjunctive therapy) reduce the insulin dose required to maintain glucose control in patients with type 1 diabetes mellitus (T1DM) while improving glycated hemoglobin A1c (HbA1c) levels without exacerbating the risk of hypoglycemia and diabetic ketoacidosis (DKA).

[0101] In some embodiments, the therapies described herein (e.g., as monotherapy or adjunctive therapy) improve insulin sensitivity and may concurrently address pathophysiological deficits in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). Patients with T1DM and T2DM typically exhibit insulin resistance in adipose, liver, and skeletal muscle tissue. They also have decreased insulin secretion and increased glucagon secretion, both of which exacerbate hyperglycemia. Patients with T1DM and T2DM exhibit increased renal glucose reabsorption and accelerated gastric emptying. Given the dual mechanism of action of the agonists described herein, the therapies have the potential to address these core deficits. In specific embodiments, the therapies described herein (e.g., as monotherapy or adjunctive therapy) improve insulin sensitivity and may concurrently address pathophysiological deficits in patients with T1DM.

[0102] In some embodiments, the therapies described herein (e.g., monotherapy or adjunctive therapy) reduce the weight of patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) in need. Approximately two-thirds of patients with T1DM and T2DM are overweight or obese. Insulin therapy can exacerbate weight gain, which risks exacerbating the negative health effects of obesity. Therefore, interventions that can reduce excess fat and improve insulin sensitivity, thereby reducing the total daily insulin load in patients with T1DM and T2DM, are an important component of the therapy. In specific embodiments, the therapies described herein (e.g., monotherapy or adjunctive therapy) reduce the weight of patients with T1DM in need.

[0103] In some embodiments, the therapies described herein (e.g., monotherapy or adjunctive therapy) reduce cardiovascular disease risk factors in patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). Patients with T1DM and T2DM who have ideal glucose control still have an increased risk of cardiovascular disease. This is believed to be caused by several precipitating factors, including hyperinsulinemia, insulin resistance, increased inflammatory atmosphere, increased atherogenic lipids, and endothelial dysfunction. Therapies that reduce insulin dosage and enhance insulin sensitivity can reduce the risk of cardiovascular disease. In specific embodiments, the therapies described herein (e.g., monotherapy or adjunctive therapy) reduce cardiovascular disease risk factors in patients with T1DM.

[0104] In some embodiments, the therapies described herein (e.g., monotherapy or adjunctive therapy) reduce blood pressure (e.g., systolic blood pressure) and / or atherogenic lipids in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). In specific embodiments, the therapies described herein (e.g., monotherapy or adjunctive therapy) reduce blood pressure (e.g., systolic blood pressure) and / or atherogenic lipids in patients with type 1 diabetes mellitus (T1DM).

[0105] I. Dual GLP-1R / GIPR agonists

[0106] GLP-1 and GIP are the main intestinal glucagons released by the gastrointestinal tract in response to food intake. Both hormones regulate glucose-dependent insulin secretion. GLP-1 also reduces glucagon secretion, slows gastric emptying, promotes satiety, and reduces food intake. Several GLP-1 mimics have been approved for the treatment of type 2 diabetes (T2DM), but their efficacy is limited by gastrointestinal side effects such as nausea, vomiting, and diarrhea.

[0107] The primary function of GIP is to stimulate glucose-dependent insulin secretion. It also regulates glucagon secretion in an inverse glucose-dependent manner to prevent hypoglycemia and does not delay gastric emptying. GIP also stimulates glucose uptake in fat cells and promotes bone strength.

[0108] The dual GLP-1R / GIPR agonist used in this therapy effectively activates the production of cyclic adenosine monophosphate (cAMP) but has almost no activity on the β-repressor signaling pathways of GLP-1R or GIPR. That is, the agonist is completely biased towards cAMP activation in both GLP-1R and GIPR, rather than partially biased (i.e., having some β-repressor activity) or unbiased (i.e., having full β-repressor activity). β-repressor activation of kinase signaling pathways also causes GLP-1R and GIPR to be shut down and internalized (Hsia et al., Curr Opin Endocrinol DiabetesObes. (2017) 24(1):73-9). This agonist does not cause internalization and thus desensitizes either GLP-1R or GIPR, thereby enhancing signal transduction efficacy.

[0109] This dual GLP-1R / GIPR agonist can have the following structural formula:

[0110]

[0111] (Formula I); (SEQ ID NO: 1),

[0112] Wherein R is a ring. In some embodiments, the ring is a cycloalkyl or heterocyclic group with a size of 4 to 8 atoms. The ring may be substituted with one or more carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro groups, but is not limited thereto. The ring may be aromatic or non-aromatic and may be fused with one or more additional rings to form a fused, bridged, or spirobicyclic moiety. In some embodiments, R is:

[0113] (Formula II).

[0114] In some embodiments, R is

[0115] (Formula III).

[0116] In some embodiments, this agonist has the following structural formula:

[0117]

[0118] (Form IV)

[0119] This compound is also referred to in this paper as CT-859, and can be described as follows:

[0120]

[0121] (Formula V); (SEQ ID NO: 1).

[0122] In some embodiments, this agonist has the following structural formula:

[0123]

[0124] (Form VI).

[0125] This compound is also referred to in this paper as CT-868, and can be described as follows:

[0126] (Formula VII); (SEQ ID NO: 1).

[0127] Pharmaceutical salts or esters of the above-mentioned compounds (e.g., compounds of formula I, IV, V, VI or VII) may also be used in the therapies described herein (e.g., as monotherapy or adjunctive therapy).

[0128] The dual GLP-1R / GIPR agonists discussed in this article, such as CT-859 and CT-868, are completely biased towards both GLP-1R and GIPR, with minimal β-repressor conjugation. Dual agonists are likely to be well-tolerated and cause fewer adverse reactions than other enterokinin mimics. For example, toxicological data showed that CT-868 was well-tolerated at the highest tested dose (100 times the pharmacologically active dose). No cases of vomiting were reported in non-human primate studies, a common adverse reaction reported with other enterokinin mimics.

[0129] The potency of this dual agonist in activating cAMP production can be measured by well-known cell-based assays. In some embodiments, this dual agonist activates cAMP at human GLP-1R and GIPR with a potency of about 0.05 to about 5 nM. In some embodiments, this dual agonist favors GLP-1R at a ratio of about 1:5 to about 1:50 (e.g., about 1:10, about 1:20, about 1:30, or about 1:40) relative to GIPR. For example, CT-868 activates cAMP at human GLP-1R and GIPR with potencies of about 0.17 nM and about 3.3 nM, respectively, and favors GLP-1R at a ratio of about 19.4 relative to GIPR.

[0130] II. Pharmaceutical compositions

[0131] The dual GLP-1R / GIPR agonist used in this therapy may be provided in a pharmaceutical composition containing an agonist and a pharmaceutical excipient. In some embodiments, the agonist (e.g., CT-868) is provided in a sterile aqueous solution suitable for subcutaneous injection. In some embodiments, the agonist (e.g., CT-868) is provided at a concentration of about 1 to about 100 mg / mL (e.g., about 5 to about 25 mg / mL, about 7.5 to about 20 mg / mL, or about 10 to about 15 mg / mL). As used herein, values ​​between the ranges and values ​​are also intended to be included as part of this disclosure. Furthermore, ranges using any combination of the listed values ​​as upper and / or lower limits are intended to be included.

[0132] In some embodiments, the agonist (e.g., CT-868) is provided at a concentration of 15 mg / mL in a solution comprising 20 mM disodium hydrogen phosphate heptahydrate / sodium dihydrogen phosphate buffer, propylene glycol, and phenol (pH 7.0). The pharmaceutical composition can be administered to a patient using, for example, a syringe or syringe such as an injection pen (e.g., an auto-injector). The pharmaceutical composition can be clear or colorless. In some embodiments, the pharmaceutical composition is a clear liquid substantially free of visible particles. In some embodiments, the level of subvisible particles ≥ 10 µm in each container is ≤ 6000. In some embodiments, the level of subvisible particles ≥ 25 µm in each container is ≤ 600. In some embodiments, the impurity-free area is ≥ 5.0% of the area. In some embodiments, the total impurities are ≤ 7.0% of the area. In some embodiments, the bacterial endotoxin is ≤ 5 EU / mL.

[0133] In some embodiments, the system for subcutaneous administration of CT-868 is a needle-based injection system having an integrated, non-replaceable 3-mL Type 1 glass cartridge. The system may be referred to as a pen. In some embodiments, each pen contains more than one dose of CT-868. In some embodiments, each pen contains a single dose of CT-868. In some embodiments, each pen contains five or more doses of CT-868.

[0134] In some embodiments, a fixed dose of CT-868 is delivered in 0.07 mL of the composition. In some embodiments, a fixed dose of CT-868 is delivered in 0.12 mL of the pharmaceutical composition. In some embodiments, a fixed dose of CT-868 is delivered in 0.17 mL of the pharmaceutical composition. In some embodiments, a fixed dose of CT-868 is delivered in 0.22 mL of the pharmaceutical composition. In some embodiments, a fixed dose of CT-868 is delivered in 0.27 mL of the pharmaceutical composition. Unless otherwise stated, the weight of CT-868 listed in this disclosure is the weight of the free base (active fraction) of CT-868.

[0135] This document also provides articles and kits that include pharmaceutical compositions or agonists. In such articles and kits, the pharmaceutical composition or agonist may be provided in a system (e.g., a pen, vial, or needle-based injection system). The articles and kits may also include instructions for using the pharmaceutical composition or agonist in the methods provided herein.

[0136] In some embodiments, the article of manufacture is an injection device, syringe, syringe, or vial. The injection device, syringe, syringe, or vial may be for single use or may include a single-dose unit of a pharmaceutical composition or agonist.

[0137] III. Diabetes therapy

[0138] The pharmaceutical composition can be used as monotherapy for patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) or as a combination therapy to assist insulin or other glycemic control therapies. The pharmaceutical composition can be administered subcutaneously at intervals deemed appropriate by a physician, such as daily, every two days, every three days, every four days, every five days, every six days, or weekly. In some embodiments, the pharmaceutical composition (e.g., containing CT-868 as the active pharmaceutical ingredient) is administered via subcutaneous injection (e.g., self-injection) once daily (QD) at a body site (e.g., abdomen, upper arm, thigh, or buttock). In some embodiments, the pharmaceutical composition is administered independently of meals.

[0139] In some embodiments, the patient has ≥25 kg / m 2 ≥27 kg / m 2 ≥30 kg / m 2 ≥35 kg / m 2 ≥40 kg / m 2 or ≥45 kg / m 2 BMI.

[0140] In some embodiments, the agonist described herein (e.g., CT-868) is administered to patients with T1DM or T2DM at a steady dose of about 1 mg to about 30 mg, such as about 1 mg to about 20 mg, about 1 mg to about 15 mg, about 1 mg to about 5 mg, about 2 mg to about 15 mg, about 5 mg to about 15 mg, or about 5 mg to 12 mg.

[0141] In some embodiments, a pharmaceutical composition containing CT-868 is administered subcutaneously to a patient with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 1.8 mg or about 2 mg of CT-868.

[0142] In some embodiments, a pharmaceutical composition containing CT-868 is subcutaneously injected into patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 2.5 mg or about 2.6 mg of CT-868.

[0143] In some embodiments, a pharmaceutical composition containing CT-868 is administered subcutaneously to a patient with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 3 mg, about 3.3 mg, or about 3.5 mg of CT-868.

[0144] In some embodiments, a pharmaceutical composition containing CT-868 is administered subcutaneously to a patient with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 4 mg, about 4.1 mg, or about 4.5 mg of CT-868.

[0145] In some embodiments, a pharmaceutical composition comprising CT-868 is administered subcutaneously to patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 5 mg, about 5.2 mg, or about 5.5 mg of CT-868. The 5.2 mg daily dose of CT-868 is administered in two divided doses of 2.6 mg each.

[0146] In some embodiments, a pharmaceutical composition comprising CT-868 is administered subcutaneously to patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 6 mg, about 6.5 mg, or about 6.6 mg of CT-868. The 6.6 mg daily dose of CT-868 is administered in two divided doses of 3.3 mg each.

[0147] In some embodiments, a pharmaceutical composition containing CT-868 is administered subcutaneously to a patient with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 7 mg or about 7.5 mg of CT-868.

[0148] In some embodiments, a pharmaceutical composition containing CT-868 is administered subcutaneously to a patient with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 8 mg or about 8.5 mg of CT-868.

[0149] In some embodiments, a pharmaceutical composition comprising CT-868 is administered subcutaneously to a patient with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 9 mg or about 9.5 mg of CT-868.

[0150] In some embodiments, a pharmaceutical composition containing CT-868 is subcutaneously injected into a patient with T1DM or T2DM at a daily dose of about 10 mg or about 10.5 mg of CT-868.

[0151] In some embodiments, a pharmaceutical composition containing CT-868 is subcutaneously injected into a patient with T1DM or T2DM at a daily dose of about 11 mg or about 11.5 mg of CT-868.

[0152] In some embodiments, a pharmaceutical composition containing CT-868 is subcutaneously injected into a patient with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 12 mg or about 12.5 mg of CT-868.

[0153] In some embodiments, a pharmaceutical composition containing CT-868 is administered subcutaneously to a patient with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 13 mg or about 13.5 mg of CT-868.

[0154] In some embodiments, a pharmaceutical composition containing CT-868 is subcutaneously injected into a patient with T1DM or T2DM at a daily dose of about 14 mg or about 14.5 mg of CT-868.

[0155] In some embodiments, a pharmaceutical composition containing CT-868 is administered subcutaneously to a patient with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) at a daily dose of about 15 mg or about 15.5 mg of CT-868.

[0156] In some embodiments, treatment with CT-868 is initiated according to an up-titration protocol, i.e., treatment begins with a low dose and gradually increases over a period of time to a higher or maximum tolerated dose. In some embodiments, titration is performed over a period of approximately 1, 2, 4, 6, 8, or 10 weeks or longer; in further embodiments, during titration, a pharmaceutical composition containing CT-868 is injected QD. In some embodiments, titration begins with a dose of 1.1 mg (starting dose) and eventually reaches a dose between 1.8 mg and 6.6 mg (e.g., 1.8 mg, 4.1 mg, 5.2 mg, or 6.6 mg) (maintenance dose or maximum dose).

[0157] In some embodiments, titration is performed according to the following protocol:

[0158] ● 1.1 mg QD, from day 1 to day 7, and

[0159] ● 1.8 mg QD, from week 1 to week 2.

[0160] In some embodiments, titration is performed according to the following protocol:

[0161] ● 1.1 mg QD, from day 1 to day 7.

[0162] ● 1.8 mg QD, from week 1 to week 2,

[0163] ● 2.6 mg QD, from week 2 to week 4.

[0164] ● 3.3 mg QD, from week 4 to week 6, and

[0165] ● 4.1 mg QD, from week 6 to week 16.

[0166] In some embodiments, titration is performed according to the following protocol:

[0167] ● 1.1 mg QD, from day 1 to day 7.

[0168] ● 1.8 mg QD, from week 1 to week 2,

[0169] ● 2.6 mg QD, from week 2 to week 4.

[0170] ● 3.3 mg QD, from week 4 to week 6,

[0171] ● 4.1 mg QD, from week 6 to week 8,

[0172] ●5.2 mg QD, from week 8 to week 10, and

[0173] ●6.6 mg QD, from week 10 to week 16.

[0174] In some embodiments, titration is performed according to the following protocol:

[0175] ● 1.0 mg QD, continued for weeks 1 and 2.

[0176] ● 2.0 mg QD, continued in weeks 3 and 4.

[0177] ● 3.0 mg QD, continued in weeks 5 and 6.

[0178] ● 4.0 mg QD, continued for weeks 7 and 8.

[0179] ● 5.0 mg QD, continued in weeks 9 and 10.

[0180] ● 6.0 mg QD, continued for weeks 11 and 12.

[0181] ● 8.0 mg QD, continued from week 12 to week 15.

[0182] ● 10.0 mg QD, continued from week 16 to week 20, and

[0183] ● 12.0 mg QD, continued from week 21 to week 48.

[0184] In some embodiments, titration is performed according to the following protocol:

[0185] ● 1.0 mg QD, continued for weeks 1 and 2.

[0186] ● 2.0 mg QD, continued in weeks 3 and 4.

[0187] ● 3.0 mg QD, continued in weeks 5 and 6.

[0188] ● 4.0 mg QD, continued for weeks 7 and 8.

[0189] ● 5.0 mg QD, continued in weeks 9 and 10.

[0190] ● 6.0 mg QD, continued for weeks 11 and 12.

[0191] ● 7.0 mg QD, continued for weeks 13 and 14, and

[0192] ● 8.0 mg QD, continued from week 15 to week 48.

[0193] In some embodiments, titration is performed according to the following protocol:

[0194] ● 1.0 mg QD, continued from week 1 to week 4.

[0195] ● 2.0 mg QD, continued from week 5 to week 8.

[0196] ● 3.0 mg QD, continued from week 9 to week 12, and

[0197] ● 4.0 mg QD, continued from week 13 to week 48.

[0198] When titrating up, if the dose is not tolerated at any stage, the administration can be reversed to the previously tolerated dose for the next dose (i.e., down-titering is permitted). In some embodiments, the patient can then attempt up-titering at a subsequent scheduled dosing time.

[0199] While receiving this adjunctive therapy, patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) may receive or continue insulin therapy. Insulin doses can be given at a basal dose, which provides a steady amount of insulin delivered throughout the day; and / or a bolus dose, which provides a dose of insulin in the diet to help transfer absorbed glucose from the blood to muscles and fat. A bolus dose is also known as a nutritional dose or mealtime dose. Insulin therapy may require the use of rapid-acting or fast-acting insulin, regular or short-acting insulin, intermediate-acting insulin, long-acting insulin, premixed or mixed insulin, or inhaled insulin as directed by a physician. Insulin dosing schedules may depend on the patient’s weight, blood glucose levels and other health conditions, type of insulin, food intake, and level of physical activity. Insulin can be administered with a syringe, pump, pen, inhaler, or injection port. For example, insulin can be administered subcutaneously via multiple daily injections (MDI) or continuously subcutaneously via a pump (continuous subcutaneous insulin infusion, CSII), where the pump can be a standalone device or a hybrid closed-loop system (e.g., coupled with a continuous glucose monitor or CGM) to provide automated insulin delivery based on sensed real-time blood glucose values. Injections can be administered in the abdomen, upper arm, thigh, and / or buttock. This adjunctive therapy can be used in conjunction with any and all types of insulin therapy. As used herein, the term "insulin dependent" refers to a patient requiring insulin therapy.

[0200] The adjunctive therapies described in this article promise to reduce the required insulin dose or dosing frequency in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM), improve insulin sensitivity, and / or reduce excess weight (e.g., by suppressing food intake). This is particularly beneficial for T1DM and T2DM patients who are prone to obesity and insulin resistance (dual diabetes). Therefore, the combined use of adjunctive non-insulin hypoglycemic agents such as CT-859 or CT-868 with insulin can provide improved diabetes management. For example, most patients with T1DM require three or more insulin injections daily, with the dosage adjusted based on self-monitored blood glucose levels and carbohydrate intake. Administering the agonists described in this article (e.g., CT-859 or CT-868) as adjunctive therapy in such patients can reduce the number of daily insulin injections required to maintain normal or near-normal blood glucose levels. Administration of agonists can also produce additional benefits such as weight loss, improved insulin sensitivity, and reduced blood pressure and / or plasma lipids.

[0201] In some embodiments, patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) receive an alternative therapy for their diabetes symptoms. For example, patients may receive diet therapy and exercise therapy. In patients with T1DM, agonists may be administered as adjuncts to insulin therapy. In patients with T2DM, agonists may be administered as adjuncts to diet or exercise and oral antidiabetic medications.

[0202] The term "adjunctive therapy" means that this agonist will be used in conjunction with another therapy. For example, in patients with type 1 diabetes mellitus (T1DM), treatment with this agonist may be adjunctive to insulin therapy. In patients with type 2 diabetes mellitus (T2DM) and / or obesity or overweight, treatment with this agonist may be adjunctive to, for example, diet or exercise.

[0203] This agonist may be used as adjunct to one or more other therapies. As used herein, "other therapies" means therapies performed in addition to treatment with this agonist. Other therapies may include, for example, insulin therapy, dietary therapy, exercise therapy, hypertension therapy, and / or lipid-lowering therapy.

[0204] In some embodiments, the above-described dosing regimen can also be used for weight management to help patients lose excess weight.

[0205] IV. T1DM patient characteristics

[0206] In some embodiments, patients treated with this method are 18 years of age or older. In some embodiments, patients have a history of a T1DM diagnosis, for example, at least one year prior to treatment. In some embodiments, the patient's body mass index (BMI) is greater than or equal to 25.0 kg / m². 2 Patients' pretreatment glycated hemoglobin A1c (HbA1c) levels may range from 7.0% to 9.5% (inclusive).

[0207] In some embodiments, prior to receiving this treatment, the patient has been treated with insulin, for example, for six months or longer prior to treatment, using an insulin pump for continuous subcutaneous insulin infusion (CSII) or with multiple daily injection (MDI) insulin. The patient may have received a stable insulin formulation, dose (i.e., within 10% of the total daily dose), and device (i.e., without switching from MDI to a pump or vice versa) for two months or longer prior to treatment.

[0208] In some embodiments, the patient does not have type 2 diabetes (T2DM) or any other type of diabetes other than T1DM. In some embodiments, the patient has not experienced diabetic ketoacidosis (DKA) or severe hypoglycemia (e.g., grade 3 as defined in the ADA Diabetes Care Standards (ADA2022)) within, for example, three months prior to treatment. The patient may not have impaired hypoglycemic perception prior to treatment (i.e., a Clarke Questionnaire score of 4 or higher (Clarke et al., Diabetes Care.(1995) 18(4):517-22)). In some embodiments, the patient receives insulin via a CSII or MDI. In some embodiments, the patient does not receive insulin via a formulation other than a CSII or MDI (e.g., inhaled insulin).

[0209] In some embodiments, the patient has not used (e.g., within the last six months or three months) any medications that may interfere with glycemic control (except insulin) (e.g., monoamine oxidase inhibitors, growth hormone); any adjunctive treatment for diabetes (e.g., pramlintide, metformin, glucagon-like peptide-1 [GLP-1] analogs [e.g., exenatide], GLP-1 receptor agonists (GLP-1RAs) [e.g., cesamagglutide, liraglutide, or dulaglutide], GLP-1 / GIP RA (Mounjaro™), sodium-glucose cotransporter-2 inhibitors, sulfonylureas, or dipeptidyl peptidase-4 inhibitors); any prescription medications and / or any over-the-counter medications, including herbal and / or dietary supplements for weight loss (e.g., orlistat, locaserin, phentermine topiramate, naltrexone-bupropion, cesamagglutide, liraglutide, Garcinia Cambogia extract, Hydroxycut®). Or glucomannan), or herbal and / or dietary supplements used for weight gain (e.g., testosterone, growth hormone, anabolic steroids, or amino acid supplements); long-term (>14 consecutive days) systemic glucocorticoid therapy (excluding topical, intraocular, intranasal, intra-articular, or inhaled formulations), or evidence of significant active autoimmune disorders (e.g., lupus or rheumatoid arthritis) that the investigator deems necessary (within the past 3 months) or likely to require concurrent treatment with systemic glucocorticoids (excluding topical, intraocular, intranasal, intra-articular, or inhaled formulations) during study participation; or any prescription or nonprescription medication known to interfere with bowel motility, including but not limited to chronic opioids, anticholinergics, antispasmodics, linaclotide, and dopamine antagonists.

[0210] In some embodiments, the patient has not undergone any surgical treatment or used any weight loss device or any other weight loss procedure (e.g., LAP-Band®, gastric balloon, duodenal cannula, skin resurfacing) for obesity (any type).

[0211] In some embodiments, the patient does not have obesity induced by other endocrine disorders (e.g., Cushing's syndrome, acromegaly, or undertreated hypothyroidism) or is diagnosed with a monogenic or syndromic form of obesity (e.g., melanocortin 4 receptor deficiency or Prader-Willi syndrome). In some embodiments, the patient does not have a clinically significant diagnosis and / or history of gastroparesis, abnormal gastric motility, abnormal gastric emptying, malabsorption disorders, chronic constipation, chronic diarrhea, inflammatory bowel disease, bowel resection, irritable bowel syndrome, or severe gastroesophageal reflux disease.

[0212] In some embodiments, the patient does not have myocardial infarction, unstable angina, coronary artery bypass grafting, percutaneous coronary intervention (including participants who may plan and / or anticipate undergoing percutaneous endovascular coronary angioplasty [PTCA] during the study period, such as participants with prior angiographic evidence that may require PTCA); transient ischemic attack, cerebrovascular accident, or hospitalization for congestive heart failure. The patient may not currently have New York Heart Association class III or IV heart failure.

[0213] In some embodiments, the patient does not have clinically significant electrocardiogram (ECG) results (e.g., QT interval [QTcF] >450 msec (male), QTcF >470 msec (female), left bundle branch block, corrected using the Fridericia formula), or any other ECG result considered indicative of active heart disease or with abnormalities that are expected to interfere with the interpretation of ECG changes or may pose safety concerns to the participant. In some embodiments, the patient does not have uncontrolled hypertension (i.e., mean sitting systolic blood pressure of 160 mmHg or higher and / or mean sitting diastolic blood pressure of 100 mmHg or higher).

[0214] In some embodiments, the patient does not have clinically significant proliferative retinopathy, macular edema, or other diabetes-related eye diseases. In some embodiments, the patient does not have any hematological conditions (e.g., hemolytic anemia, sickle cell disease, other hemoglobinopathies) or uncontrolled thyroid disease (defined as having active symptoms (e.g., palpitations, somnolence, weight gain or weight loss) and / or having thyroid-stimulating hormone (TSH) values ​​outside the normal reference range).

[0215] In some embodiments, the patient does not have acute or chronic pancreatitis, or risk factors for pancreatitis such as a history of alcoholism, gallstones (without cholecystectomy), hypercalcemia, or severe hypertriglyceridemia.

[0216] In some embodiments, the patient does not have the following: alanine aminotransferase (ALT), aspartate aminotransferase (AST), or gamma-glutamyl transferase (GGT) >3.0 × the upper limit of normal (ULN) of the reference range; alkaline phosphatase (ALP) >1.5 × the ULN of the reference range; total bilirubin > the ULN of the reference range; amylase or lipase level >2 × the ULN of the reference range; fasting triglyceride level >500 mg / dL; estimated glomerular filtration rate (eGFR) calculated by the diet-corrected equation for renal disease <45 mL / min / 1.73 m 2 If eGFR ≥ 60 mL / min / 1.73 m 2 If calcitonin is ≥ 20 ng / L; or if eGFR is < 60 mL / min / 1.73 m 2 If the following criteria apply: calcitonin ≥ 35 mg / L; hepatitis (including hepatitis B or hepatitis C); HIV; or hemoglobin level <11 g / dL (male participants) or <10 g / dL (female participants).

[0217] In some embodiments, a patient with type 1 diabetes mellitus (T1DM) is an obese patient. The patient may have a weight of ≥ 30 to < 40 kg / m². 2 The patient's BMI can be ≥ 5.7% to ≤ 6.4% HbA1c. In some embodiments, this agonist can be used as an initial body mass index (BMI) of 30 kg / m². 2 For adult patients or those of advanced age, a low-calorie diet and increased physical activity may be used for long-term weight management. In some embodiments, the patient has at least one weight-related comorbidity (e.g., hypertension, type 2 diabetes, or dyslipidemia).

[0218] Body Mass Index (BMI) is a simple height-weight ratio commonly used to classify overweight and obesity in adults. It is defined as a person's weight (in kilograms) divided by the square of their height (in meters) (kg / m²). 2 For adults, the WHO defines overweight as a BMI of 25 to 29.9 kg / m². 2 and defining obesity as a BMI ≥ 30 kg / m² 2 Obesity is typically classified into the following categories:

[0219] ● Obesity Stage I – BMI 30 to 34.9 kg / m² 2

[0220] ● Obesity Category II – BMI 35 to 39.9 kg / m² 2 ;as well as

[0221] ● Obesity Category III – BMI ≥ 40 kg / m² 2 (Also known as severe, extreme, or massive obesity)

[0222] In some embodiments, overweight and obesity in Asian and South Asian populations may be defined as follows:

[0223] ●Overweight – BMI 23 to 24.9 kg / m 2

[0224] ●Obesity – BMI ≥25 kg / m² 2

[0225] In some embodiments, the patients with type 1 diabetes mellitus (T1DM) to be treated herein are overweight. In other embodiments, the patients to be treated herein are obese, having type I, II, or III obesity.

[0226] V. T2DM patient characteristics

[0227] In some embodiments, the patient has type 2 diabetes mellitus (T2DM). In some embodiments, the patient does not have type 1 diabetes mellitus (T1DM). In some embodiments, the patient has a weight gain of >27 to ≤45 kg / m². 2 BMI. In some embodiments, the patient’s HbA1c ≤ 10.5% and FPG < 250 mg / dL. The patient may have been receiving diet and exercise therapy or stable therapy (≥ 3 months) with metformin monotherapy or a combination of metformin and sulfamethoxazole before treatment. T2DM may be defined by the 2022 ADA Standards of Medical Care in Diabetes (American Diabetes Association (ADA), “Standards of Care in Diabetes,” Diabetes Care (2024) 47:S1-S321).

[0228] In some embodiments, the patient does not have any clinically significant GI (e.g., peptic ulcer, severe gastroesophageal reflux disease (GERD), abnormal gastric emptying / gastroparesis, any malabsorption / motor disorder, chronic constipation, inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS)), cardiovascular (e.g., arrhythmia, ischemic heart disease), liver, nervous system, mental, kidney, immune, skin, endocrine, genitourinary, or hematologic system active disease. In some embodiments, the patient does not have hyperlipidemia, uncontrolled hypertension (BP) > 140 / 90 mmHg (healthy participants) or persistent systolic or diastolic BP > 160 / 90 mmHg or < 90 / 60 mmHg (participants with T2DM).

[0229] In some embodiments, the patient does not have acute or chronic pancreatitis, or risk factors for pancreatitis (e.g., gallstones (without cholecystectomy), hypercalcemia, or severe hypertriglyceridemia). In some embodiments, the patient does not have a personal or family history of medullary thyroid carcinoma (MTC) or a genetic predisposition to MTC (i.e., type 2 multiple endocrine neoplasms).

[0230] In some embodiments, the patient does not have clinically significant physical or ECG results (e.g., QTcF >450 msec for men, QTcF >470 msec for women, left bundle branch block [LBBB]). In some embodiments, the patient has not undergone prior surgical treatment for obesity (any type of bariatric surgery) or any other GI surgery that may induce malabsorption / motor problems, a history of bowel resection >20 cm, or any GI weight loss surgery (including LAP-BAND®).

[0231] In some embodiments, the patient does not use any prescription or over-the-counter medications known to interfere with glucose or insulin metabolism, including but not limited to systemic corticosteroids, testosterone, anabolic steroids, metformin, GLP-1 analogs / RAs, thiazolidinediones (TZDs), SUs, dipeptidyl peptidase-4 (DPP-4) inhibitors, insulin therapy, monoamine oxidase (MAO) inhibitors, growth hormones, or other herbal / over-the-counter preparations, including amino acids.

[0232] In some embodiments, the patient does not use any prescription or over-the-counter medications known to interfere with bowel motility, including but not limited to chronic opioids, anticholinergics, antispasmodics, serotonin (5HT3) antagonists, or dopamine antagonists.

[0233] In some embodiments, the patient does not have clinically significant abnormal clinical laboratory values, including transaminases (aspartate aminotransferase [AST], alanine aminotransferase [ALT] > 1.5 × upper limit of normal [ULN] (healthy) or ≥ 3 × upper limit of normal [ULN] (with T2DM), total bilirubin > ULN, and glomerular filtration rate (eGFR) estimated using the diet-modified renal disease [MDRD] equation < 60 mL / min / 1.73m 2 And / or calcitonin levels > 50 ng / L. Participants with type 2 diabetes mellitus were excluded if fasting serum triglycerides > 500 mg / dL or laboratory values ​​suggestive of pancreatic damage (e.g., amylase and / or lipase > 3 x ULN).

[0234] In some embodiments, the patient does not have hepatitis B, hepatitis C, or human immunodeficiency virus type 1 (HIV-1) or type 2 (HIV-2).

[0235] In some embodiments, a T2DM patient is an obese patient. The patient may have a weight of ≥ 30 to < 40 kg / m². 2 The patient's BMI can be ≥ 5.7% to ≤ 6.4% HbA1c. In some embodiments, this agonist can be used as an initial body mass index (BMI) of 30 kg / m². 2 For adult patients or those of advanced age, a low-calorie diet and increased physical activity may be used for long-term weight management. In some embodiments, the patient has at least one weight-related comorbidity (e.g., hypertension, type 2 diabetes, or dyslipidemia).

[0236] In some embodiments, the patients to be treated in this article are overweight (i.e., have a weight of 25 to 29.9 kg / m²). 2 (BMI). In other embodiments, the patients to be treated in this article are obese, having type I, II, or III obesity.

[0237] VI. Weight management

[0238] In some embodiments, the patient does not have type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM), but is overweight or obese. The patient's age can be between 18 and 65 years. In some embodiments, the patient is obese. The patient may have a weight of ≥ 30 to < 40 kg / m². 2 The patient's BMI can be ≥ 5.7% to ≤ 6.4% HbA1c. In some embodiments, this agonist can be used as an initial body mass index (BMI) of 30 kg / m². 2For adult patients or those of advanced age, a low-calorie diet and increased physical activity may be used for long-term weight management. In some embodiments, the patient has at least one weight-related comorbidity (e.g., hypertension, type 2 diabetes, or dyslipidemia).

[0239] In some embodiments, the patients to be treated herein are overweight. In other embodiments, the patients to be treated herein are obese, having type I, II, or III obesity.

[0240] Non-limiting examples of obesity (whether alone or in combination with T1DM or T2DM) include symptomatic obesity, simple obesity, childhood obesity, morbid obesity, and abdominal obesity (central obesity characterized by abdominal obesity). Non-limiting examples of symptomatic obesity include endocrine obesity (e.g., Cushing's syndrome, hypothyroidism, insulinoma, obesity-related type 2 diabetes, pseudohypoparathyroidism, hypogonadism), hypothalamic obesity, hereditary obesity (e.g., Prader-Willi syndrome, Lau-Müller-Bis syndrome), and drug-induced obesity (e.g., obesity induced by steroids, phenothiazines, insulin, sulfonylureas, or beta-blockers).

[0241] In addition to obesity, patients may also have symptoms, diseases, or disorders associated with obesity. Examples of such symptoms, diseases, or disorders include, but are not limited to, glucose intolerance, diabetes (e.g., type 2 diabetes, obesity-related diabetes), lipid metabolism disorders, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver (including non-alcoholic steatohepatitis (NASH)), coronary artery disease (e.g., myocardial infarction, angina pectoris), cerebral infarction (e.g., cerebral thrombosis, transient ischemic attack), bone or joint diseases (e.g., knee osteoarthritis, hip osteoarthritis, degenerative spondylitis, low back pain), sleep apnea syndrome, obesity-hypopnea syndrome (Pickwick syndrome), menstrual disorders (e.g., abnormal menstrual cycles, abnormal menstrual flow and cycle, amenorrhea, abnormal menstrual symptoms), visceral obesity syndrome, and metabolic syndrome. In some embodiments, the chemical entities described herein may be used to treat subjects exhibiting symptoms of both obesity and insulin deficiency.

[0242] In some embodiments, obese patients are treated with a dose of the agonist disclosed herein (e.g., CT-868) from 0.1 mg to 15.0 mg. For example, patients may be treated with CT-868 at doses of 0.5 mg, 0.75 mg, 1.5 mg, 5.0 mg, 7.5 mg, or 11 mg. Overweight or obese patients may also be treated with CT-868 at doses of 5.0 mg, 10.0 mg, or 15.0 mg. In some embodiments, such patients are treated daily with the agonist.

[0243] In some embodiments, the patient does not have any clinically significant GI (e.g., peptic ulcer, severe gastroesophageal reflux disease (GERD), abnormal gastric emptying / gastroparesis, any malabsorption / motor disorder, chronic constipation, inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS)), cardiovascular (e.g., arrhythmia, ischemic heart disease), liver, nervous system, mental, kidney, immune, skin, endocrine, genitourinary, or hematologic system active disease. In some embodiments, the patient does not have hyperlipidemia, uncontrolled hypertension (BP) > 140 / 90 mmHg (healthy participants) or persistent systolic or diastolic BP > 160 / 90 mmHg or < 90 / 60 mmHg (participants with T2DM).

[0244] In some embodiments, the patient does not have acute or chronic pancreatitis, or risk factors for pancreatitis (e.g., gallstones (without cholecystectomy), hypercalcemia, or severe hypertriglyceridemia). In some embodiments, the patient does not have a personal or family history of medullary thyroid carcinoma (MTC) or a genetic predisposition to MTC (i.e., type 2 multiple endocrine neoplasms).

[0245] In some embodiments, the patient does not have clinically significant physical or ECG results (e.g., QTcF >450 msec for men, QTcF >470 msec for women, left bundle branch block [LBBB]). In some embodiments, the patient has not undergone prior surgical treatment for obesity (any type of bariatric surgery) or any other GI surgery that may induce malabsorption / motor problems, a history of bowel resection >20 cm, or any GI weight loss surgery (including LAP-BAND®).

[0246] In some embodiments, the patient does not use any prescription or over-the-counter medications known to interfere with glucose or insulin metabolism, including but not limited to systemic corticosteroids, testosterone, anabolic steroids, metformin, GLP-1 analogs / RAs, thiazolidinediones (TZDs), SUs, dipeptidyl peptidase-4 (DPP-4) inhibitors, insulin therapy, monoamine oxidase (MAO) inhibitors, growth hormones, or other herbal / over-the-counter preparations, including amino acids.

[0247] In some embodiments, the patient does not use any prescription or over-the-counter medications known to interfere with bowel motility, including but not limited to chronic opioids, anticholinergics, antispasmodics, serotonin (5HT3) antagonists, or dopamine antagonists.

[0248] In some embodiments, the patient does not have clinically significant abnormal clinical laboratory values, including transaminases (aspartate aminotransferase [AST], alanine aminotransferase [ALT] > 1.5 × upper limit of normal [ULN] (healthy) or ≥ 3 × upper limit of normal [ULN] (with T2DM), total bilirubin > ULN, and glomerular filtration rate (eGFR) estimated using the diet-modified renal disease [MDRD] equation < 60 mL / min / 1.73m 2 And / or calcitonin levels > 50 ng / L. Participants with type 2 diabetes mellitus were excluded if fasting serum triglycerides > 500 mg / dL or laboratory values ​​suggestive of pancreatic damage (e.g., amylase and / or lipase > 3 x ULN).

[0249] In some embodiments, the patient does not have hepatitis B, hepatitis C, or human immunodeficiency virus type 1 (HIV-1) or type 2 (HIV-2).

[0250] VII. Treatment outcomes

[0251] This treatment can alter the patient's insulin secretion rate; maximum glucose drift (G) max AUC effect; fasting plasma glucose, insulin, C-peptide, GCC or HOMA-IR; gastric emptying; glucose-stimulated insulin secretion (GSIS); glucose drift; ambient glucose level; HbA1c level; or CGM indicators (TIR, time to hypoglycemia and hyperglycemia, glycemic risk index).

[0252] This treatment method can also reduce the incidence of grade 3 hypoglycemic events or DKA in patients.

[0253] This treatment method can reduce a patient's weight. In some embodiments, the method results in a 5%, 10%, 15%, or 20% weight loss. In some embodiments, this treatment method alters total lean body mass and total fat mass. Total lean body mass and total fat mass can be measured using dual-energy X-ray absorptiometry (DEXA). DEXA scans assess body composition (fat mass and lean body mass) and bone mineral density.

[0254] This treatment method can also induce changes in patients' appetite, expected food consumption, hunger, satiety, and fullness as measured by a VAS questionnaire. For the satiety questionnaire, patients are asked to complete four questions using a 100 mm VAS. Based on the first seven days, participants will be asked to rate their overall satiety / fullness (100 = completely full, 0 = not full), satiety (100 = completely full, 0 = not full), hunger (100 = never hungrier, 0 = not hungry at all), and expected food consumption (100 = very much, 0 = not at all). The overall appetite score will be calculated as the average of the following four individual scores: [satiety + fullness + (100 - expected food consumption) + (100 - hunger)] ÷ 4.

[0255] The methods described in this article can also modify (e.g., reduce) food and calorie intake during free-eating periods.

[0256] This treatment method can also alter the absolute and relative changes in basal, pulse, and total daily insulin use, measured in units per day and units per kg per day.

[0257] VIII. Exemplary embodiments

[0258] The following non-limiting exemplary embodiments are provided to further illustrate this disclosure.

[0259] 1. A method, said method for

[0260] Adjunctive therapy for type 1 diabetes (T1DM)

[0261] To improve glycemic control in patients with type 1 diabetes mellitus (T1DM)

[0262] Increase insulin sensitivity in patients with type 1 diabetes mellitus (T1DM).

[0263] Increase insulin-independent glucose management in patients with type 1 diabetes mellitus (T1DM).

[0264] Reduce the need for therapeutic insulin in patients with type 1 diabetes mellitus (T1DM).

[0265] To alleviate hypertension in patients with type 1 diabetes mellitus (T1DM)

[0266] Reduce atherogenic lipids in patients with type 1 diabetes mellitus (T1DM), and / or

[0267] Weight management for patients with type 1 diabetes mellitus (T1DM)

[0268] The method involves administering a dual agonist of glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor via subcutaneous injection to a patient in need.

[0269] The agonist described herein comprises the following structures or their pharmaceutical salts or esters:

[0270]

[0271] (Formula I),

[0272] Wherein R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, said cycloalkyl or heterocyclic group optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and

[0273] The agonist is administered at a dose of 1 mg to 20 mg, independent of body weight.

[0274] 2. The method according to Example 1, wherein the agonist comprises the following structure or a pharmaceutical salt or ester thereof:

[0275]

[0276] (Form V).

[0277] 3. The method according to Example 1, wherein the agonist comprises the following structure or a pharmaceutical salt or ester thereof:

[0278]

[0279] (Form VII).

[0280] 4. The method according to any one of Examples 1 to 3, wherein the application step is repeated at intervals of one to seven days.

[0281] 5. The method according to Example 4, wherein the application steps are repeated once a day.

[0282] 6. The method according to any one of the foregoing embodiments, wherein the dose is a dose that activates both the GLP-1 receptor and the GIP receptor.

[0283] 7. The method according to any one of the foregoing embodiments, wherein the dose is about 1.8 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg or 15 mg.

[0284] 8. The method according to any one of the foregoing embodiments, wherein the patient has a weight of 43 kg / m². 2 Or a higher BMI.

[0285] 9. The method according to any one of the foregoing embodiments, wherein the patient receives additional therapy for T1DM.

[0286] 10. The method according to Example 9, wherein the additional therapy is insulin therapy, diet therapy, exercise therapy, hypertension therapy and / or lipid-lowering therapy.

[0287] 11. A dual agonist of glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor for use in accordance with any one of Examples 1 to 10.

[0288] 12. Use of a dual agonist of glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor for the manufacture of a medicament for use in any one of Examples 1 to 10.

[0289] An article for use in the method according to any one of Examples 1 to 10, wherein the article comprises one or more of the dosage units.

[0290] 13. The article of embodiment 13, wherein the article is a syringe or injector, optionally a disposable syringe or injector.

[0291] 14. A method, said method for

[0292] Adjunctive therapy for insulin-dependent patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM)

[0293] Treatment of patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus who require this.

[0294] To improve glycemic control in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM)

[0295] Increase insulin sensitivity in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

[0296] Increase insulin-independent glucose management in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

[0297] Reduce the need for therapeutic insulin in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

[0298] To relieve hypertension in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM)

[0299] Reduce atherogenic lipids in patients with T1DM or T2DM, and / or

[0300] Weight management for patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM)

[0301] The method involves administering a dual agonist of glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor via subcutaneous injection to a patient in need.

[0302] The agonist described herein comprises the following structures or their pharmaceutical salts or esters:

[0303]

[0304] (Formula I),

[0305] Wherein R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, said cycloalkyl or heterocyclic group optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and

[0306] The agonist is administered at a dose of 1 mg to 20 mg, independent of body weight.

[0307] 15. The method according to Example 14, wherein the agonist comprises the following structure or a pharmaceutical salt or ester thereof:

[0308]

[0309] (Form V).

[0310] 16. The method according to Example 14, wherein the agonist comprises the following structure or a pharmaceutical salt or ester thereof:

[0311]

[0312] (Form VII).

[0313] 17. The method according to any one of Examples 14 to 16, wherein the application step is repeated at intervals of one to seven days.

[0314] 18. The method according to Example 17, wherein the application steps are repeated once a day.

[0315] 19. The method according to any one of Examples 14 to 18, wherein the dose is a dose that activates both the GLP-1 receptor and the GIP receptor.

[0316] 20. The method according to any one of Examples 14 to 19, wherein the dose is about 1.8 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg or 15 mg.

[0317] 21. The method according to any one of Examples 14 to 20, wherein the patient has a body temperature of 25 kg / m². 2 Or a higher BMI.

[0318] 22. The method according to any one of Examples 14 to 21, wherein the patient receives additional therapy for T1DM or T2DM.

[0319] 23. The method according to Example 22, wherein the additional therapy is insulin therapy, diet therapy, exercise therapy, hypertension therapy and / or lipid-lowering therapy.

[0320] 24. A dual agonist of glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor for use in any one of Examples 14 to 23.

[0321] 25. Use of a dual agonist of glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor for the manufacture of a medicament for use in any one of Examples 14 to 23.

[0322] 26. An article for use in the method according to Examples 14 to 23, wherein the article comprises one or more of the dosage units.

[0323] 27. The article of embodiment 26, wherein the article is a syringe or injector, optionally a disposable syringe or injector.

[0324] 28. A method, said method for

[0325] Adjunctive therapy for insulin-dependent patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

[0326] Treatment of patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus who require this.

[0327] To improve glycemic control in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM)

[0328] Increase insulin sensitivity in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

[0329] Increase insulin-independent glucose management in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

[0330] Reduce the need for therapeutic insulin in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

[0331] To relieve hypertension in patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM)

[0332] Reduce atherogenic lipids in patients with T1DM or T2DM, and / or

[0333] Weight management for patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM)

[0334] The method involves administering a dual agonist of glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor via subcutaneous injection to a patient in need.

[0335] The agonist described herein comprises the following structures or their pharmaceutical salts or esters:

[0336]

[0337] (Formula I),

[0338] Wherein R is a cycloalkyl or heterocyclic group of 4 to 8 atoms, said cycloalkyl or heterocyclic group optionally substituted with a carbonyl, hydroxyl, methyl, phenyl, isopropyl, trifluoromethyl, or nitro group; and

[0339] The agonist is administered at a dose of 1 mg to 20 mg, independent of body weight.

[0340] 29. The method according to Example 28, wherein the agonist comprises the following structure or a pharmaceutical salt or ester thereof:

[0341]

[0342] (Form V).

[0343] 30. The method according to Example 28, wherein the agonist comprises the following structure or a pharmaceutical salt or ester thereof:

[0344]

[0345] (Form VII).

[0346] 31. The method according to any one of Examples 28 to 30, wherein the application step is repeated at intervals of one to seven days.

[0347] 32. The method according to Example 31, wherein the application steps are repeated once a day.

[0348] 33. The method according to any one of Examples 28 to 32, wherein the dose is a dose that activates both the GLP-1 receptor and the GIP receptor.

[0349] 34. The method according to any one of Examples 28 to 33, wherein the dose is about 1.5 mg, 1.8 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20 mg.

[0350] 35. The method according to any one of Examples 28 to 34, wherein the patient has a weight of 25 kg / m². 2 Or a higher BMI.

[0351] 36. The method according to any one of Examples 28 to 35, wherein the patient receives additional therapy for T1DM or T2DM.

[0352] 37. The method according to Example 36, wherein the additional therapy is insulin therapy, diet therapy, exercise therapy, hypertension therapy and / or lipid-lowering therapy.

[0353] 38. A dual agonist of glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor for use in the method according to any one of Examples 28 to 37.

[0354] 39. Use of a dual agonist of glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor for the manufacture of a medicament for use in any one of Examples 28 to 37.

[0355] 40. An article for use in the method according to any one of Examples 28 to 37, wherein the article comprises one or more of the dosage units.

[0356] 41. The article of embodiment 40, wherein the article is a syringe or injector, optionally a disposable syringe or injector.

[0357] 42. A compound for treating type 1 diabetes mellitus (T1DM) in patients with this need, wherein the compound comprises the following structure or a pharmaceutical salt or ester thereof:

[0358]

[0359] (Formula VII) (SEQ ID NO: 1),

[0360] The compound is to be administered subcutaneously to the patient in need at a dose of 1 mg to 20 mg, independent of body weight.

[0361] 43. A compound for treating type 2 diabetes mellitus (T2DM) in patients with this need, wherein the compound comprises the following structure or a pharmaceutical salt or ester thereof:

[0362]

[0363] (Formula VII) (SEQ ID NO: 1),

[0364] The compound is to be administered subcutaneously to the patient in need at a dose of 1 mg to 20 mg, independent of body weight.

[0365] 44. The compound used according to Example 42 or 43, wherein the compound is used for

[0366] Improve the patient's blood glucose control.

[0367] Increase the patient's insulin sensitivity,

[0368] Increase the insulin-independent glucose management of the patients.

[0369] Reduce the patient's need for therapeutic insulin.

[0370] Relieve the patient's high blood pressure, and / or

[0371] Reduce atherosclerotic lipids in the patients.

[0372] 45. The compound used according to any one of Examples 42 to 44, wherein the patient is overweight or obese, and the method reduces the patient's weight.

[0373] 46. ​​A compound for weight management in patients in need, wherein the compound comprises the following structure or a pharmaceutical salt or ester thereof:

[0374]

[0375] (Formula VII) (SEQ ID NO: 1),

[0376] The compound is to be administered subcutaneously to the patient in need at a dose of 1 mg to 20 mg, independent of body weight.

[0377] 47. The compound used according to Example 46, wherein the patient has type 1 or type 2 diabetes.

[0378] 48. The compound used according to any one of Examples 42 to 47, wherein the administration of the compound is in conjunction with one or more other therapies.

[0379] 49. The compound used according to Example 48, wherein one or more additional therapies include insulin therapy, diet therapy, exercise therapy, hypertension therapy and / or lipid-lowering therapy.

[0380] 50. The compound used according to any one of Examples 42 to 49, wherein the application steps are repeated at intervals of one to seven days.

[0381] 51. The compound used according to any one of Examples 42 to 49, wherein the application steps are repeated once daily.

[0382] 52. The compound used according to any one of Examples 42 to 51, wherein the compound is administered at a dose that activates both the GLP-1 receptor and the GIP receptor.

[0383] 53. The compound used according to any one of Examples 42 to 52, wherein the dose is about 1.5 mg, 1.8 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20 mg.

[0384] 54. The compound used according to any one of Examples 52 to 53, wherein the dose is 1.77 mg, 3.25 mg or 4 mg.

[0385] 55. The compound used according to any one of Examples 42 to 54, wherein the dose is 1.1 mg, 1.8 mg, 2.6 mg, 3.3 mg, 4.1 mg, 5.2 mg or 6.6 mg.

[0386] 56. The compound used according to any one of Examples 42 to 55, wherein the patient has a blood pressure of 25 kg / m². 2 Or a higher BMI.

[0387] 57. The compound used according to any one of Examples 42 to 56, wherein the compound is to be administered to the patient in a pharmaceutical composition comprising:

[0388] The compound at a concentration of 15 mg / mL,

[0389] Disodium hydrogen phosphate heptahydrate / sodium dihydrogen phosphate buffer, optionally at a concentration of 20 mM.

[0390] Propylene glycol, and

[0391] Phenol, and optionally, the pharmaceutical composition is at pH 7.0.

[0392] 58. A pharmaceutical composition comprising:

[0393] (i) Compounds containing the following structures or their pharmaceutical salts or esters at a concentration of 15 mg / mL:

[0394]

[0395] (Formula VII) (SEQ ID NO: 1),

[0396] (ii) Sodium dihydrogen phosphate heptahydrate / sodium dihydrogen phosphate buffer, optionally at a concentration of 20 mM.

[0397] (iii) Propylene glycol, and

[0398] (iv) Phenol, further optionally, wherein the pharmaceutical composition is at pH 7.0.

[0399] 59. The compound described herein used according to any one of Examples 42 to 57, which provides, in the pharmaceutical composition according to claim 58

[0400] 60. An article comprising a compound used according to any one of Examples 42 to 57 and 59 or a pharmaceutical composition according to Example 58, wherein the article comprises one or more dose units, optionally a single dose unit.

[0401] 61. An article comprising a compound used according to any one of Examples 42 to 57 and 59 or a pharmaceutical composition according to Example 58, wherein the article comprises five dose units.

[0402] 62. The article of use according to Example 60 or 61, wherein the article is a syringe, pen or syringe, and optionally wherein the article is for single use.

[0403] 63. The article of use according to any one of Examples 60 to 62, wherein the article of use comprises a needle-based injection system having an integrated, non-replaceable 3-mL Type 1 glass cartridge and a pharmaceutical composition comprising 3 mL of an agonist at a concentration of 15 mg / mL.

[0404] Unless otherwise defined herein, scientific and technical terms related to this invention shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although similar or equivalent methods and materials may also be used in practice or testing of this disclosure. In case of conflict, this specification (including definitions) shall prevail. Furthermore, unless the context otherwise requires, singular terms shall include plurals, and plural terms shall include singulars. Throughout the specification and embodiments, the words “having” and “comprising” or variations such as “has” or “having” or “comprises” shall be understood to imply inclusion of the stated integers or groups of integers, but not to exclude any other integers or groups of integers. All publications and other references mentioned herein are incorporated in their entirety by reference as if each individual reference were specifically and individually indicated to be incorporated in their entirety by reference. Although numerous references are cited herein, such citations do not constitute an admission that any of these references constitutes part of common general knowledge in the art. As used herein, the terms “about” or “approximately,” when applied to one or more values ​​of interest, refer to values ​​similar to the stated reference values. In some embodiments, the term refers to a range of values ​​of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less falling in either direction of the reference value, unless otherwise stated or otherwise apparent from the context.

[0405] According to this disclosure, reverse references in dependent claims are intended to directly and explicitly disclose the abbreviation of each combination of claims indicated by the reverse reference. Any compound disclosed herein may be used in any treatment method described herein, wherein the individual to be treated is as defined anywhere herein. Furthermore, the headers herein are created for organizational convenience and are not intended to limit the scope of the claimed invention in any way.

[0406] The following examples are provided to better understand the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0407] Example

[0408] Example 1: Uncoupled, biased GLP-1R and GIPR agonists produced sustained glucose reduction, decreased food intake, and weight loss in a rodent obesity model.

[0409] To elucidate the importance of signal transduction bias, a dual GLP-1 / GIP receptor agonist, CT-859, was designed that is completely biased towards cAMP (Figures 1A and 1B). CT-859 does not exhibit β-repressor protein coupling at either receptor. Figure 2A (and 2B). The effects of CT-859 on glucose (GLUC) homeostasis, food intake (FI) regulation, and weight loss (WL) were evaluated in rodent models.

[0410] method

[0411] Using the HitHunter® cAMP Assay for Small Molecules (catalog number 90-0075SM) kit, cAMP generated by GLP-1, GIP, liraglutide, CT-859, exenatide-4, and exenatide-Phe1 was measured in mammalian cell lines overexpressing GLP-1R or GIPR. For mouse recipients, stably transfected U2OS (GLP-1R – DiscoverX 95-0179C3) and CHO (GIPR – DiscoverX 95-0154C2) cells were used (Figures 1A and 1B). The PathHunter® assay kit was used with PathHunter® CHO-K1 GLP-1R β-repressor protein (DiscoverX93-0300C2), PathHunter® CHO-K1 GIPR β-repressor protein (DiscoverX 93-1095C2), HEK293mGLP1R and / or HEK293 mGIPR β-repressor protein cell lines to measure β-repressor protein-2 recruitment using a β-galactosidase complementation assay to measure GLP-1, GIP, liraglutide, CT-859, exenatide-4 and exenatide-Phe1. Figure 2A (and 2B).

[0412] Male GLP-1R+ / + (WT) and GLP-1R- / - (GLP-1R-KO) mice aged 12–13 weeks from Taconic were housed individually and fed a standard diet. For the intraperitoneal glucose tolerance test (ipGTT), mice were randomly assigned by weight within their respective genotypes to the following groups: WT and GLP-1R-KO mediator (phosphate-buffered saline + Tween; n = 6), WT and GLP-1R-KO CT-859 (20 nmol / kg; n = 6), and WT and GLP-1R-KO CT-859 (200 nmol / kg; n = 6). The ipGTT was performed five hours after fasting and four hours after compound administration. After fasting, glucose was measured, blood was collected, and glucose (2 g / kg) was administered via intraperitoneal injection. Blood glucose levels were measured after administration, and blood samples were collected at 15, 30, 60, and 120 minutes post-glucose injection for insulin measurement. Insulin was measured using the MSD Insulin Kit. Results were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons (Figure 3).

[0413] Untreated 10-week-old male C57BL / 6J mice from Jackson Laboratory were housed individually and fed a standard diet. Mice were randomly assigned by body weight to the following groups: mediator (phosphate-buffered saline + Tween; n = 6 / time point), liraglutide (20 nmol / kg; n = 6 / time point), and CT-859 (20 nmol / kg; n = 6 / time point). Intraperitoneal glucose tolerance test (ipGTT; 2 g / kg) was performed at 4, 24, and 48 hours after compound administration. Mice were fasted for five hours prior to the ipGTT. Glucose was measured after the fasting period, followed by intraperitoneal injection of glucose (2 g / kg). Blood glucose levels were measured at 20, 40, 60, and 120 minutes after glucose injection. Results were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons (Figures 4A and 4B).

[0414] Pharmacokinetic studies and bioanalyses were performed by BioDuro Inc. and are briefly described here. Male CD-1 mice aged 7 to 9 weeks received liraglutide (1 mg / kg) or CT-859 (1 mg / kg) via a single subcutaneous (SC) injection. Subsequently, blood was collected from K2-EDTAmicrovettes® via saphenous vein punctures at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 32 punctures after drug administration. Blood was placed on ice until centrifuged at 4600 rpm for 5 minutes at 4°C. Plasma was stored at -80°C until analysis. Compounds were extracted from plasma and quantified according to a standard curve using an AB Sciex 7500+ LC-MS / MS system (Figure 5).

[0415] Untreated 24-week-old male DIO C57BL / 6J mice from the Jackson laboratory were housed individually and fed a 60% high-fat diet. Mice were randomly assigned by body weight to the following groups: mediator (phosphate-buffered saline + Tween; n = 8), liraglutide (20 nmol / kg; n = 8), and CT-859 (20 nmol / kg; n = 8). Compounds were administered daily via a single subcutaneous injection for 21 days. Body weight and food consumption were measured daily (Figs. 8 and 9). On the last day of the study, mice were fasted for 5 hours. At the end of the fasting period, glucose was measured (Fig. 6), and plasma was collected for insulin analysis. Plasma insulin was measured using an MSD kit. The log(HOMA-IR) was calculated using fasting glucose and insulin (Fig. 7). Body weight and cumulative food consumption were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons. Fasting blood glucose, insulin, and log(HOMA-IR) were analyzed using univariate ANOVA and the Holm-Sidak test for multiple comparisons.

[0416] Untreated 23-week-old male DIO C57BL / 6J mice from the Jackson laboratory were housed individually and fed a 60% high-fat diet. Mice were randomly assigned by body weight to the following groups: mediator (phosphate-buffered saline + Tween; n = 8), liraglutide (200 nmol / kg; n = 8), and CT-859 (200 nmol / kg; n = 8). The compounds were administered daily via a single subcutaneous injection for 21 days. Body weight was measured daily (Fig. 11). Food consumption was measured at days 1, 7, and 14 (Fig. 12A) or day 8 (Fig. 12B) after the start of treatment. On the last day of the study, glucose was measured, and plasma was collected for insulin analysis. Plasma insulin was measured using an MSD kit. The log(HOMA-IR) was calculated using glucose and insulin (Fig. 10). Body weight and food consumption were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons. Plasma insulin and log(HOMA-IR) were analyzed using univariate ANOVA and the Holm-Sidak test for multiple comparisons.

[0417] WT mice were randomly assigned to the following groups based on body weight: mediator (phosphate-buffered saline + Tween; n = 9), exenatide-4 (0.025 nmol / kg; n = 8), and exenatide-Phe1 (0.025 nmol / kg; n = 10). The compounds were administered once via intraventricular injection (ICV), and body weight and food consumption were measured at 4, 24, 48, and 72 hours thereafter (Figures 13 and 14). Body weight and cumulative food consumption were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons.

[0418] result

[0419] In GLP-1R knockout mice, a 20 nmol / kg dose of CT-859 was established as the dose for GLP-1R conjugation only, as it had no effect on GLUC AUC during the intraperitoneal glucose tolerance test (IPGTT) and, similar to the veh, indicated a lack of GIPR activation at this dose (Fig. 3). In lean mice, a 20 nmol / kg dose of CT-859 caused a dramatic decrease in glucose (GLUC) AUC, similar to the dramatic decrease achieved four hours after administration with a 20 nmol / kg dose of unbiased GLP-1RA (liraglutide) (Figs. 4A and 4B), although plasma exposure was reduced by 82% four hours after CT-859 administration (Fig. 5). Notably, CT-859 remained effective up to 48 hours (GLUC AUC decreased by 45%, compared to veh, p<0.001), and liraglutide-treated mice were indistinguishable from those treated with the vector (Figs. 4A and 4B). Compared to DIO mice administered 20 nmol / kg liraglutide for 20 days, diet-induced obese (DIO) mice administered 20 nmol / kg CT-859 for 20 days showed reduced fasting GLUC (146.5 vs. 210.6 mg / dL; p<0.0001) (Fig. 6) and insulin resistance (log(HOMA-IR); 0.74 vs. 1.08; p=0.001) (Fig. 7), and achieved greater weight loss (WL) (-16.5 vs. -9.1%; p=0.001) (Fig. 8), while there was no significant difference in cumulative food intake (FI) (Fig. 9). In contrast, DIO mice treated with a GIPR-binding dose of CT-859 at 200 nmol / kg for 20 days showed reduced insulin resistance (non-fasting log(HOMA-IR); 1.17 vs. 1.42, p<0.05) (Fig. 10), produced more WL (-30.1% vs. -19.2%; p<0.0001) (Fig. 11), and induced significant FI inhibition (day 7: 1.2 g vs. 1.9 g; p=0.0003; day 14: 1.7 g vs. 2.2 g; p<0.05) (Fig. 12A), suggesting that these differences may be a result of central GIPR activation.

[0420] In lean mice, administration of exenatide-4 and exenatide-Phe1 via ICV injection resulted in a significant reduction in cumulative food consumption, which was associated with weight loss. However, compared with exenatide-4, administration of exenatide-Phe1 resulted in a greater reduction in food intake and a much longer period of weight loss (Figures 13 and 14).

[0421] In summary, these results demonstrate that CT-859, as a fully biased dual GLP-1 / GIPR agonist (GLP-1 / GIPRA), possesses GLP-1R-favorable unbalanced activity, producing sustained and greater GLUC reduction, FI inhibition, and WL compared to liraglutide, as an unbiased GLP-1R agonist (GLP-1RA).

[0422] Example 2: Biased signal transduction enhances the glucose-lowering and weight-loss effects of dual GLP-1R / GIPR agonists

[0423] GLP-1 and GIP are hormones that promote insulin secretion and inhibit food intake in a glucose-dependent manner. This example describes CT-859, as a biased dual GLP-1 / GIPR agonist, exhibiting reverse receptor internalization agonistic effects. At lower concentrations, CT-859 lowers glucose levels for up to 48 hours via GLP-1R activation, while liraglutide's effect lasts only 24 hours. Furthermore, CT-859 is more potent than liraglutide in reducing weight. We also demonstrate that biased GIPR agonists are more effective than unbiased agonists in lowering glucose levels. Central administration of both biased GLP-1R and GIPR agonists resulted in greater food consumption and weight loss than unbiased agonists, and their combination was more effective than the combination of a biased GLP-1R agonist and an unbiased GIPR agonist. These results demonstrate that glucagon-based therapies that stimulate biased signaling are more effective than unbiased therapies, and that biased signaling should be considered when designing new glucagon-based therapies.

[0424] method

[0425] The sources of the selected reagents and resources are shown in Table 1 below.

[0426] Table 1. Sources of Selected Reagents and Resources

[0427]

[0428] Mice

[0429] C57BL / 6J and diet-induced obese (DIO) male mice were obtained from The Jackson Laboratory. GLP-1R mice were obtained from Taconic Biosciences. + / + GLP-1R - / - GIPR + / + and GIPR - / - Male mice. Unless otherwise specified, mice were housed individually under standard environmental conditions (22°C, 12h:12h light:dark cycle) with free access to water and conventional diet (C57BL / 6J; Teklad rodent diet 2920x, Inotiv) or HFD (DIO C57BL / 6J; 60% kcal from fat, Research Diets #D12492). Lean mice between 8 and 10 weeks of age were used. DIO mice were kept in HFD for at least 18 weeks prior to experiments. All studies were approved by the FibroGen and Explora BioLabs Institutional Animal Care and Use Committee.

[0430] Peptide synthesis

[0431] Peptides were synthesized using microwave-assisted solid-phase peptide synthesis (SPPS) on a Liberty Blue microwave peptide synthesizer (CEM Corporation) via the Fmoc / t-Bu strategy. Fmoc deprotection was performed using 20% ​​piperidine in 0.1 M Oxyma / DMF solution. Amino acid coupling was performed using a five-fold excess of the reagent; Fmoc-amino acids (0.2 M DMF solution), DIC (0.5 M or 1.0 M DMF solution), and Oxyma (0.5 M or 1.0 M DMF solution) were used at scales of 0.05 mmol or 0.1 mmol on Rink Amide ProTide resin (LL) or pre-loaded Wang (LL) resin.

[0432] Cleavage and post-treatment conditions: Side-chain protecting groups were removed from the resin by accompanying cleavage in a TFA / TIS / H2O / PhOH (88:2:5:5 v / v / v / v) solution (10 mL / 0.05 mmol) at room temperature for 3 hours. The peptide was precipitated using cold diethyl ether (30 mL / 0.05 mmol) and separated by centrifugation (3000 rpm, 10 min).

[0433] Purification conditions: The crude peptide was iteratively purified by RP-HPLC until a purity >95% was obtained. The purification conditions are listed in Tables 2 to 5 below; appropriate fractions were combined and lyophilized. Peptide purity was determined by analytical RP-HPLC and its identity was confirmed by LCMS.

[0434] Table 2. Purification Procedure P1

[0435]

[0436] Table 3. Purification Procedure P2

[0437]

[0438] Table 4. Purification Procedure P3

[0439]

[0440] Table 5. Purification Procedure P4

[0441]

[0442] Analytical HPLC conditions: The purity of the peptides was checked by analytical RP-HPLC, and identity was confirmed by LCMS under the conditions shown in Tables 6 to 8.

[0443] Table 6. Analytical RP-HPLC Conditions A1

[0444]

[0445] Table 7. Analytical RP-HPLC Conditions A2

[0446]

[0447] Table 8. LC-MS Conditions

[0448]

[0449] Preparation of CT-859: The solid-phase preparation of CT-859 is shown below:

[0450] ,

[0451] Where K* is:

[0452] .

[0453] The N-terminal modification of compound CT-859 was prepared according to the following synthesis:

[0454] .

[0455] R-1 is coupled with the above-mentioned carboxylic acid 3, and then cleaved from the resin according to the following procedure:

[0456] .

[0457] Resin-bound peptide intermediate R-1 was prepared from Rink amide resin using a standard solid-phase peptide synthesis method with Fmoc chemist's α-Fmoc and a standard amino acid building block with side-chain protection. In addition to the standard amino acid building block, Fmoc-(α-aminoisobutyric acid) and N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((S)-5-(tert-butoxy)-5-oxo-4-palmitamidopentanoyl)-L-lysine were also used.

[0458] DIPEA (34.8 mL, 4.0 equiv.) was added to a slurry of hydrochloride 1 (9.83 g, 50 mmol) and thioglycolic anhydride 2 (6.61 g, 50 mmol, 1.0 equiv.) in DMF (150 mL) at 23 °C. The reaction mixture was stirred at ambient temperature for 30 min, at which point the reaction was considered complete by HPLC analysis. PyBOP (26.0 g, 1.0 equiv.) was added, followed by additional DIPEA (17.4 mL, 2.0 equiv.). After stirring at ambient temperature for 5 min, the reaction mixture was added to resin R-1 (14 mmol). The slurry was stirred at ambient temperature for 4 h and filtered. The resin was washed with DMF (3x), IPA (3x), and isopropyl ether (2x) and dried under vacuum to give 128.6 g of product resin. The resin was treated with TFA / TIS / DOT / H2O (90 / 5 / 2.5 / 2.5 v / v / v / v) for 3 hours and the reaction mixture was filtered. The resin was washed with TFA, and the filtrate and washes were combined and diluted with pre-cooled isopropyl ether. The precipitated solid was collected by filtration and dried under vacuum to give 44 g of crude peptide.

[0459] The crude peptide was purified using a 2'' C18 column with a gradient of 0.05M AcOH in H2O / MeCN (flow rate = 100 mL / min), followed by salt exchange to hydrochloride using 0.01N HCl buffer (flow rate = 100 mL / min) on the 2'' C18 column. CT-859 hydrochloride (6 g) was obtained as a white powder (analytical HPLC purity 97.0%), with an ESI-MS value of 1543.9. 212 H 320 N 47 O 65 S2 [M+3H] 3+ The required value is 1543.9.

[0460] Preparation of Ex-Phe1

[0461] The sequence of Ex-Phe1 is:

[0462] FG-EGT-FTS-DLS-KQM-EEE-AVR-LFI-EWL-LAG-GPS-SGA-PPPS-NH2 (SEQ ID NO:2).

[0463] Exenatide-Phe1 (Ex-Phe1) was prepared in two fractions (0.1 mmol and 0.05 mmol) at a scale of 0.15 mmol using the standard Fmoc-SPPS method. Resin from the 0.1 mmol fraction was subjected to lysis conditions for three hours using 6 mL of TFA / TIS / PhOH / H2O (88:2:5:5 v / v / v / v). The resin was filtered and washed with TFA (2 x 2 mL). The filtrate and wash were combined and diluted with ice-cold Et2O (30 mL) to precipitate the peptide. The slurry was centrifuged at 2000 rpm for 10 min, and the supernatant was discarded. The crude peptide was dissolved in 10 mL of DMSO / AcOH (1:1 v / v). Resin from 0.05 mmol batches was also subjected to lysis conditions using TFA / TIS / DODT / H2O (90:5:2.5:2.5 v / v / v / v) for three hours and treated as described for previous batches. The crude peptide solutions from both batches were combined for HPLC purification. Initial purification was performed using HPLC method P1, followed by a second purification using HPLC method P2 to obtain 27.7 mg Ex-Phe1 (96.0% purity, 214 nm) by analytical HPLC (condition A2). ESI-MS values ​​were 1050.0, C0.0. 187 H288 N 48 O 60 S (M+4H) 4+ The required value is 1050.2.

[0464] cAMP production

[0465] Cells were kept in cell-specific medium in a 37°C incubator with 5% CO2. cAMP production was measured using the HitHunter® cAMP assay kit. For both GLP-1R and GIPR, 10,000 cells per well were plated 24 hours prior to assay in either GIPR or GLP-1R Complete Cell Plating Reagent 2 in 384-well small-volume tissue culture plates. Before starting the assay, the medium was replaced with 5 mL of anti-cAMP antibody at a 1:2 ratio: 1x HBSS / 10 mM HEPES / 625 mM IBMX. The compound was diluted 1:1 in DMSO and then 5 nL was transferred to the wells using an ECHO acoustic liquid processor (Labcyte). The plated cells were incubated at 37°C for 30 min, and the cell suspension was incubated at room temperature with shaking for 30 min. The cAMP assay reagent was added according to the manufacturer's specifications, and luminescence was measured after the specified incubation time.

[0466] Beta-arrestin 2 recruitment

[0467] To measure GLP-1R and GIPR-mediated β-repressor protein recruitment, we used Promega's NanoBiT® technology or NanoLuc® binary technology. NanoLuc luciferase is divided into two subunits, called LgBiT and SmBiT, which are expressed in HEK293 cells as a fusion protein at the C-terminus of GLP-1R or GIPR and the N-terminus of β-repressor-2. Twenty-four hours prior to the β-repressor protein NanoBiT® assay, cells were extracted with cell dissociation medium and seeded at 10,000 cells per well in TC-treated 384-cell microplates. The next day, the medium was removed and the cells were resuspended in 10 mL of Opti-MEM. TMReplace the Nano-Glo® live cell substrate with a 1:100 dilution and equilibrate to room temperature for 10 minutes. Measure background luminescence before adding 10 nL of the compound using the ECHO acoustic liquid processor. Measure luminescence for 30 minutes at 1.5-minute intervals using an EnVision multimode plate reader (PerkinElmer). All assays were configured such that each row of the 384-well plate contained a single dilution series and, for normalization purposes, a single low control well (medium treatment) and a single high control well (GLP-1 or GIP treatment).

[0468] GLP-1R internalization

[0469] GLP-1R and GIPR internalization were measured using Promega's Nano-Glo® HiBit extracellular assay system (Promega Corporation; Madison, WI). Hek293 cells were transiently transfected with HiBiT-labeled hGLP-1R or hGIPR plasmids (Promega Corporation; Madison, WI). Cells were extracted using TrypLE expression enzyme (ThermoFisher Scientific) and seeded in 96-well plates at 80,000 to 100,000 cells per well. Cells were then treated with Nano-Glo HibiT extracellular buffer (1:100) containing LgBiT protein and Nano-Glo HiBiT extracellular substrate (1:50). Next, the test compound in DMSO was added to the cells, and the plates were read at two-minute intervals for 120 minutes on an EnVision multimode plate reader (Perkin Elmer; Waltham, MA).

[0470] Intraperitoneal glucose tolerance test

[0471] Liraglutide and CT-859 were administered via a single subcutaneous injection (SC) at the doses provided in each graph 4 to 48 hours prior to the intraperitoneal glucose tolerance test (ipGTT). Mice were fasted for five hours on the day of the ipGTT. Baseline blood glucose was determined from whole blood collected from the tail using an AlphaTrakII® glucometer. Subsequently, glucose 2 g / kg dextran (as a 20% solution in physiological saline) was injected via a single intraperitoneal injection. Blood glucose was determined at the time intervals specified in each graph. When plasma insulin concentration was required, mice were warmed under a heat lamp, blood glucose concentration was determined, and approximately 20 μL of whole blood was collected in a K2-EDTA microvette (Sarstedt). Area under the curve (AUC) analysis was performed using GraphPad Prism software, with the baseline set to Y = 0.

[0472] Food consumption

[0473] Short-term food intake: Mice were weighed, glucose levels were measured using a NOVA Biomedical glucometer (DataSciences International), and treatment was administered via a single SC injection or intraventricular injection into the lateral ventricle. Mice were returned to their cages with a pre-weighed amount of food on the cage floor. Body weight, food consumption, and glucose levels were measured at 24, 48, and up to 72 hours after treatment administration.

[0474] Intracerebroventricular cannulation

[0475] Male C57BL / 6J, GLP-1RKO, wild-type littermates, and C57BL / 6J DIO mice were implanted with a 26-gauge guide cannula with a 5 mm base. The cannula was incised 1.6 mm below the base using the following coordinates: anterior-posterior (AP); -0.4 mm, R / L; 1 mm, dorsoventral (DV); -1.3 mm. Mice were allowed to recover for one week. After one week, the mice were manually restrained and injected with a 33-gauge internal cannula syringe with a 0.8 mm protrusion.

[0476] Weight loss

[0477] DIO mice were acclimatized to daily weighing and treatment for approximately one week until their weight stabilized. Based on current weight, mice were subcutaneously injected once daily with an indicated dose of peptide or mediator. Injection was performed six hours before the start of the dark cycle to reach Tmax approximately at the start of the dark cycle. At the indicated time, a 24-hour food consumption study was conducted as described above. The percentage of body weight relative to initial body weight was calculated daily by dividing the daily body weight by the body weight collected before the first peptide dose and then multiplying by 100%. At the end of the study, blood glucose was determined, and blood was collected to quantify plasma insulin concentration. Mice were sedated with isoflurane and euthanized by decapitation. Whole-truncal blood was collected in a K2-EDTA microvette and kept on ice until centrifuged at 5000 rcf for 10 minutes at 4°C. Plasma was stored at -80°C until analysis. Subcutaneous and inguinal fat, as well as the liver, were removed and weighed.

[0478] Pharmacokinetics

[0479] Pharmacokinetic (PK) analysis of CT-859 and liraglutide was performed in male CD-1 mice by BioDuro Inc.

[0480] Plasma insulin

[0481] Whole blood collected in a K2-EDTA microvette was kept on ice until centrifuged at 5000 rcf for 10 minutes at 4°C. Plasma was removed and stored at -80°C until analysis. Plasma insulin concentration was determined using the U-PLEX mouse insulin assay (Meso Scale Discovery).

[0482] Statistical data

[0483] Results are expressed as mean (± standard error). Differences in glucose tolerance were assessed using one-way ANOVA and the Bonferroni test for multiple comparisons. All other comparisons were performed using two-way ANOVA with grouping and time as between-subject factors. Multiple comparisons were performed with Bonferroni correction. Statistical significance was set at p < 0.05. Statistical analysis was performed using GraphPad Prism 9.2.0 (GraphPad Software, Boston, MA).

[0484] result

[0485] CT-859 is a biased dual GLP-1R / GIPR agonist.

[0486] To investigate the cAMP accumulation, β-repressor protein coupling, and internalization properties of CT-859 and related tool compounds, recombinant cell lines expressing GLP-1R or GIPR were used. CT-859 is a complete agonist of cAMP accumulation at mouse GLP-1R, with potency 1 / 113 that of the natural ligand GLP-1 and 1 / 12 that of liraglutide (EC50: GLP-1 3 pM, liraglutide 29 pM, CT-859 343 pM, Fig. 15A). However, it exhibits pattern bias as it induces negligible (<5%) recruitment of β-repressor protein-2 to mouse GLP-1R, compared to GLP-1 (99%) and liraglutide (98%) (Fig. 15B). At the mouse GIPR, CT-859 is a partial agonist (44% Emax) for cAMP accumulation, with a potency driving cAMP accumulation that is 1 / 48th that of GIP (1.6 nM vs. 32 pM, Figure 15F). Similar to its effect at GLP-1R, it also exhibits negligible (< 5%) recruitment of β-repressor-2 to the mouse GIPR compared to GIP (Figure 15G). Next, the effects on human receptor internalization will be investigated, which may mimic the internalization characteristics at the mouse receptor, as the coupling properties of ligand cAMP and β-repressor are similar at both receptors. Figure 16 These receptors are highly conserved across species (92% identical to mouse GLP-1R, 94% similar, and 81% identical to mouse GIPR, 85% similar). Unlike GLP-1 and liraglutide, which drive human GLP-1R internalization (EC50 1.7 and 10.2 nM, respectively, Emax 56% and 75%, respectively), CT-859 does not induce receptor internalization (Emax -16%; Fig. 15C and 15D). Instead, two hours after exposure to CT-859, GLP-1R expression was higher than in mediator-treated cells (Fig. 15D). CT-859 exhibits a similar effect at human GIPR, with stimulated surface expression being greater than that of the mediator (Fig. 15H and 15I). This suggests that CT-859 acts as an antagonist of β-repressor protein coupling, inducing a reciprocal agonist effect that induces receptor internalization. In fact, CT-859 inhibits native ligand-mediated recruitment of β-repressor protein-2 at GLP-1R and GIPR (Figures 15E and 15J).

[0487] CT-859 normalizes postprandial glucose levels primarily through GLP-1R activation

[0488] The glucose-lowering effect of CT-859 was characterized in lean C57BL / 6J mice. During the intraperitoneal glucose tolerance test (ipGTT), CT-859 dose-dependently improved postprandial glucose with an ED50 of 0.46 nmol / kg (Figs. 17A and 17B), while liraglutide improved postprandial glucose with an ED50 of 7.3 nmol / kg (Figs. 17C and 17D). Since CT-859 is a dual GLP-1 / GIPR agonist, the contribution of each receptor to the glucose-lowering effect of CT-859 was assessed. For this purpose, ipGTT was performed in GLP-1RKO, GIPRKO mice and their wild-type (+ / +) littermates. + / + In mice, CT-859 (20 and 200 nmol / kg) reduced postprandial glucose levels equally and significantly compared to the mediator (Figures 18A and 18C). In another aspect, in GLP-1R... - / - In mice, 20 nmol / kg of CT-859 was ineffective in lowering glucose levels, while a dose of 200 nmol / kg reduced glucose levels by 46% compared to the mediator, and CT-859 (200 nmol / kg) reduced glucose levels by 75% in GLP-1R+ / + mice (Figures 18B and 18C). This suggests that the glucose-lowering effect at 20 nmol / kg is primarily mediated by GLP-1R, while at 200 nmol / kg, GIPR may be involved. - / - In mice, both doses of CT-859 (20 and 200 nmol / kg) were effective in lowering glucose levels. (Compared to GIPR) + / + In comparison, in GIPR - / - In mice, 20 nmol / kg CT-859 maintained glucose improvement (Figures 18D, 18E, and 18F). These results demonstrate that the glucose-lowering effect of 20 nmol / kg CT-859 is primarily driven by GLP-1R activation and does not require GIPR conjugation. This dose is referred to as the "non-GIPR conjugation dose".

[0489] At non-GIPR binding doses, CT-859 exhibited a prolonged glucose-lowering effect compared to liraglutide. To assess whether there was a difference in glucose-lowering effect between biased and unbiased GLP-1R agonist activity, GIPR... - / -In mice, ipGTT was performed using the maximum glucose-lowering dose of CT-859 (20 nmol / kg), a non-GIPR conjugate dose, and liraglutide (20 nmol / kg). CT-859 and liraglutide improved postprandial glucose 4 hours after a single dose. However, liraglutide no longer differed from the mediator at 24 hours post-treatment, while CT-859 maintained its activity at 24 and 48 hours (Figures 19A–19D). Similar studies were conducted in lean C57BL / 6J mice and diet-induced obese (DIO) mice to confirm the effect of GIPR conjugate doses. - / - Observations in mice. Results largely correlated with GIPR. - / - The results in mice were consistent. In lean C57BL / 6J mice, as shown by the area under the curve (AUC), there was no difference in glucose levels between CT-859 and liraglutide 4 hours after drug administration (Fig. 19E and 19H); however, in DIO mice, CT-859 was more potent than liraglutide (Fig. 19I and 19L). (Compared to GIPR) - / - The results in mice were consistent, finding that CT-859 remained effective up to 48 hours after administration, while in both lean C57BL / 6J and DIO mice, liraglutide was no longer distinct from the mediator (Figs. 19F, 19G, 19H, 19J, 19K, and 19L). To rule out the possibility of prolonged glucose reduction due to differences in drug exposure, the pharmacokinetics of CT-859 and liraglutide in mice were compared. Although CT-859 had a longer half-life, the exposure to liraglutide was 6 times that of CT-859 (Fig. 20). These results suggest that the observed differences in glucose reduction are inherent to CT-859 and may be caused by biased signaling of GLP-1R. To provide further evidence, exenatide-Phe1 (Ex-Phe1) was characterized and confirmed as a cAMP-biased agonist that prevents receptor internalization (Figs. 21A–21D). A single dose of Ex-Phe-1 showed that the biased GLP-1R agonist remained effective in the ipGTT eight hours after drug administration, while the unbiased GLP-1R agonist exenatide-4 (Ex-4) was no longer effective (Figures 21E and 21F). These results are consistent with the observations of Jones et al.

[0490] Central administration of CT-859 at non-GIPR engaging doses is more potent than liraglutide in reducing body weight

[0491] Studies have shown that GLP-1R agonists inhibit food intake through a centrally mediated mechanism. To characterize the central effects of biased and unbiased GLP-1R activation on food consumption and weight loss, compounds were administered to the lateral ventricles via intraventricular (ICV) injection. In lean C57BL / 6J mice, ICV administration of CT-859 (0.025 nmol) reduced food consumption by 63% and 38% at 24 and 48 hours post-treatment, respectively, compared to the mediators, while liraglutide (0.025 nmol) reduced food consumption by 33% and 15% at 24 and 48 hours post-treatment, respectively. Compared to liraglutide, CT-859 reduced food consumption by 45% and 27% at 24 and 48 hours post-treatment, respectively. Figure 22B Consistent with the reduction in food consumption, CT-859 resulted in a 9% and 5% reduction in body weight at 24 and 48 hours post-treatment, respectively. Liraglutide resulted in a 4% reduction in body weight at 24 hours post-treatment. Compared to liraglutide, CT-859 resulted in a 5% and 5% reduction in body weight at 24 and 48 hours post-treatment, respectively (Figure 22A). Next, the results were compared with those obtained in GLP-1R. - / - ICV-injected CT-859 in mice and their WT littermates was evaluated to characterize how each receptor promoted the effects observed in lean C57BL / 6J mice. CT-859 (0.025 nmol) inhibited GLP-1R. + / + Food consumption and weight reduction were observed in mice treated with CT-859, but it had no effect on GLP-1R- / - mice (Figs. 22C and 22D). This suggests that at this dose, CT-859 inhibits food consumption and reduces weight by activating GLP-1R and does not bind to GIPR at this dose. To confirm that these effects are mediated solely by biased signaling at GLP-1R, the effects of ICV-administered Ex-phe1 and Ex-4 were evaluated in lean C57BL / 6J mice. ICV-administered Ex-4 and Ex-Phe1 equally reduced food intake and weight at 24 hours post-treatment (Figs. 21G and 21H). However, by 48 hours, Ex-4 began to fail, while Ex-phe1 remained effective (Figs. 21G and 21H). The data showed that a single dose of biased GLP-1 administered by ICV was more effective in inhibiting food intake and reducing weight compared to unbiased GLP-1.

[0492] Long-term CT-859 is more potent than liraglutide in reducing body weight.

[0493] To further evaluate the differences between biased and unbiased GLP-1R agonists, a long-term study was conducted in the obese and hyperglycemic model C57BL / 6J DIO mice. First, CT-859 (20 nmol / kg) (the non-GIPR conjugating dose) was compared with liraglutide (20 nmol / kg). CT-859 and liraglutide initially resulted in similar weight loss. However, CT-859 began to differ from liraglutide after one week of administration. At the end of the study, CT-859 resulted in a 20% reduction in body weight compared to the conjugate. Liraglutide resulted in a 13% reduction in body weight compared to the conjugate. Furthermore, CT-859 resulted in an 8% reduction in body weight compared to liraglutide (Figure 23A). Interestingly, there was no difference in food intake between CT-859 and liraglutide (Figure 23B), suggesting the potential to utilize other mechanisms involved in weight loss through biased GLP-1R activation. At the end of the study, body composition was examined to determine the source of weight loss. The CT-859 group had 39% less groin fat weight than the mediator group; however, liraglutide showed no difference compared to the mediator group (Figure 24A). There was no difference in epididymal fat weight between the two groups. Figure 24B Compared to the mediator, both liraglutide and CT-859 reduced liver weight (Fig. 24C). CT-859 was more effective than liraglutide in reducing fasting glucose; however, both treatments were more effective than the mediator in reducing plasma insulin levels (Figs. 23C and 23D); therefore, CT-859 had a lower Log(HOMA-IR) compared to liraglutide. Figure 23ENext, CT-859 (200 nmol / kg, GIPR conjugate dose) and liraglutide (200 nmol / kg) were administered to DIO mice for 19 days to assess the full pharmacological potential for weight loss. Following a pattern similar to the non-GIPR conjugate dose, CT-859 and liraglutide initially reduced body weight equally. However, CT-859 began to differentiate from liraglutide after 3 days of administration, and by the end of the study, CT-859 resulted in a 33% reduction in body weight compared to the conjugate. Liraglutide resulted in a 22% reduction in body weight compared to the conjugate. Furthermore, CT-859 resulted in a 14% reduction in body weight compared to liraglutide (Figure 23F). Compared to the non-GIPR conjugate dose, CT-859 resulted in a 39% reduction in food consumption compared to liraglutide after 7 days of administration (Figure 23G). At this dose, GIPR conjugation may have altered the dynamic effects of CT-859 on food intake and the mechanisms involved in weight loss. At study end, CT-859 was more effective than liraglutide in reducing inguinal and epididymal fat weight, and both reduced liver weight equally (Figs. 24D–24F). CT-859 was more effective than the conjugate in reducing postprandial glucose levels, while liraglutide had no effect (Fig. 23H). CT-859 was more effective than the conjugate in reducing postprandial insulin levels (Fig. 23I). Compared to the conjugate, both CT-859 and liraglutide reduced Log(HOMA-IR) (…). Figure 23J These data indicate that while CT-859 is more potent than liraglutide at non-GIP binding doses, its superior efficacy is more pronounced at GIP binding doses.

[0494] discuss

[0495] In this study, the GLP-1R / GIPR agonist CT-859 was engineered to exhibit a bias towards G protein coupling at both receptors. CT-859 does not induce receptor internalization at either receptor; and it inhibits β-repressor protein coupling via its natural ligands GLP-1 and GIP. These characteristics appear to translate into improved in vivo efficacy, as evidenced by the following observations: CT-859 improved glucose tolerance, where ED... 50 The ED is 0.46 nmol / kg, while the ED of liraglutide is... 50The concentration was 7.3 nmol / kg. These results are surprising because CT-859's potency in GLP-1R cAMP accumulation assays is less than one-hundredth that of the natural ligand GLP-1 and less than one-tenth that of liraglutide. When liraglutide and CT-859 were tested at the maximum glucose reduction dose, CT-859 was shown to have a prolonged improvement in glucose tolerance in lean and DIO mice compared to liraglutide. This prolonged efficacy was not due to additional GIPR binding, as the tested dose does not bind GIPR, and the circulating drug concentration cannot explain it, since the drug exposure to liraglutide is six times that of CT-859. These results may suggest that the improving efficacy of CT-859 may be mediated by biased signaling at GLP-1R. To support this research, it has been demonstrated that loss of β-repressin-2 drastically reduces the insulin secretory response to GLP-1R agonists (Bitsi et al., Science Advances.(2023) 9:eadf7737). Nevertheless, long-term exposure to GLP-1R agonists resulted in better glucose reduction in these mice, suggesting that loss of β-repressin-2 signaling severely impairs the insulin response to GLP-1R agonists. However, long-term exposure to these agonists increases their effectiveness in lowering glucose levels by increasing insulin secretion (Bitsi, ibid.). Furthermore, studies have demonstrated that biased GLP-1R agonists are more effective than unbiased GLP-1R agonists in glucose regulation (Pickford et al., British Journal of Pharmacology.(2020) 177:3905-23; Lucey et al., Molecular Metabolism.(2020) 37:100991; Jones et al., Nat Comm.(2018) 9:1602; Zhang et al., Nat Comm.(2015) 6:8918). In summary, these results suggest that the absence of β-repressor protein coupling increases the glucose-lowering efficacy of GLP-1 receptor agonists.

[0496] Short-term observations translated into better efficacy in long-term studies. In a 3-week DIO mouse study, non-GIPR-binding doses of CT-859 were compared with liraglutide; CT-859 was more effective than liraglutide in reducing fasting BG and improving HOMA-IR. More importantly, treatment with CT-859 produced more weight loss than liraglutide. In this study, both treatments equally suppressed food consumption. These results suggest that appetite suppression may contribute to the efficacy of both treatments, but additional mechanisms may explain the observed difference in weight loss between CT-859 and liraglutide. Consistently, GLP-1R agonists have been shown to stimulate thermogenesis and adipocyte browning in brown adipose tissue (BAT), independent of nutrient intake in mice. Furthermore, a longitudinal study involving obese type 2 diabetes (T2D) patients demonstrated that long-term treatment with liraglutide increased energy expenditure (Beiroa et al., Diabetes. (2014) 63:3346-58).

[0497] In contrast to peripheral administration, central administration of CT-859 was more effective than liraglutide in suppressing food consumption. Evaluation of biased GLP-1R agonists was primarily conducted after peripheral administration, and enhanced efficacy in glucose regulation was consistently observed. However, the effects on appetite and weight regulation were inconsistent with different biased GLP-1R agonists (Jones et al., Nat Comm. (2018) 9:1602; Zhang et al., Nat Comm. (2015) 6:8918).

[0498] One possible explanation is the varying bioavailability of these agonists in the brain. GLP-1R agonist peptides are typically derivatized by lipid modifications on lysine residues that extend their half-life, thereby prolonging their persistence. These lipid modifications can alter the bioavailability of GLP-1R agonists in the brain, as the length of such lipid modifications contributes to the ability of these agonists to cross the blood-brain barrier (BBB). Short-acting GLP-1R agonists such as Ex-4 are primarily detectable outside the BBB; however, GLP-1R agonists with extended half-life modifications are detected in brain regions outside the BBB, and their diffusion is correlated with the length of the lipid modification (Skovbjerg et al., Neuropharmacology.(2023) 238:109637). The ability of GLP-1R agonists to cross the BBB is essential, as studies on inhibiting or knocking down / knockout neuronal GLP-1R have demonstrated that the anorexia and weight loss effects of GLP-1R agonists are primarily mediated by the central nervous system (CNS) (Sisley et al., J Clin Investigation. (2014) 124:2456-463; Secher et al., J Clin Investigation. (2014) 124:4473-88; Fortin et al., Sci Trans Med. (2020) 12:eaay8071). Many factors can alter the bioavailability of GLP-1R agonists in the brain, such as GLP-1R expression, blood glucose, and obesity (Imbernon et al., Cell Metabolism. (2022) 34:1054-63.e1057; Bakker et al., Cell Reports. (2022)). 41). Therefore, central administration is more suitable for assessing the effects of biased and unbiased GLP-1R agonists on food intake inhibition and weight loss. Indeed, in lean mice, non-GIPR-binding doses of CT-859 administered via ICV were more effective than liraglutide in inhibiting food intake and reducing weight. To confirm the results for CT-859 and liraglutide, Ex-phe1 and Ex-4 were administered via ICV, and Ex-phe1 was shown to be more effective than Ex-4 in inhibiting food intake and reducing weight. These effects were not observed with peripheral administration (Jones et al., Nat Comm. (2018) 9:1602; Pickford et al., British Journal of Pharmacology. (2020) 177:3905-3923).Proximity to the CNS GLP-1R may be key, which could also explain the difference in appetite suppression between central and peripheral administration of CT-859. Our data demonstrate that, in comparison to unbiased GLP-1R agonists (liraglutide and Ex-4), the use of biased GLP-1R agonists (CT-859 and Ex-phe1) yields superior glucose and weight regulation efficacy.

[0499] Next, the efficacy of GIPR-binding doses of CT-859 was evaluated and compared with equimolar doses of liraglutide. Treatment with both molecules resulted in significant weight loss, but treatment with CT-859 resulted in a greater weight loss, approximately 35% less than the mediator, compared to approximately 25% for liraglutide. The additional 10% weight loss from CT-859 is likely a result of biased GLP-1R agonism and GIPR activation through inhibition of food intake. At this dose, CT-859 was more effective than liraglutide in inhibiting food intake. These results demonstrate that GIPR activation regulates energy expenditure and weight loss, and that enhanced efficacy can be obtained with the use of biased GIPR agonists. Regarding the synergistic effect between GLP-1 and GIP, biased GIPR agonists are best combined with biased GLP-1R agonists for optimal efficacy. As CT-859 is a biased dual GLP-1R / GIPR agonist, it possesses all the characteristics of a highly effective molecule. In fact, when GIPR is administered in combination, superior efficacy is achieved in terms of food intake suppression and weight loss.

[0500] in conclusion

[0501] In this study, CT-859 was described as a dual GLP-1R / GIPR agonist with cAMP bias at both receptors. CT-859 was used as a tool compound to examine the importance of biased signaling in glycemic control, food intake, and weight. The biased dual GLP-1R / GIPR agonist was demonstrated to be more effective than the unbiased GLP-1R agonist in glycemic regulation, food intake inhibition, and weight loss. The superior efficacy of the biased dual GLP-1R / GIPR agonist was demonstrated to be due to the fact that both biased GLP-1R and GIPR agonists alone are more effective than their unbiased counterparts in glycemic control, food intake inhibition, and weight loss, and that the combination of the two biased agonists exhibits positive synergy. These results suggest that biased signaling should be considered when designing more effective enterohepatic receptor agonists.

[0502] Example 3: Biased GLP-1 enhances weight loss and has additional benefits for glucose homeostasis in a diabetic rodent model.

[0503] CT-868 and CT-859 are biased dual GLP-1 and GIP receptor modulators, exhibiting no β-repressor protein coupling at either receptor. The effects of these two compounds on weight loss (WL) and glucose (GLUC) homeostasis were evaluated in relevant rodent models.

[0504] method

[0505] Untreated 9-week-old male Lep from Jackson Laboratory ob (ob / ob) mice were housed individually and fed a standard diet. Mice were randomly assigned by body weight to the following groups: mediator (phosphate-buffered saline + Tween; n = 8), liraglutide (200 nmol / kg; n = 8), and CT-868 (30 nmol / kg; n = 8). The compounds were administered daily via a single subcutaneous injection. Results were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons (Figure 25).

[0506] Untreated male GIPR-KO mice aged 11 to 15 weeks from Taconic were housed individually and fed a standard diet. Mice were randomly assigned by body weight to the following groups: mediator (phosphate-buffered saline + Tween; n = 5 / 6), liraglutide (20 nmol / kg; n = 6), and CT-859 (20 nmol / kg; n = 6). Intraperitoneal glucose tolerance test (ipGTT; 2 g / kg) was performed 5 hours after fasting and 4 hours after compound administration. One week after washout, the compound was administered subcutaneously. Subsequently, ipGTT was performed in the same mice at 24 and 48 hours after compound administration. Results were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons (Figure 26).

[0507] A seven-week-old male C57BL / 6-Ins2 from Jackson Laboratory Akita / J (Akita) mice were housed individually and fed a standard diet. Mice were randomly assigned by body weight to the following groups: mediator (phosphate-buffered saline + Tween; n = 7), liraglutide (20 nmol / kg; n = 6), and CT-868 (20 nmol / kg; n = 6). The compounds were administered daily via a single intraperitoneal injection. Body weight was recorded daily, and postprandial glucose was measured at baseline, 7 days after treatment initiation, and 14 days after treatment. Results were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons (Figure 27).

[0508] 9- to 10-week-old male C57BL / 6-Ins2 from Jackson Laboratory Akita / J (Akita) mice were housed individually and fed a standard diet. Approximately two weeks prior to the start of liraglutide and CT-868 treatment, mice were implanted with half a LinBit insulin pill. Mice were randomly assigned to the following groups based on postprandial blood glucose: mediator (phosphate-buffered saline + Tween; n = 7), liraglutide (200 nmol / kg; n = 8), and CT-868 (200 nmol / kg; n = 8). The compounds were administered daily via a single intraperitoneal injection. Body weight was recorded daily, and postprandial glucose was measured at baseline, 7 days after treatment initiation, and 14 days after treatment. Results were analyzed using a two-way ANOVA and the Holm-Sidak test for multiple comparisons. Figure 28 ).

[0509] At Jackson Laboratory, 24-week-old C57BL / 6J diet-induced obese (DIO) mice received intraperitoneal injections of streptozotocin (STZ; 50 mg / kg) daily for 5 days. Mice were received from Jackson's laboratory at 27 weeks of age. Mice were implanted with one LinBit insulin pill and monitored for 18 days. A second LinBit insulin pill was then implanted before the start of liraglutide and CT-868 treatment. Mice were then randomly assigned by body weight to the following groups: mediator (phosphate-buffered saline + Tween; n = 7), liraglutide (200 nmol / kg; n = 7), and CT-868 (200 nmol / kg; n = 7). The compounds were administered daily via a single intraperitoneal injection. Body weight and postprandial blood glucose were recorded daily. Results were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons. Figure 29 ).

[0510] An 8-week-old male C57BL / 6-Ins2 from Jackson Laboratory Akita / J (Akita) mice were housed individually and fed a standard diet. Mice were randomly assigned by weight to receive either insulin or a blank insulin pellet. Mice were anesthetized with isoflurane, and a blank pellet or one or more LinBit insulin pellets were implanted subcutaneously in the interscapular region. The day after pellet implantation, mice were again randomly assigned to the following groups: mediator (phosphate-buffered saline + Tween) + 0.5 LinBit insulin pellets (low insulin; n = 7), mediator + 1.5 LinBit insulin pellets (high insulin; n = 7), CT-868 (300 nmol / kg) + blank pellets (n = 7), and CT-868 (300 nmol / kg) + 0.5 LinBit insulin pellets (CT-868 + low insulin; n = 7). CT-868 was administered daily via a single intraperitoneal injection for 14 days. Body weight and postprandial blood glucose were recorded daily. On the last day of the study, mice were fasted for 4 hours; blood glucose was then measured, and blood was collected for insulin. Plasma insulin was measured using the MSD kit. The results were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons (Figures 30 and 31).

[0511] Male GLP-1R-KO mice aged 26–27 weeks from Taconic were housed individually and fed a standard diet. For the intraperitoneal glucose tolerance test (ipGTT), mice were randomly assigned by body weight to either the mediator (PBS; n = 6) or CT-868 (300 nmol / kg; n = 7). Mice were fasted overnight (18 hours). After overnight fasting, the compound was administered subcutaneously. One hour after compound administration, glucose was measured, blood was collected, and glucose (2 g / kg) was administered intraperitoneally. Subsequently, blood glucose levels were measured, and insulin was measured at 10, 20, 30, 40, 60, and 120 minutes after glucose injection. Insulin was measured using the MSD insulin kit. Results were analyzed using an unpaired t-test (Figure 32). Non-treated 7-week-old male C57BL / 6-Ins2 mice from Jackson Laboratory were also included. Akita / J (Akita) mice were housed individually and fed a standard diet. Mice were randomly assigned by body weight to the following groups: mediator (phosphate-buffered saline + Tween) + insulin glargine (3 U / kg; n = 6), CT-868 (10 nmol / kg) + insulin glargine (3 U / kg; n = 6), CT-868 (30 nmol / kg) + insulin glargine (3 U / kg; n = 6), and CT-868 (100 nmol / kg) + insulin glargine (3 U / kg; n = 5). Mice were fasted for 5 hours prior to the pyruvate tolerance test. After 3 hours of fasting, insulin glargine and CT-868 were administered subcutaneously separately. At the end of fasting, glucose was measured, and pyruvate (1 g / kg) was administered intraperitoneally. Blood glucose levels were then measured at 20, 40, 60, and 120 minutes after pyruvate injection. The results were analyzed using one-way ANOVA and the Holm-Sidak test for multiple comparisons (Figure 33).

[0512] Untreated 15-week-old male GLP-1R-WT (WT) and GLP-1R-KO (KO) mice from Taconic were housed individually and fed a standard diet. Mice were randomly assigned by body weight to the following groups: WT-mediator, WT-CT-868 (30 nmol / kg), WT-CT-868 (100 nmol / kg), KO-mediator, KO-CT-868 (30 nmol / kg), and KO-CT-868 (100 nmol / kg). Mice were fasted overnight (17 hours). After overnight fasting, glucose was measured, and the compound was administered subcutaneously. Next, glucose was measured 2 hours after compound administration, and pyruvate (1 g / kg) was administered intraperitoneally. Blood glucose levels were then measured at 20, 40, 60, and 120 minutes after pyruvate injection. Results were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons (Figure 34).

[0513] GLP-1R-WT (wild-type) and GLP-1R-KO (GLP-1R - / -) mice from Taconic were housed individually and fed a standard diet. Mice were randomly assigned by body weight to the following groups: mediator (phosphate-buffered saline + Tween; n = 6), CT-859 (0.025 nmol / kg; n = 6), and CT-859 (1 nmol / kg; n = 6). The compound was administered via a single ICV injection. Body weight (BW), total food consumption (F), and plasma glucose were measured at 24-hour intervals after drug administration until 72 hours (Figures 35 and 36). Results were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons.

[0514] GLP-1R-WT (wild-type) mice from Taconic were housed individually and fed a standard diet. Mice were randomly assigned by body weight to the following groups: mediator (phosphate-buffered saline + Tween; n = 9), CT-859 (0.025 nmol / kg; n = 10), and liraglutide (0.025 nmol / kg; n = 9). The compounds were administered via a single ICV injection. Body weight (BW) and total food consumption (F) were measured 19 hours after drug administration (Figures 37A and 37B). Results were analyzed using two-way ANOVA and the Holm-Sidak test for multiple comparisons.

[0515] result

[0516] In ob / ob mice, at non-GIPR conjugated doses, CT-868 at a dose of 30 nmol / kg achieved greater weight loss (19.8% vs. 11.5%, p = 0.002) compared to 200 nmol / kg of the unbiased GLP-1 receptor agonist liraglutide, indicating that biased GLP-1 improved weight loss compared to unbiased GLP-1 (Figure 25). In GIPR- / - mice, the GLUC tolerance test (GTT) showed similar reductions in GLUC AUC 4 hours after administration of 20 nmol / kg of liraglutide and CT-868 (53% vs. 62%, p = NS), but only CT-868 maintained a significant reduction at both 24 and 48 hours, suggesting that biased GLP-1 induces a sustained reduction in GLUC (Figure 26).

[0517] In Akita mice, after 14 days of administration of CT-868 at a non-GIPR-conjugated dose of 20 nmol / kg, body weight (BW) decreased by 4% and blood glucose (BG) decreased by 30% compared to a dose of liraglutide (p=0.01), indicating an enhanced effect of biased GLP-1 on glycemic control (Fig. 27). In Akita (↓35%, p=0.009) (Fig. 28) and DIO-STZ (↓32%, p<0.0001) (Fig. 29) mice, a GIPR-conjugated dose of CT-868 significantly reduced BG on an insulin (INS) background compared to a dose of liraglutide at 200 nmol / kg. Furthermore, in DIO-STZ mice, CT-868, acting as an adjunct to insulin, was more effective than liraglutide in reducing BW (Fig. 29).

[0518] BG in Akita mice treated for 14 days with a combination of CT-868 at a dose of 300 nmol / kg and low INS (0.5 LinBit INS pellets) was normalized to the same extent as with mediator + high INS (1.5 LinBit INS pellets), but with INS levels 68% lower (p < 0.0001) (Figures 30 and 31).

[0519] In GLP-1R- / - mice, CT-868 at a dose of 300 nmol / kg reduced GLUC AUC by 38% compared to the mediator (p<0.0001), without any associated INS drift or hyperinsulinemia, indicating that GIP enhances INS-independent GLUC treatment or INS sensitivity (Figure 32).

[0520] In response to the pyruvate challenge, CT-868 at a dose of 100 nmol / kg on an INS-treated background was used to deliver Akita (Figure 33) and GLP-1R, respectively. - / - (Figure 34) GLUC AUC in mice was inhibited by 36% (p<0.05) and 14% (p=0.006), suggesting that GIPR activation may help inhibit endogenous glucose production.

[0521] In GLP-1R-WT mice, compared with the mediator control, administration of CT-859 via ICV injection resulted in a dose-dependent decrease in cumulative food consumption, which was associated with a dose-dependent decrease in body weight. Weight loss persisted for up to 72 hours. Higher doses of CT-859 were also associated with decreased blood glucose (Figure 35). In contrast, administration of CT-859 via ICV injection to GLP-1R-KO mice had no significant effect on food consumption, body weight, or blood glucose compared with the mediator control (Figure 36).

[0522] In a head-to-head comparative study, administration of CT-859 via ICV injection resulted in significantly greater reductions in food intake and body weight compared to an equivalent dose of liraglutide (Figures 37A and 37B).

[0523] In summary, these data demonstrate the importance of biased cAMP signaling of GLP-1RA in the central nervous system for long-term inhibition of food intake and weight loss in mouse models. These data also demonstrate that CT-859 and CT-868 provide enhanced weight loss via biased GLP-1 and enhance GLUC homeostasis via biased GIP compared to unbiased GLP-1 receptor agonists such as liraglutide.

[0524] Example 4: The weight-independent effects of CT-868 on glucose homeostasis in overweight and obese diabetic adults

[0525] method

[0526] CT-868 is a biased dual GLP-1 and GIP receptor modulator that does not exhibit β-repressor protein coupling or receptor internalization at either receptor. In a randomized, double-blind, crossover study, its effects on insulin (INS) secretion were investigated in 20 overweight and obese adults with type 2 diabetes mellitus (T2DM) via graded glucose infusion (GGI), glucose (GLUC) homeostasis in response to the mixed dietary tolerance test (MMTT), gastric emptying (GE), and food intake (FI). In a previous phase 1 study, CT-868 was tested in healthy and overweight / obese participants at single doses up to 11 mg and up to 5 mg / day for 14 days without any up-titration and was found to be safe and well-tolerated.

[0527] Subjects were divided into two groups: CT-868 and placebo, n = 7; and CT-868 and placebo versus liraglutide, n = 13; the latter served as a fixed third group. The average age and BMI were 52.2 years and 32.7 kg / m², respectively. 2 Of these, 55% were male. Treatment was administered for 4 days to minimize weight loss, with a 14-day washout interval in between.

[0528] Purpose

[0529] The primary objective of this study was to evaluate the effects of CT-868 on insulin secretion rate (ISR) and glucose homeostasis in overweight and obese adults with type 2 diabetes mellitus (T2DM) compared to placebo and liraglutide.

[0530] Research Design

[0531] This is a phase 1, double-blind, placebo-controlled, randomized, single-center, crossover trial involving 20 adults aged 18 to 65 years diagnosed with type 2 diabetes mellitus (T2DM).

[0532] a) Diet and exercise alone, or stable therapy (≥ 3 months) using metformin monotherapy or a combination of metformin and sulfonylurea (SU) prior to screening. Wash off SU ≥ 7 days prior to randomization.

[0533] b) Body Mass Index (BMI) >27 and ≤45 kg / m² 2

[0534] c) Baseline HbA1c ≤ 10.5%; and fasting plasma glucose < 250 mg / dL

[0535] Group 1 (n = 13) included a three-way crossover design evaluating CT-868 (5 mg, 3.25 mg, and 3.25 mg doses), placebo, and liraglutide (0.6 mg, 1.2 mg, and 1.2 mg doses) as an activity comparison, evaluated over three internal periods (Figure 38A). Group 2 (n = 7) included a two-way crossover design evaluating CT-868 (5 mg and 3.25 mg doses) and placebo over two internal periods (Figure 38B).

[0536] During each period, subjects received a randomized study drug via subcutaneous injection (SC) on days 1, 2, and 3; pharmacokinetic (PK) and pharmacodynamic (PD) blood samples were collected. Assessments included graded glucose infusion (GGI) on day 3; mixed diet tolerance test (MMTT), gastric emptying (GE), and free food intake assessment on day 4; and appetite and satiety ratings on days 1, 2, 3, and 4 using the hunger mimicry scale (VAS).

[0537] end

[0538] The primary endpoint of this study was to assess the insulin secretion rate (ISR) relative to ambient glucose (ISR / G).

[0539] The secondary endpoint of this study was:

[0540] ●Assess changes in glucose, insulin, C-peptide, and glucagon levels during the Mixed Diet Tolerance Test (MMTT);

[0541] ●Gastric emptying was assessed based on paracetamol absorption;

[0542] ●Appetite, hunger, and satiety were assessed using a hunger simulation scale and free food intake; and

[0543] ●Assess CT-868 plasma exposure [maximum plasma concentration (C]]. max Time to maximum plasma concentration (t) max ); Area under the concentration-time curve (AUC) within the dosing interval 0-tau Terminal half-life (t) 1 / 2 )).

[0544] Safety assessments in this study included treatment-induced adverse events (TEAEs), serious adverse events (SAEs), adverse events of particular concern (AESIs), vital signs, 12-lead electrocardiogram (ECG), clinical laboratory assessments, and physical examinations.

[0545] result

[0546] A total of 20 participants with type 2 diabetes mellitus were randomly assigned to different groups in the study. The participant demographics are shown in Table 9 below.

[0547] Table 9. Demographic and Baseline Characteristics

[0548]

[0549] The endpoints of graded glucose infusion (GGI) are shown in Table 10 below.

[0550] Table 10. PD-Stage Glucose Infusion (GGI) Endpoint

[0551]

[0552] GGI data showed that insulin secretion was robust against CT-868 compared to placebo. maxChanges in ISR / GLUC (pmol / kg / min): CT-868 1.1 (0.1), placebo 0.1 (0.2), liraglutide 1.0 (0.3)]. The data demonstrate that patients with T2DM treated with liraglutide or CT-868 had a robust insulin secretion response compared to participants treated with placebo (Figure 39).

[0553] MMTT data and insulin attack data are shown in Table 11 below.

[0554] Table 11. MMTT and insulin drift

[0555]

[0556] These data show that the MMTT-based GLUC iAUC for CT-868 0-240 The INS iAUC was significantly lower than placebo [72(46) vs 392(48) mmol / L*min] and numerically lower than liraglutide [187(55) mmol / L*min]. Furthermore, compared to both placebo and liraglutide, the INS iAUC was significantly lower. 0-240 Significant reductions: CT-868 1178 (846), placebo 5833 (873), liraglutide 4613 (1253) mU / L*min (Figure 40). Therefore, compared with liraglutide, CT-868 reduced glucose and significantly reduced insulin drift during the mixed diet tolerance test (MMTT) in patients with T2DM.

[0557] Since both CT-868 and liraglutide exhibited similar gastric emptying delay (GE) to placebo, the observed decrease in glucose (GLUC) and INS drift with CT-868 indicated improved INS sensitivity or enhanced INS-independent GLUC management. CT-868 also demonstrated minimal inhibition of glucagon during the MMTT compared to liraglutide, which showed a decrease in glucagon. Food intake (FI) was lower with CT-868 compared to placebo, along with reduced hunger and appetite as assessed by VAS. No significant weight changes were observed during any treatment period. GI side effects were mostly mild and transient.

[0558] Based on these data, the accompanying decrease in plasma glucose and insulin drift may be a result of enhanced glucose handling, for example, promoted by insulin sensitization mechanisms. Glucose handling with less / minimum glucagon inhibition compared to liraglutide may reduce the risk of hypoglycemia with CT-868. These data support the robust, weight-independent effect of CT-868 on glucose handling (compared to unbiased liraglutide) with minimal glucagon inhibition.

[0559] In addition, CT-868 tends to reduce appetite and hunger scores, which translates into a significant suppression of food intake (absolute amount and calorie consumption) during free eating, as shown in Table 12 below.

[0560] Table 12. Weight, Appetite / Hunger Score, and Food Intake

[0561]

[0562] Plasma exposure to CT-868 in patients with type 2 diabetes mellitus (T2DM) is shown in Figure 41 and Table 13 below. These data confirm that key pharmacodynamic assessments (i.e., GGI, MMTT, and GE) are typically performed at the point of maximum CT-868 plasma concentration.

[0563] Table 13. Plasma exposure to CT-868 in patients with type 2 diabetes mellitus (T2DM)

[0564]

[0565] The security studies summarized in Table 14 below show that:

[0566] 1. CT-868 is well tolerated;

[0567] 2. Most TEAEs are grade 1 (mild);

[0568] 3. GI TEAEs with nausea, vomiting, and constipation are grade 1 in severity, transient, and resolve spontaneously without the need for any concomitant medication;

[0569] 4. Grade 1 hypoglycemic events were observed in 7 participants after CT-868 administration (primarily during GGI surgery), and in 2 and 3 participants, respectively, after placebo and liraglutide administration; and

[0570] 5. One subject discontinued the study due to a Grade 1 injection site reaction to RUQ ecchymosis (4cm x 3cm) (Phase 1 / 4 days before discharge).

[0571] Table 14. Security Data – AE Overview

[0572]

[0573] in conclusion

[0574] The results of this study showed that: during the study period, there was no significant change in body weight after either treatment; both CT-868 and liraglutide delayed gastric emptying compared to placebo; CT-868 demonstrated robust insulin secretion from β-cells in patients with T2DM compared to placebo; CT-868 treatment reduced appetite and hunger scores, accompanied by a significant reduction in food intake, compared to placebo; during the MMTT, patients with T2DM treated with CT-868 showed lower blood glucose levels, accompanied by a significant reduction in insulin drift, compared to both placebo and liraglutide. The associated reduction in glucose and insulin drift indicates enhanced insulin sensitivity and / or insulin-independent glucose handling induced by CT-868, independent of weight loss. Finally, the study indicates that CT-868 was well-tolerated in patients with T2DM with no significant adverse reactions.

[0575] Example 5: Pancreatic and extrapancreatic effects of the signal transduction-based dual GLP-1 / GIP receptor agonist CT-868 on metabolic homeostasis in preclinical models and in participants with or without type 2 diabetes.

[0576] The dual GLP-1 / GIP (glucagon-like peptide-1 / glucose-dependent insulinotropic peptide) receptor agonist CT-868 exhibits selectivity for the GLP-1 receptor (GLP-1R) relative to the GIP receptor (GIPR) and complete cAMP signaling bias on both receptors. CT-868 has a once-daily dosing pharmacokinetic (PK) profile, effectively enhancing glucose-stimulated insulin secretion in mice. In a mouse model of obesity, CT-868 positively affects glucose metabolism and reduces food intake and body weight, demonstrating potential advantages over unbiased GLP-1R agonists such as liraglutide.

[0577] Results from two Phase 1 clinical trials indicate that single-dose (0.1–11 mg) and multiple-dose (up to 5.0 mg / day) CT-868 (without up-titering) administration is generally safe and well-tolerated in participants with and without type 2 diabetes who are overweight / obese. Compared with placebo and liraglutide, CT-868 demonstrated robust glucose reduction, reduced insulin drift, and preserved glucagon secretion, and induced dose-dependent weight loss. These results support further evaluation of the long-term effects of CT-868 on glucose levels, weight, and related parameters in overweight and / or diabetic individuals.

[0578] method

[0579] The sources of key reagents and resources are shown in Table 15.

[0580] Table 15. Sources of Selected Resources

[0581]

[0582] Animal experiments

[0583] All rodent experiments were subject to the necessary approvals from the FibroGen and Explora BioLabs Institutional Animal Care and Use Committee.

[0584] Drug substances and structures

[0585] CT-868 DS is a novel synthetic hydrochloride manufactured as a lyophilized white powder. The structure, molecular formula, and molecular weight of CT-868 are as follows:

[0586] CT-868 Structure: 2-(2-piperidinone-1-yl)-ethylcarbamoylmethylthioacetyl-Glu-Gly-ThrPhe-Thr-Ser-Asp-Tyr-Ser-lle-Tyr-Leu-Asp-Lys-Gln-Ala-Ala-Aib-Glu-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(γ-Glu-palmitoyl)-NH2 hydrochloride (SEQ ID NO: 3)

[0587] Molecular formula: C 214 H 323 N 47 O 65 S (free base)

[0588] Molecular weight: 4610.3 g / mol (free base)

[0589] Cell lines and maintenance

[0590] Cells were maintained in a 37°C incubator at 5% CO2 using standard laboratory practices and in cell-specific media. For human GLP-1R and GIPR cAMP assays, internally prepared stable clones of SNAP-human GLP-1R and SNAP-human GIPR CHO-K1 were used. For mouse GLP-1R and GIPR cAMP assays, DiscoverXHithunter U2OS and CHO-K1 cells were used, respectively. For the β-repressor-2 NanoBiT® assay, equal amounts of the indicated GPCR-LgBit and SmBiT-β-repressor-2 plasmids were co-transfected into HEK293 cells, and stable plasmid DNA expression was measured 48 hours post-transfection or after antibiotic selection. For internalization assays, 6–12 ng of plasmid DNA containing an N-terminal HiBiT-tagged human GLP-1R or GIPR open reading frame and 6–12 µg of vector DNA were co-transfected. All transfections were performed in T75 flasks using 6–12 μg DNA, Fugene 6 at a 1:3 ratio, and 10 million to 12.5 million HEK293 cells. Measurements were performed 48 hours post-transfection.

[0591] cAMP production

[0592] The production of cAMP in cells was measured using the HitHunter® cAMP Assay Kit for Biologics. For human receptors, cells were extracted with cell dissociation medium, counted, rotated, and resuspended in a 1:2 ratio of anti-cAMP antibody: 1× HBSS / 10 mM HEPES / 625 µM IBMX. 5 µL of 10,000 cells were added to each well of a 384-well small-volume assay plate. For mouse GLP-1R and GIPR cAMP assays, 3 to 10,000 cells per well were plated 24 hours prior to assay in whole cell plating reagent 2 (mouse GIPR cells) or 5 (mouse GLP-1R cells) in a 384-well small-volume tissue culture treatment plate. Before starting the assay, the medium was replaced with 5 µL of 1:2 ratio anti-cAMP antibody: 1× HBSS / 10 mM HEPES / 625 mM IBMX. The compound was diluted 1:1 in DMSO, and then 5 µL was transferred to wells using an ECHO acoustic liquid processor (Labcyte). After incubation for 30 minutes, cAMP detection reagent was added according to the manufacturer's instructions, and luminescence was measured after the recommended incubation time.

[0593] Beta-arrestin 2 recruitment

[0594] To measure GLP-1R and GIPR-mediated β-repressor protein recruitment, Promega's NanoBiT® technology or NanoLuc® binary technology was used. Both allow for the detection and quantification of proteins in live cells: protein interactions (Samms et al., Trends Endocrinol Metab 31:410-21). NanoLuc luciferase is divided into two subunits, called LgBiT and SmBiT, which are expressed in HEK293 cells as fusion proteins at the C-terminus of GLP-1R or GIPR and the N-terminus of β-repressor-2, respectively. Twenty-four hours prior to the assay, cells were extracted with cell dissociation medium and seeded at 10,000 cells per well in TC-treated 384-cell microplates. The next day, the medium was removed and replaced with a 1:100 dilution of 10 µL Nano-Glo® live cell substrate from Optimem, and the plates were equilibrated to room temperature for ten minutes. Measure background luminescence before adding 10 nL of compound using the ECHO acoustic liquid processor. Measure luminescence for 30 minutes at 1.5-minute intervals.

[0595] GLP-1R internalization

[0596] GLP-1R and GIPR internalization were measured using Promega's Nano-Glo® HiBit extracellular assay system (Promega Corporation; Madison, WI). HEK293 cells were transiently transfected with a small amount of HiBiT-labeled hGLP-1R or hGIPR plasmid (Promega Corporation; Madison, WI). The next day, cells were extracted using TrypLE expression enzyme (ThermoFisher Scientific) and seeded in 96-well plates at 80,000 to 100,000 cells per well. Forty-eight hours after transfection, the medium was replaced with Nano-Glo HiBiT extracellular buffer (1:100) containing LgBiT protein and Nano-Glo HiBiT extracellular substrate (1:50). After 15 minutes of equilibration and reading of background luminescence, the test compound was added to the cells and read at two-minute intervals on an EnVision multimode plate reader (Perkin Elmer; Waltham, MA) for 120 minutes.

[0597] Equipment and data analysis

[0598] Luminescence was measured using an EnVision multi-mode plate reader (PerkinElmer). All assays were configured such that each row of the 384-well plate contained a single dilution series, and for normalization purposes, a single low control well (medium treatment) and a single high control well (GLP-1 or GIP treatment) were included. GraphPad Prism version 8.4.3 was used to normalize the data, generate nonlinear regression curves, and produce graphs. Each dilution series was normalized to adjacent high (100%) and low (0%) wells on the plate. The dose-response data were fitted to the curve using nonlinear regression analysis with the “log(agonist) vs. response-variable slope” setting, where Y = baseline response + (maximum response - baseline response) / (1 + 10^(LogEC)). 50 -X)*HillSlope)), and Hillslope is restricted to 1. Compound potency (EC) is extracted from this regression analysis. 50 ) and efficacy (E) maxWhen the maximum activity is less than 10%, the curve is not fitted. For NanoBiT® assays, a single time point during a 30-minute activity measurement is selected to obtain the maximum luminescence response. Each well at this time point is then normalized to the background signal along with the low (DMSO) and high (500 nM GLP-1 / GIP) controls in each row.

[0599] Mice

[0600] Lean C57BL / 6J, diet-induced obese (DIO) C57BL / 6J, and leptin-deficient male mice (ob / ob) were obtained from Jackson Laboratory. Unless otherwise specified, mice were housed individually under standard environmental conditions (22°C, 12h:12h light:dark cycle) with free access to water and conventional diets (C57BL / 6NJ and ob / ob; Laboratory Rodent Diet 5001, LabDiet) or HFD (DIO C57BL / 6J; 60% kcal from fat, Research Diets #D12492). Eight-week-old lean mice were used. DIO mice were maintained on HFD for at least 18 weeks prior to experiment and used at approximately 23 weeks of age. Ob / ob mice were used at nine weeks of age. Ventilation ratio studies were conducted at RenaSci Ltd. In this study, seven- to eight-week-old C57BL / 6J mice were ordered from Charles River. Mice were housed in groups (n=3 per cage) and fed a high-fat diet (D12451 diet, 45% kcal of which was fat; Research Diets) for 21 weeks.

[0601] Intraperitoneal glucose tolerance test

[0602] Intraperitoneal glucose tolerance test (ipGTT) was performed in lean C57BL / 6J mice. Five hours prior to the ipGTT, the mediator and CT-868 were administered via a single subcutaneous injection (SC) at the doses provided in each figure. On the day of the ipGTT, mice were fasted for five hours. Baseline blood glucose was determined from whole blood collected from the tail using an AlphaTrak glucometer. Subsequently, glucose 2 g / kg dextrose (as a 20% solution in physiological saline) was administered via a single intraperitoneal injection. Blood glucose was determined at the time intervals specified in each figure. Additionally, whole blood was collected in K2-EDTA microvettes (Sarstedt) and kept on ice until centrifuged at 5000 rcf for ten minutes at 4°C. Plasma was removed and stored at -80°C until insulin analysis.

[0603] Diet tolerance test

[0604] Mixed diet tolerance test (MMTT) was performed in 23-week-old C57BL / 6J DIO mice. The mediator, liraglutide (30 nmol / kg), and CT-868 (30 nmol / kg) were administered via SC injection (10 mL / kg) 24 hours prior to the MMTT. Mice were fasted for 16 hours. At the end of the fasting period, blood glucose was measured from the tail using an AlphaTrak glucometer. Subsequently, a liquid diet (Ensure Plus; Abbott Nutrition #64905) was administered orally via gavage (10 mL / kg). Blood glucose was determined at the time intervals specified in each graph, and whole blood was collected in K2-EDTA microvettes (Sarstedt) and kept on ice until centrifuged at 5000 rcf for 10 minutes at 4°C. Plasma was removed and stored at -80°C until insulin analysis.

[0605] Weight loss

[0606] DIO and ob / ob mice were acclimatized to daily weighing and treatment for approximately one week until weight stability was achieved. Based on current body weight, mice were subcutaneously injected once daily with an indicated dose of CT-868, liraglutide, or the carrier. Injection was performed six hours prior to the start of the dark cycle to reach T0 approximately at the start of the dark cycle. max In ob / ob studies, 24-hour food consumption studies were conducted, if instructed, by adding a pre-weighed amount of food to the cage floor and measuring the amount of food remaining the following day. In DIO and ob / ob weight loss studies, the percentage of body weight relative to initial body weight was calculated daily by dividing the daily body weight by the body weight collected before the first peptide dose and then multiplying by 100%. Blood glucose was determined at the end of the study, and blood was collected to quantify plasma insulin concentration. Mice were sedated with isoflurane and euthanized by decapitation. Whole-truncation blood was collected in a K2-EDTA microvette and kept on ice until centrifuged at 5000 rcf for 10 minutes at 4°C. Plasma was stored at -80°C until insulin analysis. Subcutaneous and groin fat, as well as the liver, were removed and weighed.

[0607] Ventilation ratio

[0608] DIO mice were acclimatized to solitary housing for two weeks before being introduced into the PhenoMaster system (TSE Systems, Bad Homburg, Germany). Baseline parameters were recorded three days prior to the start of CT-868 administration. Mice were assigned to treatments using body weight, food intake, water intake, oxygen consumption (VO2), carbon dioxide production (VCO2), and activity as variables. The medium and CT-868 (20 nmol / kg) were administered daily for two weeks via a single subcutaneous injection at approximately 15:55. Body weight (BW) was recorded daily during administration, while food intake, water intake, VO2, CO2, and activity were recorded by the PhenoMaster system. Results are presented as daily averages for light and dark cycles.

[0609] Plasma insulin

[0610] Plasma insulin concentrations were determined using the U-PLEX mouse insulin assay or the mouse / rat insulin assay (Meso Scale Discovery) and read on a MESO Quickplex Q 60MM instrument.

[0611] Pharmacokinetics

[0612] The pharmacokinetic (PK) level of CT-868 was measured in male CD-1 mice. Blood samples were collected up to 32 hours after a single SC injection. Plasma concentrations were measured using an API 6500 LC-MS / MS system.

[0613] Quantitative and statistical analysis

[0614] Results are expressed as mean (± standard error). One-way ANOVA and Tukey's test for multiple comparisons were used to detect differences between glucose homeostasis groups. All others were performed using two-way ANOVA with grouping and time as between-subjects factors. Multiple comparisons were performed with Tukey correction when significant differences were observed. Statistical significance was set at p < 0.05. Statistical analysis was performed using GraphPad Prism 9.2.0 (GraphPad Software, Boston, MA).

[0615] First-in-human study of safety and tolerability of Ct-868 in healthy participants and participants with obesity (single ascending dose and multiple ascending dose study) Study design

[0616] This is a first-in-human (FIH) randomized, double-blind, placebo-controlled, dose-escalation study conducted between December 2018 and January 2020 at a research center in Victoria, Australia, in accordance with the Declaration of Helsinki (Ethical Principles for Medical Research Involving Human Subjects) and the 2007 NHMRC National Declaration on Ethical Conduct in Human Research (updated 2018). (Registered with the Australia New Zealand Clinical Trials Registry [ANZCTR], identifier: ACTRN12618001988246).

[0617] Eligible participants must be between 18 and 65 years of age (inclusive) and have a BMI between 27 and 45 kg / m². 2 Or 20 to 25 kg / m 2 (The "lean" cohort) consisted of participants in Part 2 with a waist circumference ≥ 102 cm (male) or ≥ 88 cm (female), a weight loss of ≤ 5% in the previous 3 months, normal blood pressure or well-managed hypertension (only if the blood pressure medication dose was stable for ≥ 2 months), normal lipid profile or well-managed dyslipidemia (only if the lipid-lowering medication dose was stable for ≥ 2 months), fasting plasma glucose ≤ 100 mg / dL (6.0 mmol / L), and demonstrated health through a comprehensive clinical assessment (detailed medical history, complete physical examination, and comorbidities allowing for higher weight (e.g., mild impaired glucose tolerance, mild hypertension, mild hyperlipidemia)). Clinical laboratory parameters were within the normal range or considered clinically insignificant; however, serum creatinine, ALP, liver enzymes (AST, ALT), and total bilirubin (unless the participant had Gilbert's syndrome) could not exceed ≥ 2 (i.e., AST, ALT, or ALP) or ≥ 1.5 × Participants were required to have the upper limit of normal (ULN) (total bilirubin) and no history of cardiovascular disease or any other major medical condition other than well-managed hypertension or dyslipidemia in the past three years. All participants provided written informed consent before undergoing any study-specific tests or procedures.

[0618] Study procedures

[0619] This study aimed to systematically evaluate the safety, tolerability, pharmacokinetic (PK), and PD of CT-868 administered in single (SAD) and multiple escalation (MAD) doses in healthy and obese participants. In Part 1 (SAD), participants were randomized on the morning of Day 1, with two sentinel participants receiving their first dose of the study drug (one participant received CT-868, and the other received a placebo). During this period, in the absence of clinically significant safety signals in the sentinel participants, the remaining participants received a single SC injection of the study drug (CT-868 or matched placebo), except for cohorts S6 and S9, who received two (S6 participants) or three (S9 participants) SC injections to reach the specified dose level because their dose was higher than 5.0 mg (Figure 46A).

[0620] In Part 2 of the study (MAD), participants were randomized on the morning of Day 1 to receive either CT-868 via SC injection or placebo. Daily doses were administered from Day 2 to Day 14. The highest dose level for cohort M1 was determined based on safety and pharmacokinetic data from Part 1 (SAD) above, and the doses for cohorts M2 and M3 were subsequently reduced (Figure 46B).

[0621] Primary endpoint

[0622] Part 1 (SAD) participants were admitted to the treatment unit on Day -1 and were restricted from discharge upon completion of all Day 3 assessments and in the absence of clinically significant safety signals. Similarly, Part 2 (MAD) participants were admitted on Day -2 and remained in the unit until completion of all Day 16 assessments.

[0623] Following administration of the study drug, Part 1 (SAD) participants underwent safety and other assessments on days 3 and 8 (± 1 day, outpatient visit) and were followed up for at least 30 days after the last dose of study drug. Part 2 (MAD) participants underwent safety and other assessments after completing all day 16 assessments and were discharged without clinically significant safety signals. All participants were followed up for at least 30 days after the last dose of study drug. Participants were followed up for safety and other assessments on days 21 (± 2 days, outpatient visit) and 44 (± 3 days, conference call).

[0624] Secondary endpoints

[0625] The primary endpoints were the incidence, nature and severity of adverse events (AEs), safety of laboratory analytes, vital signs, electrocardiogram (ECG), and incidence of events of particular concern (AESI).

[0626] Quantitative and statistical analysis

[0627] Characterization of PK profile: PK parameters include maximum concentration (Cmax), time to maximum concentration (Tmax), and area under the plasma concentration-time curve (AUC). For pre-dose samples, blood samples were collected within two hours before administration and within ±10 minutes after administration at the nominal / planned time. For Part 1 (SAD) participants, samples were collected at -2 to 0 hours (pre-dose), and at 1, 2, 3, 4, 6, 8, 12, 16, 20 hours (day 1), 24, 30, 36 hours (day 2), 48 hours (day 3), and day 8 (±1 day, follow-up). For Part 2 (MAD) participants, samples were collected at -2 to 0 hours (pre-dose), 2, 3, 4, 6, 8, 12 hours (day 1), 24 hours (day 2), 48, 60 hours (day 3), 72 hours (day 4), and 96 hours (day 5). For days 7, 8, 11, and 14, samples were collected at -2 to 0 hours (before administration), 12 hours after administration (day 7), 2, 4, 6, 8, 12 hours (day 14), 24, 36 hours (day 15), 48 hours (day 16), and day 21 (± 1 day, follow-up).

[0628] Characterization of PD profile: In Part 1 (SAD) participants, plasma glucose was assessed at -2, -1 hour (day -1, fasting), -2 to 0 hours (before administration), and 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 hours (day 1), 24 hours (day 2), 48 hours (day 3), and day 8 (± 1 day, follow-up) after administration. Part 2 (MAD) Participants were assessed at 0, 1 hour (day -1, fasting), -2 to 0 (before administration), 1, 2, 3, 4, 6, 8, 12, 16 hours (day 1); before administration (fasting) and 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 96 hours (day 5) before lunch and dinner, and -2 to 0 hours (before administration, fasting, days 6, 8, 10, and 12); before administration and before lunch and dinner (days 6 to 13); before administration and 1, 2, 3, 4, 6, 8, 12, 16 hours (day 14), 24 hours (day 15), and day 21 (± 1 day, follow-up).

[0629] For Part 1 (SAD, starting from cohort S2), participants underwent a dietary tolerance test (MTT) at 0 hours (day -1) and 24 hours (day 2). Participants ingested 237 mL of a standardized liquid meal (Ensure Plus) served chilled or at room temperature, within 5 minutes. ® Samples were collected 60 minutes before the MTT and 20 and 40 minutes (± 2 minutes) after the start of the test meal intake, and at 1, 1.5, 2, 2.5, 3, 3.5, and 4 hours (± 10 minutes). After the MTT, blood samples were collected to measure endpoints (Cavg and AUC for glucose, insulin, and C-peptide). Part 2 (MAD) participants were assessed at 2 hours on day -1 and 3 hours on days 7 and 14 (post-dose). Additionally, data on their weight, waist circumference, total LDL-cholesterol, high-density lipoprotein (HDL-cholesterol), and triglycerides were collected.

[0630] Exploratory outcome measures included insulin, HbA1c, GLP-1, gastric inhibitory peptide [GIP], glucagon, adiponectin, fibroblast growth factor 21 [FGF-21], and C-terminal telopeptide of type I collagen [CTX] (also known as a C-terminal marker of cross-linking of collagen, leptin, and ghrelin). PD biomarkers were collected from day 1 (SAD) cohort S2 onwards at 2 to 0 hours (before dosing) and 4 hours after dosing. Biomarkers for the MAD cohort were collected at 2 to 0 hours (before dosing) and 3 hours (3 hours), 48 hours (before dosing) and 51 hours (3 hours), 96 hours (before dosing) and 99 hours (5 hours), and 2 to 0 hours (before dosing) and 3 hours (3 hours) (4 days), 48 hours (before dosing) and 51 hours (5 days), 96 hours (before dosing) and 99 hours (5 days), and 2 to 0 hours (before dosing) and 3 hours (3 hours) (5 days) (6 days).

[0631] PK-PD modeling

[0632] The sample size selected for the study was chosen without statistical justification but was deemed sufficient to assess the study objectives. The ITT cohort included all randomly assigned participants. The safety cohort included all randomly assigned participants who received any amount of the investigational drug (CT-868 or placebo). The PK cohort included all randomly assigned participants who received any amount of the active investigational drug (CT-868) and had sufficient plasma concentration-time data to determine at least one PK parameter. The PD cohort included all randomly assigned participants who received any amount of the investigational drug (CT-868 or placebo), with results derived from baseline and PD assessments at ≥ 1 baseline.

[0633] Adverse events were coded using the Medical Dictionary of Regulatory Activities (MedDRA® version 21.1). The number of participants experiencing treatment-related adverse events (TEAEs) and the number of individual TEAEs were summarized by treatment, state of emergency (SOC), and physical examination (PT). TEAEs were also summarized by severity and relationship to the investigational drug. AESIs were listed and summarized by treatment group. Where applicable, a list of AEs, SAEs, and deaths leading to study interruption was provided.

[0634] Laboratory assessments, vital sign evaluations, and ECG parameters are listed by participant and summarized by treatment and protocol-specified collection time points. Summary of changes relative to baseline and counts of out-of-normal values ​​at each protocol-specified time point are also presented by treatment. Physical examination abnormalities, including the incidence of ISRs, are summarized by treatment, time point, and overall.

[0635] Placebo-controlled and comparator-controlled crossover study of CT-868 in participants with obesity and participants with T2DM.

[0636] Individual and mean CT-868 concentration-time data were tabulated by cohort / dose level. PK parameters were calculated from individual plasma concentrations of CT-868 using a non-compartmental method. PK parameter estimates were tabulated and summarized by descriptive statistics (mean, standard deviation [SD], median, minimum and maximum, coefficient of variation [CV%], geometric mean, and geometric CV%). Dose equilibrium was analyzed using a power model with log-transformed PK parameter values ​​and log-transformed doses. Slope parameters and 90% confidence intervals (CIs) of the slope were estimated using a power model. Dose equilibrium was generally concluded if the 90% CI around the slope estimate included a value of 1.

[0637] All individual PD and exploratory biomarker data are listed in the data list and summarized by nominal sampling time point and treatment group, with descriptive statistics presented by CT-868 dose group, all CT-868 dose combination group, and placebo combination group. At each protocol-specified time point, the observed change relative to baseline and percentage change relative to baseline for the summarized PD biomarker parameters by treatment are presented. A generalized linear mixed model approach is used. Where the endpoint is a summarized statistical statistic over time, such as Cavg or AUC of glucose, insulin, or C-peptide measures during a dietary tolerability test, the model includes treatment as a fixed factor and baseline values ​​as covariates (i.e., using an analysis of covariance [ANCOVA] model). When the endpoint is an outcome of change from baseline to each time point, the model includes treatment, time point, and the interaction between treatment and time point as fixed factors, baseline values ​​as covariates, and participant identification as a random or replicate factor to satisfy the replicated measure nature of the data. Estimates of the overall and individual time point treatment means and 95% CIs, as well as differences between treatments, are presented. A p-value < 0.05 will be declared significant.

[0638] Patient inclusion criteria Study design

[0639] The two-part Phase 1 clinical trial design (Clinicaltrials.gov ID: NCT04973111) was conducted at a center in the United States to evaluate the pharmacokinetics (PK), pharmacodynamics (PD), and safety of CT-868 in participants with obesity (“obese participants”) and participants with type 2 diabetes mellitus (T2DM) (“T2DM participants”) in accordance with the International Council for Harmonisation of Protocols (ICH) Good Clinical Practice (GCP): Combined Guideline (E6) and applicable regulatory requirements, including clinical trial guidelines developed in accordance with the fundamental principles defined in US 21 CFR Parts 50, 56, and 312 and the principles of the Declaration of Helsinki (2013 Fortaleza revision); with the aim of providing greater protection for individuals.

[0640] Study procedures

[0641] All participants were between 18 and 65 years old at the time of randomization. Obese participants had a BMI ≥ 30 to < 40 kg / m². 2 Patients with type 2 diabetes mellitus (T2DM) are diagnosed with T2DM (but not type 1 diabetes) according to disease-specific diagnostic criteria (WHO classification of diabetes), have a duration of T2DM ≥ 6 months, and have a BMI > 27 to ≤ 45 kg / m². 2 HbA1c ≤10.5% and FPG < 250 mg / dL. Participants must have been on diet and exercise only, or on stable therapy (≥ 3 months) with metformin monotherapy or a combination of metformin and SU prior to screening. Participants were not receiving any GLP-1 analog / GLP-1 RA and DPP-4 inhibitor therapy. All participants were nonsmokers (> 6 months prior to study initiation). Male participants consented to true abstinence or the use of condoms plus effective contraception, and female participants of childbearing potential (WOCBP) consented to appropriate contraception. If postmenopausal (absence of menstruation > 12 months), status was confirmed by testing for FSH levels above the normal range in amenorrhea participants < 60 years of age.

[0642] Exclusion criteria include:

[0643] ● Any clinically significant GI (peptic ulcer, severe gastroesophageal reflux disease (GERD), abnormal gastric emptying / gastroparesis, any malabsorption / motor dysfunction, chronic constipation, inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS)), cardiovascular (including arrhythmias, history of ischemic heart disease), liver, nervous system, mental, kidney, immune, skin, endocrine, genitourinary or hematologic system active disease, uncontrolled hyperlipidemia or uncontrolled hypertension (BP) >140 / 90 mmHg (healthy participants) or persistent BP systolic or diastolic >160 / 90 mmHg or <90 / 60 mmHg (participants with T2DM);

[0644] ● History of acute or chronic pancreatitis or current diagnosis of acute or chronic pancreatitis or risk factors for pancreatitis, such as gallstones (without cholecystectomy), hypercalcemia or severe hypertriglyceridemia.

[0645] ● Personal or family history of medullary thyroid carcinoma (MTC) or genetic predisposition to MTC (i.e., type 2 multiple endocrine neoplasms);

[0646] ● Clinically significant physical or ECG results at the time of screening (e.g., QTcF > 450 msec for men, QTcF > 470 msec for women, left bundle branch block [LBBB]), which may interfere with any aspect of study behavior or result interpretation, or expose that particular participant to safety issues;

[0647] ● Any history of weight control treatment, including use of approved weight loss drug therapy and / or dietary supplements within three months prior to screening;

[0648] ●Previous surgical treatment for obesity (any type of weight loss surgery) or any other GI surgery that may induce malabsorption / motor problems, history of bowel resection >20 cm or any GI weight loss surgery (including LAP-BAND®).

[0649] ● Historical or recent weight changes of ≥ ±5% within 30 days (based on self-reporting or medical records);

[0650] ● Currently using any prescription or over-the-counter medications known to interfere with glucose or insulin metabolism, including but not limited to systemic corticosteroids, testosterone, anabolic steroids, metformin, GLP-1 analogs / RAs, thiazolidinediones (TZDs), SUs, dipeptidyl peptidase-4 (DPP-4) inhibitors, insulin therapy, monoamine oxidase (MAO) inhibitors, growth hormones, or other herbal / over-the-counter preparations, including amino acids. For participants currently treated for type 2 diabetes mellitus (T2DM), SUs were washed out ≥ 7 days prior to first dose and metformin monotherapy was only administered during the study period (participants were not receiving any GLP-1 analog / GLP-1 RA and DPP-4 inhibitor therapy), and participants receiving stable (≥ 2 months) therapy with lipid-lowering and / or antihypertensive medications were permitted to use up to 2 different antihypertensive medications.

[0651] ● Currently using any prescription or over-the-counter medications known to interfere with bowel motility, including but not limited to chronic opioids, anticholinergics, antispasmodics, serotonin (5HT3) antagonists, or dopamine antagonists;

[0652] ● Clinically significant abnormal laboratory values, including transaminases (aspartate aminotransferase [AST], alanine aminotransferase [ALT] > 1.5 × upper limit of normal [ULN] (healthy) or ≥ 3 × upper limit of normal [ULN] (with T2DM), total bilirubin > ULN, and glomerular filtration rate (eGFR) estimated using the dietary correction for kidney disease [MDRD] equation < 60 mL / min / 1.73 m 2 And / or calcitonin levels > 50 ng / L. Participants with type 2 diabetes mellitus were excluded if fasting serum triglycerides > 500 mg / dL or laboratory values ​​suggestive of pancreatic damage (e.g., amylase and / or lipase > 3 x ULN).

[0653] ●A history of any serious adverse reaction or hypersensitivity to any research drug component or drug with a similar chemical structure, a history of obvious multiple and / or severe allergies, or an existing allergic reaction or obvious intolerance to prescription or non-prescription drugs or food.

[0654] ● History of hepatitis B surface antigen (HBsAg) or positive test, or positive hepatitis C test, or presence of human immunodeficiency virus type 1 (HIV-1) or type 2 (HIV-2) antibody;

[0655] ● Any active or clinically significant infection, including coronavirus disease (COVID-19) within 15 days prior to screening, or any COVID-19 vaccine administration within 5 days prior to the scheduled administration period, or planned for such vaccination during any period of residence (in-home treatment). COVID-19 vaccination is permitted during the study period provided that at least 5 days have passed since the vaccine administration and before any other administration, and the participant is symptom-free for at least 48 hours after vaccination. Participants must follow the research center-specific COVID-19 protocol throughout the study period;

[0656] ● A history of alcohol abuse within the past year; or a history of illicit drug (including cannabis) abuse within the past 3 months; or evidence of current use or a positive drug test (including cannabis) at the time of screening; and

[0657] ● Participate in a study drug / device study within 30 days or 5 half-lives (whichever is longer) after the last administration of any IP, donate or lose >500 mL of blood within 56 days, or undergo any major surgery within 6 months.

[0658] Exclusion criteria for participants with type 2 diabetes mellitus (T2DM) also included evidence of significant active neuropsychiatric disorders or chronic seizures or major neurological disorders. Participants who were stable prior to screening and controlled by a stable dose of selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), antipsychotics, and lithium for ≥ 6 months may be allowed. In addition, participants with osteoporosis, osteopenia, recurrent fractures, anemia, or hemoglobinopathies; acute proliferative retinopathy or macular degeneration and / or severe neuropathy, especially autonomic neuropathy; recurrent severe hypoglycemia or hypoglycemic altered consciousness or recent ketoacidosis; cardiac disease defined as symptomatic heart failure (New York Heart Association Class III or IV), myocardial infarction, coronary artery bypass grafting or angioplasty, unstable angina requiring medication, transient ischemic attack, cerebral infarction or cerebral hemorrhage; significant liver disease other than fatty liver disease; or a history of neoplastic disease within the past 5 years (exceptions: participants with adequately treated non-melanoma skin cancer or other malignancies who have been successfully treated for ≥10 years prior to screening and are highly unlikely to experience recurrence during the study period).

[0659] Quantitative and statistical analysis

[0660] Participants with obesity were assigned to either Group 1 or Group 2 according to their enrollment order. In Group 1, participants were randomly assigned to a crossover drug sequence of CT-868, placebo, or liraglutide in a 1:1 ratio (n=3 for CT-868:placebo:liraglutide sequence and n=3 for placebo:CT-868:liraglutide sequence). Liraglutide was administered in an open-label manner. In Group 2, participants were randomly assigned to either CT-868 or placebo in a 1:1 ratio (Figure 46B).

[0661] Participants with type 2 diabetes mellitus (T2DM) were also assigned to either Group 1 or Group 2 based on their enrollment order. In Group 1, participants were randomly assigned to a crossover sequence of CT-868, placebo, or liraglutide at a ratio of n=7 for CT-868:placebo:liraglutide, and n=6 for placebo:CT-868:liraglutide. Liraglutide was administered in an open-label manner. In Group 2, participants were randomly assigned to a crossover sequence of CT-868 or placebo at a ratio of n=3 for CT-868:placebo, and n=4 for placebo:CT-868.

[0662] Chemical structure of CT-868

[0663] Participants with obesity underwent a screening visit (day -30 to day 1, before administration), two (Group 1) or three (Group 2) 4-day internal treatment periods (day -1 to day 3), a washout period of ≥14 days between internal treatment periods (maximum allowed 21 days), and a follow-up visit (F / U, day 10 ± 3, after last administration). Participants received a SC (surname-selective) dose of the randomized investigational drug on the evenings of days 1 and 2 of the treatment period (see drug allocation table). All participants were released home on the evening of day 3.

[0664] Subjects with type 2 diabetes mellitus (T2DM) underwent a screening visit (day -30 to day 1, before dosing), two (Group 1) or three (Group 2) 5-day in-treatment periods (day -1 to day 4), a washout period of ≥14 days between in-treatment periods (maximum allowed 21 days), and a follow-up visit (F / U, day 10 ± 3, after last dosing). Participants receiving metformin and sulfonylurea (SU) combination therapy may have experienced an introductory period in which their SU was washed off at least 7 days prior to in-treatment period 1 and continued to receive metformin monotherapy throughout the study. During treatment, participants received a randomized SC dose of the investigational drug on the evenings of days 1, 2, and 3 (see drug allocation table). Participants in Group 2 were released home on the afternoon / evening of day 4. Participants in Group 1 continued into in-treatment period 3.

[0665] Table 16. Drug Allocation Table

[0666]

[0667] Primary endpoint: The primary endpoint was the rate of insulin secretion (ISR) relative to glucose (G). Following short-term administration of CT-868, the relationship of ISR at any given blood glucose level was assessed by a graded glucose infusion (GGI) procedure performed two days after administration (day 3) to fully investigate the glucose metabolism effects of CT-868 relative to placebo and liraglutide before its weight-loss induction effect materialized.

[0668] Secondary endpoints: Secondary endpoints include maximum glycemic drift (G). max The area under the curve of ISR during the 150-minute time interval during GGI, estimated based on C-peptide levels (AUEC[0-t]), AUEC(0-t) of ISR x G and ISR / G during the 150-minute time interval during GGI, estimated based on C-peptide levels, and AUEC(0-t) and time-averaged PD response (AUEC[0-t] / t) of insulin, C-peptide, glucose and GLG during GGI.

[0669] Blood samples were collected before and after administration during the internal treatment period to assess PD and PK endpoints. In healthy participants, PK parameters (maximum plasma concentration [C]) were assessed on days 2 and 3. max Time to reach maximum plasma concentration [T] max AUC within the dosing interval 0-tauTotal AUC and incremental AUC (e.g., AUC) (0-20h) ) and terminal half-life [t 1 / 2 Samples were collected for PD parameters (glucose, homeostasis model assessment of insulin resistance [HOMA-IR], insulin, C-peptide, GCG). Additionally, fasting plasma glucose (FPG), insulin, C-peptide, GCG, HOMA-IR, and VAS were assessed on days 1, 2, and 3 after an overnight fast of ≥10 hours.

[0670] In participants with type 2 diabetes mellitus (T2DM), blood samples were collected on days 2, 3, and 4 to assess pharmacokinetic (PK) profiles, and on days 2 and 3 to assess PD profiles. Fasting plasma glucose (FPG), insulin, C-peptide, GCG, and HOMA-IR were measured after an overnight fast of ≥10 hours on days 1, 2, 3, and 4. A mixed diet tolerance test (MMTT) and expected dietary consumption (free-flow diet) were administered on day 4. Appetite and food intake were assessed using the VAS questionnaire (appetite, hunger, and satiety) on days 1, 2, 3, and 4. A gastric emptying / paracetamol test was also administered on day 4, and the results were assessed using the AUC parameter. 0-60min AUC 0-300min , average concentration of paracetamol, C max and T max To assess the effect of CT-868 on gastric emptying.

[0671] Safety assessments conducted throughout the study included monitoring the incidence and severity of adverse events (AEs), treatment-induced AEs (TEAEs) and AEs of particular concern (AESIs) (e.g., GI events, hypoglycemia), clinical laboratory abnormalities (ECG, chemistry, hematology, and urinalysis), and changes in vital sign measurements (BP, heart rate, respiratory rate, and ear temperature).

[0672] Preclinical models

[0673] The inclusion set was defined as all participants who signed informed consent forms and were used in the treatment analysis. The safety set was defined as all randomized participants who received at least one dose of the study drug, as determined both overall and for each internal treatment period. The safety set was used for all safety analyses, demographic and baseline characteristics, prior / concomitant medications, and protocol deviations. The PK set used for all PK analyses was defined as all participants in the safety sets determined both overall and for each internal treatment period who received the study drug, with evaluable PK data suitable for assessing PK profiles within each internal treatment period. The PD set used for all PD analyses was defined as all participants in the safety sets determined both overall and for each internal treatment period who received the study drug, with evaluable PD data suitable for assessing PD parameters within each internal treatment period.

[0674] Pre-hepatic ISR was calculated based on the deconvolution of peripheral C-peptide concentrations during the GGI period. The effects of CT-868 on the relationship between ISR and G (ISR x G and ISR / G) were assessed using two mixed-effects models in the PD population compared to placebo and liraglutide, with each component and comparison attributed to the study design. The CT-868 vs. placebo comparison model included the treatment group, treatment sequence, and in-sequence treatment period as fixed effects, and in-sequence participants as random effects. The CT-868 vs. liraglutide comparison model included the treatment group, treatment sequence, and treatment group-sequence interaction as fixed effects, and in-sequence participants as random effects. Response variables were the slopes of the ISR x G curves and the slope of the ISR / G curve in both independent models. The least-squares (LS) mean for each treatment group and the differences in the LS mean between CT-868 and placebo and between CT-868 and liraglutide were calculated, and relevant 95% confidence intervals (CIs) were constructed. The Kenward-Roger approximation is used for the denominator degrees of freedom. Time points are defined as 10-minute intervals from time 0 to 150 minutes.

[0675] Except for whether or not the diet was free of charge, each exploratory endpoint for PD used the same analytical method (mixed-effects model) as the primary endpoint analysis, using the applicable time points described in each section. All exploratory PD and PK parameters were summarized by study section, treatment, and time point, or by study section for GGI or by treatment for Part 2. Each parameter had descriptive statistics (n, mean, SD, coefficient of variation [%CV], minimum, 1st quartile, median, 3rd quartile, and maximum).

[0676] All statistical analyses were two-tailed and assessed at a 5% significance level. Tables were generated for appropriate demographic, baseline, and safety parameters. For categorical variables, summary tables of the number and percentage of participants in each category of the parameter (and the category with missing data) were presented. For continuous variables, the number of participants (n), mean, standard deviation (SD), median, minimum (min), and maximum (max) were presented. Coefficients of variation for some analyses were presented.

[0677] Investigator-recorded adverse events (AEs) were coded into Systemic Organ Classifications (SOCs) and Preferred Terms (PTs) using the MedDRA version 24.0. Severity was collected using the Common Terminology Criteria for AEs (CTCAE) version 5.0 toxicity grading. Events with missing severity were aggregated into three levels. For each participant, a count of TEAEs aggregated by severity was performed for the maximum severity within each SOC and PT for each intra-treatment period and overall. TEAEs marked as definite, probable, possible, or missing in relation to the study treatment were considered relevant TEAEs. Events with missing relation to the study treatment were aggregated into relevant events. The number and percentage of participants with TEAEs were aggregated for each SOC and PT coded by MedDRA.

[0678] result

[0679] CT-868 enhances glucose homeostasis through GLP-1R and GIPR mediated mechanisms.

[0680] CT-868 is a modified 39-amino acid peptide. While the hybrid GLP-1 / GIP peptide sequence provides optimal activity against both GLP-1R and GIPR, the N-terminal modification with a 2-((2-oxo-2-((2-(2-oxoperidin-1-yl)ethyl)amino)ethyl)thio)acetyl group aims to stabilize the molecule for dipeptidyl peptidase-4 cleavage, thereby conferring a bias towards G protein coupling relative to β-repressor protein recruitment. Acylation of the C-terminal lysine side chain with a γ-glutamylpalmitoyl group extends the molecule's circulating half-life via albumin binding, facilitating once-daily dosing.

[0681] Figure 43D

[0682] CT-868 is a cAMP-biased dual agonist targeting the GLP-1 / GIP receptor with a selective imbalance towards GLP-1R.

[0683] In vitro experiments were conducted using recombinantly expressed receptors to evaluate the potency, efficacy, and signal transduction properties of CT-868 at GLP-1R and GIPR. In human GLP-1R, CT-868 acts as a potent, complete agonist of cAMP accumulation. The potency of CT-868 is one-sixth that of its natural ligand GLP-1, similar to liraglutide (Figure 42A and Table 17).

[0684] Table 17. Intrinsic potency of CT-868 in cells expressing GLP-1R or GIPR

[0685]

[0686] In contrast, at the human GIPR, CT-868 exhibits 1 / 16th the potency of the natural ligand GIP and is a partial agonist of cAMP accumulation (Figure 24B and Table 17). These data suggest that CT-868's GLP-1R selectivity relative to the GIPR contrasts with the GIPR selectivity of the dual agonist tesipatide (E1 et al., Nat Metab. (2023) 5:945-54). However, unlike the natural ligand or liraglutide and tesipatide, there was no β-repressor recruitment at either receptor in response to CT-868 stimulation (Figures 24C to 24D, Table 17).

[0687] Further analysis revealed that while the natural ligand promotes receptor internalization, CT-868 effectively inhibited GLP-1R and GIPR internalization, as evidenced by greater surface expression at 60 to 120 minutes compared to the mediator-treated control (Figures 42E–42F and Table 17). This is consistent with CT-868's limited ability to recruit β-repressor proteins. These results indicate that CT-868 is selective for GLP-1R and is entirely biased towards binding cAMP signaling relative to β-repressor protein recruitment and internalization. CT-868 demonstrated considerable potency and efficacy in mouse cells expressing GLP-1R and GIPR, as validated by cAMP accumulation assays (Table 17), thus supporting its use in preclinical studies.

[0688] Long-term CT-868 reduces body weight in a dose-dependent manner

[0689] In mice, a glucose tolerance test (GTT) was performed to assess the effects of CT-868 on glucose-stimulated insulin secretion (GSIS) and glucose drift. During the intraperitoneal glucose tolerance test (IPGTT), a single subcutaneous injection of CT-868 significantly and substantially inhibited glucose drift, even at the lowest tested dose (3 nmol / kg), compared to the mediator group (Figure 43A). Area under the curve (AUC) analysis of plasma insulin revealed no significant difference between the mediator and the administered CT-868 dose; however, postprandial plasma insulin levels at 15 minutes after CT-868 (10 nmol / kg) were significantly higher than those after mediator treatment (Figure 43B).

[0690] To assess GIPR-mediated glucose control activity, CT-868 was administered to GLP-1R knockout (KO) mice prior to IPGTT. At a dose of 30 nmol / kg, CT-868 exhibited limited glucose inhibition via GIPR; however, a significantly greater glucose-lowering effect was observed at higher doses (i.e., 300 nmol / kg). Figure 44B At higher GIPR-binding doses, CT-868 achieved glucose reduction without increasing insulin secretion. These results indicate that the glucose-lowering effect at this dose is insulin-independent. Therefore, a pyruvate tolerance test (PTT) was performed to determine whether inhibition of endogenous glucose production contributed to the observed effect. In wild-type mice, CT-868 at 30 nmol / kg and 300 nmol / kg reduced glucose levels during the PTT; however, in GLP-1R KO mice, only the 300 nmol / kg dose reduced glucose levels during the PTT (Figures 43C to 43D), suggesting that GIPR activation may promote glucose reduction by inhibiting endogenous glucose production.

[0691] Previous studies have reported improved efficacy of biased GLP-1R agonists (Willard et al., JCIInsight (2020) 5). To confirm this finding, the glucose-lowering effect of the minimum GIPR-binding dose of CT-868 compared to the unbiased GLP-1R agonist liraglutide was investigated in diet-induced obese (DIO) mice. For this purpose, a mixed diet tolerance test (MMTT) was performed 24 hours after equimolar administration (30 nmol / kg) of CT-868, liraglutide, or the mediator. AUC analysis showed that both liraglutide and CT-868 significantly reduced blood glucose levels compared to the mediator-treated group. Furthermore, the minimum GIPR-binding dose of CT-868 demonstrated greater efficacy than liraglutide in inhibiting glucose drift following the dietary challenge (Figure 43H). Mice treated with CT-868 had higher insulin levels 60 minutes after treatment; however, AUC analysis showed only a non-significant trend toward higher insulin levels compared to the mediator and liraglutide (Figure 43I).

[0692] To better interpret these results and help determine the dosing regimen for CT-868 in preclinical studies, pharmacokinetic (PK) studies were conducted in mice. The PK studies revealed that liraglutide exposure was three times that of CT-868, with an AUC of [missing value]. 0-Inf The concentrations were 22286 and 7921 h*nM / L, respectively, with circulating half-lives of 4 hours and 7 hours, respectively. This suggests that the improved glucose disposal resulting from CT-868 treatment compared to liraglutide treatment in previous studies is likely not due to differences in PK properties, but rather to the unique signaling properties of CT-868. These PK properties support once-daily administration of CT-868 in mice.

[0693] Phase 1 single and multiple ascending dose clinical study of CT-868 safety, tolerability, and pharmacokinetics

[0694] A series of in vivo studies were conducted to investigate the effects of long-term CT-868 treatment on body weight and body composition in mice. DIO mice receiving daily subcutaneous injections of CT-868 (3, 10, or 30 nmol / kg) showed a dose-dependent decrease in body weight over 14 days, with mean reductions of 6.8 ± 1.2% (SD), 10.7 ± 0.6% (SD), and 18.4 ± 1.0% (SD) compared to their baseline body weight, respectively (Figure 44A). In contrast, mice treated with the medium showed a mean increase of 10.0 ± 2.2% (SD) in their baseline body weight during the same treatment period.

[0695] Body composition was analyzed at the end of the study. Compared with the control group treated with the medium, CT-868 treatment caused a dose-dependent decrease in inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and liver weight. Pharmacokinetic profile of CT-868 Specifically, at 30 nmol / kg, compared with the medium-treated control, CT-868 caused significant reductions in iWAT, eWAT, and liver weight of 61%, 58%, and 52%, respectively. This demonstrates that the weight loss observed during CT-868 treatment was primarily due to a reduction in fat mass.

[0696] In a subsequent series of long-term in vivo administration studies, it was determined whether CT-868 was also effective in a mouse model of hereditary obesity. Treatment with CT-868 (10 or 30 nmol / kg), liraglutide (200 nmol / kg), or a mediator was used to treat leptin deficiency (Lep). ob / ob The mice, hereinafter referred to as ob / ob mice, were administered CT-868 at 10 nmol / kg and 30 nmol / kg, respectively, after 24 days of daily administration. Compared with their counterparts treated with liraglutide and the vehicle, the mice showed significant dose-dependent weight loss (3.1 ± 2.8% (SD) and 8.4 ± 1.6% (SD), respectively) (Figure 44C). In contrast, the mice treated with liraglutide and the vehicle showed increases in their baseline body weight of 2.2% ± 2.2% (SD) and 15.4% ± 2.9% (SD), respectively, during the same treatment period. These results indicate that CT-868 is more potent than liraglutide in reducing body weight and achieves its maximum effect at significantly reduced doses.

[0697] Compared to the mediator-treated control, all treatment groups showed significant reductions in food intake after the first dose (day 1) and the eighth dose (day 8). However, only the CT-868 group continued to show lower food intake on day 15 after the fifteenth dose (Figure 3D). These results indicate that CT-868 treatment was associated with a consistent and sustained reduction in food consumption, while the effect of liraglutide treatment on food consumption was reduced by the final measurement.

[0698] Having established the weight-loss effect of CT-868, the next step was to investigate the underlying mechanisms driving these changes. Indirect calorimetry was used to gain a deeper understanding of the ventilation ratio (RER), a key indicator of fuel utilization and energy expenditure. DIO mice received subcutaneous injections of either CT-868 (20 nmol / kg) or the medium daily. Oxygen consumption (VO2), carbon dioxide production (VCO2), and dynamic activity were monitored twice daily for 14 days to capture potential diurnal variations. During the baseline period, the RER (VCO2 / VO2 ratio) was similar in the treatment groups, ranging from 0.81 to 0.86 (to the left of the red boundary in Supplementary Figure S1A). A value of 0.8 indicates mixed utilization of carbohydrates and fats, which is typical, and in the dark phase, the RER tended to be slightly higher in both groups.

[0699] Notably, during the first nine days of the testing phase, the relative energy expenditure (RER) of the CT-868-treated animals was significantly lower than that of the vector-treated groups (to the right of the red boundary in Figure 45A). This decrease in RER suggests a shift towards increased fat utilization as an energy source after CT-868 administration. This is unlikely to be due to increased physical activity, as there was no difference in dynamic activity between the two groups after CT-868 administration (to the right of the red boundary in Figure 45B).

[0700] These in vitro and animal studies indicate that further clinical research is needed to explore CT-868.

[0701] Figure 46C

[0702] A double-blind, randomized, placebo-controlled phase 1 clinical trial was conducted to evaluate the safety, tolerability, and pharmacokinetics (PK) of CT-868 administered in single or multiple doses. In the single escalation dose (SAD) phase, 56 patients with overweight / obesity (BMI 27–45 kg / m²) participated. 2 Participants were sequentially enrolled in cohorts S1–7, with 8 lean participants (BMI 20–25 kg / m²) included. 2 The enrollment cohort S8 (Figure 46A) included eight participants in each cohort, with two receiving placebo and six receiving CT-868. In the multiple escalation dose (MAD) phase, 23 overweight / obese participants were enrolled in cohorts M1–M3 and randomized (1:3) to receive either placebo or CT-868 (Figure 46B). All 64 participants completed the SAD study, and 22 participants completed the MAD study.

[0703] CT-868 demonstrates exposure-dependent effects on glycemic parameters and weight loss in participants with overweight / obesity.

[0704] Following a single dose of CT-868, the mean maximum plasma concentration (C) max Increases with increasing dose level () CT-868 is safe and well-tolerated and does not require up-titration at single doses of 0.1 mg to 7.5 mg or multiple doses of 0.75 mg to 1.5 mg. ), and the maximum concentration (T) max The median time ranged from 3 hours (cohort S2: 0.5 mg CT-868) to 20 hours (S6: 7.5 mg CT-868), but was predominantly between 3 and 12 hours. Median t 1 / 2 The duration of action decreased with increasing CT-868 dose levels, ranging from 77.3 hours (S3: 1.5 mg CT-868) to 19.7 hours (S9: 11 mg CT-868).

[0705] At a dose of 1.5 mg CT-868 (S3 and S7 [lean participants]), lean participants reached C earlier. max Median T in lean participants max The median T for participants with a duration of 6.0 hours and who were overweight / obese was 6.0 hours. max The duration was 8.0 hours. Geometric mean (% CV) of lean participants C max (7.79 [42.4%] ng / mL) was higher than that of participants who were overweight / obese (5.62 [28.1%] ng / mL). Similarly, the CT-868 AUC trended upward in lean participants (AUC0-t: 167.2 [67.5%] h*ng / mL [lean] vs 154.2 [45.7%] h*ng / mL; AUC 0-inf : 209.2 [50.5%] h*ng / mL [lean] vs 184.9 [47.6%] h*ng / mL). Other PK parameters (t ½ There was no significant difference in CL / F or Vz / F between lean participants and participants who were overweight / obese.

[0706] In the case of repeated daily dosing (MAD phase), plasma CT-868 concentrations were detectable from 2 hours post-dose (Day 1) to 240 hours post-dose (Day 11) for all doses (0.75–5.0 mg), reaching a plateau several days after administration (Figure 46D). Plasma concentrations reached after 14 days of administration were dose-related but not dose-proportional. The pharmacokinetic profile supports once-daily dosing in humans.

[0707] Phase 1 placebo-controlled and comparator-controlled crossover clinical study of CT-868 in participants with overweight / obesity with and without T2DM (CT-868-003 study) CT-868 pharmacokinetics in participants with obesity and participants with T2DM

[0708] Dietary tolerance testing (MTT) in the SAD cohort showed a decreasing trend in mean AUC values ​​for glucose, insulin, and C-peptide with varying drug dosage. However, the AUC values ​​for insulin and C-peptide were statistically significant when analyzed as a function of CT-868 exposure (Figure 46E).

[0709] Weight loss was observed in all MAD cohorts and maintained until the end of dosing in all cohorts. The mean change in body weight relative to baseline on day 13 was -2.62% in the 1.5 mg cohort and -3.37% in the 5.0 mg cohort (Table 18). CT-868 concentration (AUC) on day 14 0 τ It was significantly associated with weight loss (Figure 46F).

[0710] Table 18. Average Changes in Body Weight

[0711]

[0712] CT-868 is more potent than liraglutide in lowering glucose and reducing insulin excursion. CT-868 and liraglutide demonstrate similar gastric emptying delay compared to placebo in participants with T2DM.

[0713] CT-868 was generally safe and well-tolerated in lean and overweight / obese participants at single doses up to 7.5 mg and repeated doses up to 1.5 mg / day for up to 14 days, without requiring any up-titration. No serious adverse events (SAEs) or drug-induced hypoglycemic events were observed during either phase of the study. In the SAD phase, the most common treatment-related adverse events (TEAEs) were gastrointestinal symptoms, which occurred most frequently in the highest dose cohort (11.0 mg); twelve events were reported in six (100%) participants, all of which were considered drug-related. At 7.5 mg, considering the maximum tolerated single dose, three participants (50%) reported three drug-related TEAEs (two nausea events and one diarrhea event).

[0714] During the MAD phase, all 23 (100%) participants reported ≥ 1 TEAE and ≥ 1 drug-related TEAE. Of the 84 reported TEAEs, 58 (69%) were considered related to the study drug. One participant in the highest dose cohort (5.0 mg) reported two drug-related TEAEs (mild nausea and vomiting), leading to withdrawal from the study drug and subsequent study interruption; at this dose level, all six participants reported mild to moderate gastrointestinal symptoms. In the 1.5 mg dose cohort, 4 / 6 participants reported gastrointestinal symptoms, including constipation (4 / 6), nausea (2 / 6), vomiting (2 / 6), abdominal pain (1 / 6), and diarrhea (1 / 6). In the lowest dose cohort (0.75 mg), 2 / 6 participants reported only nausea.

[0715] ​ ​

[0716] A second phase 1 study was conducted to evaluate the pharmacokinetic and pharmacodynamic effects of CT-868 on insulin secretion rate and environmental glucose levels in twelve obese but non-T2DM male participants (“Obese Participants”; Part 1) and 20 overweight / obese and T2DM participants (“T2DM Participants”; Part 2) compared to placebo and the active comparative liraglutide. This was a single-center controlled crossover study (Figures 47A–47B). The demographic and baseline characteristics of the participants in the CT-868-003 study are shown in Table 19.

[0717] Table 19. Demographic and baseline characteristics of participants in the placebo-controlled and comparator-controlled crossover study (CT-868-003 study) among overweight and T2DM participants.

[0718]

[0719] For Parts 1 and 2, participants in Group 1 received a crossover treatment of CT-868, placebo, and liraglutide for two days each, with a 14-day washout period between treatments. In Group 2, participants received a crossover treatment of CT-868 and placebo for two days each, with a 14-day washout period in between (Figures 47A-47B). In Part 1, two participants (16.7%) withdrew from the study, and the remaining 10 participants (83.3%) completed the study. In Part 2, 16 participants (80.0%) completed the study. Of the four participants (20.0%) who discontinued, two discontinued due to adverse events (AEs), one discontinued due to protocol violation, and one discontinued for other reasons.

[0720] A total of 12 participants with obesity completed the CT-868 treatment period (Groups 1 and 2), ten completed the placebo period (Groups 1 and 2), and four completed the liraglutide period (Group 1 only). For participants with T2DM, 20 completed the CT-868 treatment period, and 18 and 10 completed the placebo and liraglutide treatment periods, respectively. Due to the crossover design, differences in demographic and baseline characteristics between groups, as well as the treatment sequence, were not expected to affect the interpretation of the results. Data from each participant group were pooled for analysis based on the treatment received.

[0721] The endpoints evaluated in this study included the short-term effects of CT-868 on the relationship between insulin secretion rate (ISR) and ambient glucose levels in the MMTT, PK and blood glucose parameters, gastric emptying rate, free food intake, and hunger, appetite, and satiety parameters. These short-term assessments were conducted to thoroughly investigate the glucose-dependent effects of CT-868 on weight loss, i.e., before any weight loss effect occurs.

[0722] ​

[0723] Participants with obesity and those with type 2 diabetes mellitus (T2DM) showed similar CT-868 plasma concentration-time curves two days after CT-868 administration. The key pharmacokinetic parameter, Cp, was observed in both obese and T2DM participants. max (247.0 and 273.8 ng / ml respectively), T maxThe insulin secretion response (t½, 1,190 min vs. 655.8 min), AUC (209,126 vs. 231,699 ng / mL*min), incremental AUC (135,840 vs. 128,267 ng / mL*min), and terminal half-life (t½, 1,190 vs. 1,190 min) appeared similar. CT-868 demonstrated a robust insulin secretory response in both obese and T2DM participants.

[0724] The relationship between insulin secretion rate [ISR] and plasma glucose levels (ISR / G and ISR × G) serves as an indicator of β-cell health and was therefore assessed in graded glucose infusion (GGI) procedures two days after administration of CT-868, liraglutide, or placebo. CT-868 treatment increased β-cell responsiveness to glucose in both obese and T2DM participants (as indicated by a steeper ISR / G slope) (Figures 48A–48B). In obese participants, the ISR / G slope was steeper in those receiving CT-868 than in those receiving placebo (mean least squares [LS] difference in ISR / G slope: 5.691 [95% CI: 4.227, 7.154]) and in those receiving liraglutide (2.750 [95% CI: -0.368, 5.869]) (Figure 48A). For participants with T2DM, the ISR / G slope was also steeper in those receiving CT-868 than in those receiving placebo (LS mean difference in ISR / G slope: 1.922 [95% CI: 1.232, 2.612]) (Figure 48B). There was no difference in ISR / G slope between CT-868 and liraglutide in participants with T2DM.

[0725] ​

[0726] The effects of CT-868 on postprandial glucose, insulin, and glucagon in participants with type 2 diabetes mellitus (T2DM) were evaluated in the MMTT (Figure 49, Table X). Both CT-868 and liraglutide showed robust glucose-lowering effects compared to placebo. CT-868 was associated with smaller insulin drift compared to liraglutide and placebo. Glucagon responses were similar between CT-868 and placebo, while liraglutide delayed glucagon secretion at lower levels at 15 and 60 minutes after MMTT initiation.

[0727] During the MMTT, the mean insulin iAUC of CT-868 was LS.0-240min Significantly lower than placebo or liraglutide (Figure 49, Table 20). For glucose, the mean iAUC of CT-868 at LS was significantly lower than that of placebo or liraglutide (Figure 49, Table 20). 0-240min Significantly lower than placebo (LS mean difference: -320.40 [95% CI: -455.41, -185.39]), but the difference between CT-868 and liraglutide was not significant (LS mean difference: -110.60 [95% CI: -241.54, 20.34]). For glucagon, the difference between CT-868 and liraglutide was not significant in LS mean iAUC. 0-240min There was no significant difference in this aspect (LS mean difference: 351.349 [95% CI: -673.85, 1376.55]), or no difference in this aspect between CT-868 and placebo (LS mean difference: -799.29 [95% CI: -2482.44, 883.85]). For CT-868, the insulin-to-glucose product (I×G) was one-tenth that of liraglutide and 1 / 27th that of placebo, and for liraglutide, the I×G was 1 / 2.6th that of placebo (data not shown).

[0728] Table 20 shows the mean incremental AUC between CT-868 and placebo or liraglutide. 0-240min LS Mean. Least Squares (LS) means were derived using two mixed-effects models. The first model, comparing only CT-868 and placebo, included the treatment group, sequence, and period as fixed effects, and the in-sequence participants as random effects. The second model, comparing only CT-868 and liraglutide, included the treatment group, sequence, and group-sequence interactions as fixed effects, and the in-sequence participants as random effects. The denominator degrees of freedom used the Kenward-Roger approximation, and the random effects used the variance component covariance structure. Abbreviations: iAUC = area under the curve; LS = least squares; T2DM = type 2 diabetes.

[0729] Table 20. Mean incremental AUC between CT-868 and placebo or liraglutide 0-240min LS mean difference

[0730]

[0731] ​ ​

[0732] The effect of CT-868 on gastric emptying was assessed via paracetamol absorption assays. Based on paracetamol plasma concentration-time curves, administration of CT-868 and liraglutide produced gastric emptying delays similar to placebo (Figure 50, Table 21).

[0733] Table 21. Paracetamol plasma concentration-time curve

[0734]

[0735] Compared with placebo, CT-868 reduced food intake in participants with type 2 diabetes mellitus (T2DM).

[0736] Food intake and total calorie intake during the free-feeding period were assessed in participants with type 2 diabetes mellitus (T2DM) three days after administration of CT-868, liraglutide, or placebo. Mean food intake and total calorie expenditure were similar in participants administered CT-868 or liraglutide (LS mean difference -48.7 g [95% CI: -142.6, 45.3] (food intake), -202.3 kcal [95% CI: -478.1, 73.6] (total calories)) and were significantly different between CT-868 and placebo (Table 22). There were no significant differences in weight change among CT-868, liraglutide, and placebo three days after administration (Day 4; Table 22). Compared with day 1 (before the first dose of CT-868), pre-breakfast hunger and appetite scores on day 4 (3 days after administration) were lower in the CT-868 and liraglutide groups and higher in the placebo group (Table 22).

[0737] Table 22. Effects of CT-868 on hunger and appetite scores, as well as food intake and total calorie intake during free-eating periods, in participants with T2DM before and three days after drug administration (CT-868-003 study).

[0738]

[0739] CT-868 was safe and well-tolerated in participants with obesity and those with type 2 diabetes mellitus (T2DM).

[0740] In the crossover studies, CT-868 generally showed a good tolerability and safety profile (Table 23).

[0741] Table 23. Adverse events occurring during treatment in placebo and comparator-controlled crossover studies (CT-868-003 study)

[0742]

[0743] In Part 2 (Participants with T2DM), 14 out of 20 (70.0%) participants who received CT-868 reported >1 TEAE, and all TEAEs were considered mild (Grade 1), comparable to those who received liraglutide. One participant reported a TEAE that led to treatment discontinuation. 9 out of 20 participants (45.0%) reported CT-868-related TEAEs. The majority of these were gastrointestinal symptoms (nausea, gastroesophageal reflux disease, and vomiting). TEAEs were reported in 7 out of 18 participants (38.9%) in the placebo group and 3 out of 10 participants (30.0%) in the liraglutide group (Table 24).

[0744] Table 24. Summary of TEAEs (CT-868-003 study) by system organ classification and preferred terminology

[0745]

[0746] The incidence of hypoglycemia during GGI was similar in the CT-868 and liraglutide groups. No participants reported any SAEs. No clinically significant CT-868-related changes in vital signs, ECG parameters, or laboratory values ​​were reported.

[0747] discuss

[0748] The effects of CT-868, a signal transduction-based dual receptor agonist engineered to enhance signal transduction efficacy at both GLP-1R and GIPR, on insulin sensitivity, glucose homeostasis, and body weight were characterized in vitro, in animal models, and in two Phase 1 clinical studies.

[0749] In vitro studies have shown that CT-868 acts as a dual GLP-1 / GIP receptor agonist, exhibiting complete signaling bias at both receptors, preferentially accumulating cAMP without recruiting or internalizing β-repressor proteins (Figures 42A–42H). In animal studies assessing the physiological relevance of these properties, the minimum GIPR-binding dose of CT-868 was found to reduce glycemic drift in response to dietary challenge more effectively than the unbiased GLP-1R monoagonal liraglutide (Figures 43A–43I). Furthermore, these improvements in glycemic control are not a result of increased insulin secretion from CT-868, but rather mediated through GIP signaling, such as GLP-1R… - / - This has been demonstrated in experiments on mice.

[0750] CT-868 not only avoids receptor internalization but also acts as a reverse agonist, maintaining the receptor at the cell surface. Studies of cAMP-biased GLP-1R agonists have demonstrated enhanced insulinotropic and weight-loss effects relative to unbiased GLP-1R agonists. Animal studies in this application support these observations, where dose levels of CT-868 showed limited GIPR activation, reduced postprandial blood glucose levels, and induced weight loss more effectively than the unbiased GLP-1R monoagonal liraglutide (Figures 44A-44D). Unlike liraglutide, the initial weight loss from CT-868 was maintained after the dosing period and attributed to a sustained reduction in food intake without decreasing the animals' activity levels. Furthermore, metabolic changes (VO2, VCO2, RER) indicated increased utilization of fat as an energy source. To what extent these effects can be primarily attributed to biased cAMP signaling rather than GIPR activation warrants further investigation. Nevertheless, it is evident that additional weight loss exists at higher doses (e.g., 300 nmol / kg), which is likely due to GIPR activation.

[0751] In a first-in-human Phase 1 study, CT-868 was tested in participants of normal weight and overweight / obese individuals with single doses up to 11.0 mg and multiple doses up to 5.0 mg / day for 14 days without stepwise titration, and was found to be safe and well-tolerated. Plasma CT-868 exposure was dose-dependent, supporting once-daily administration. In participants receiving multiple doses of CT-868, pharmacokinetic profiles showed that continuous systemic exposure to CT-868 throughout the 14-day dosing period resulted in peak plasma concentrations within 48 to 96 hours after the first dose. In a crossover study, pharmacokinetic analyses of participants with obesity and type 2 diabetes mellitus (T2DM) showed peak plasma concentrations within 11 to 12 hours after administration, with a terminal half-life of approximately 20 hours. This further supports the once-daily dosing regimen of CT-868 and confirms the importance of performing key pharmacodynamic assessments (GGI, MMTT, gastric emptying assay) at the point of maximum CT-868 exposure. The highest dose (5.0 mg / day) was not associated with any SAE, drug-induced hypoglycemia, or severe TEAE, but in one participant it was considered intolerance due to a TEAE that led to that individual's drug withdrawal and study interruption. In both clinical studies, most TEAEs were mild and GI-related (nausea, vomiting), generally consistent with expectations for drugs targeting GLP-1R. In this dose escalation study, no severe or serious TEAEs or drug-related hypoglycemia were reported with CT-868.

[0752] The pancreatic and extrapancreatic effects of CT-868 in the context of type 2 diabetes mellitus (T2DM) and obesity were investigated in a placebo-controlled and comparator-controlled crossover study. In participants with T2DM, CT-868 robustly enhanced glucose-stimulated insulin responses, similar to liraglutide. This effect was more pronounced in obese participants (without T2DM), partly due to the preservation of β-cell function compared to those with T2DM, as indicated by higher baseline C-peptide levels in this group. In obese participants, CT-868 appeared to stimulate insulin secretion more strongly than liraglutide, possibly due to the additional GIPR agonist effect of CT-868 or GLP-1R desensitization induced by liraglutide. While CT-868 and liraglutide showed similar robust glucose-lowering effects after mixed meals, CT-868 was associated with less insulin drift (iAUC). 0-240minThese effects were correlated with, but did not inhibit, glucagon. These effects were not caused by significant differences in gastric emptying or weight loss, which were largely similar during the treatment periods of CT-868 and liraglutide. Instead, CT-868 appears to potentially promote insulin sensitivity and / or insulin-independent glucose disposal (relative to liraglutide) by modulating GIPR signaling (Hammoud et al., Nat Rev Endocrinol (2023) 19:201-16). GIPR can also affect energy metabolism through other insulin-independent mechanisms, such as regulating lipid storage via its activity in adipose tissue (Samms, ibid.). In type 1 diabetes or advanced type 2 diabetes, where β cells cannot produce the necessary insulin, the ability to target insulin-independent mechanisms for glucose control, such as GIP stimulation of peripheral glucose uptake, may have greater therapeutic importance.

[0753] In these studies, the minimum GIPR-binding dose of CT-868 generally showed greater effects on both blood glucose and weight loss than the unbiased monoagonal liraglutide, highlighting the physiological relevance of CT868 signaling bias. Furthermore, the additional GIPR agonistic effect of CT-868 compared to monoagonals may contribute to enhanced glucose reduction, as reported by two other dual GLP-1R / GIPR agonists (Finan et al., Sci Transl Med (2013) 5:209ra151). These results suggest that some of the observed GIPR-mediated effects of CT-868 may be insulin-independent. Simultaneous GIPR targeting has also been proposed to potentially help mitigate the proemetic effects of GLP-1R activation (Borner et al., Diabetes (2021) 70: 2545-53), thereby improving treatment tolerability. The results of the recently completed Phase 2 study (Clinical Trial ID: NCT05110846) will provide further insights into optimizing CT-868 dosing and up-titering to support maximizing weight loss and glycemic improvement, as well as acceptable tolerability.

[0754] in conclusion

[0755] These results further reveal the effects of dual GLP-1R / GIPR agonism and how the balance between GLP-1R / GIPR activation and the simultaneous bias in cAMP signaling at both GLP-1R and GIPR favorably influences metabolic homeostasis. In two Phase 1 clinical studies, the signal transduction-based dual GLP-1 / GIP receptor agonist CT-868 was observed to be safe and well-tolerated. CT-868 exhibited a robust glucose-lowering effect independent of weight change, but with reduced insulin drift and preserved glucagon secretion compared to liraglutide. These results support further evaluation of the long-term effects of CT-868 on glucose, weight, and other parameters in individuals with overweight / obesity and type 1 or type 2 diabetes.

[0756] Example 6: A phase 2, double-blind, randomized, placebo-controlled study evaluating the efficacy, safety, tolerability, and pharmacokinetics of CT-868 administered for 16 weeks in overweight and obese adult participants with type 1 diabetes.

[0757] This example outlines the protocol of a Phase 2 study to evaluate the efficacy, safety, tolerability, and pharmacokinetics of CT-868 in overweight or obese adult participants with a T1DM diagnosis record of ≥ 1 year and who have been treated with insulin via continuous subcutaneous insulin infusion (CSII) or multiple daily insulin injections (MDI) for ≥ 6 months.

[0758] Goals and End Points

[0759] The primary objective of this study was to compare the effects of titration up to a maximum of 6.6 mg of CT-868 (arm 4) versus placebo (arm 1) on the change (%) of HbA1c from baseline (day 1) to week 16, and the primary endpoint was the mean change (%) of HbA1c from baseline (day 1) to week 16 in arm 4 compared with arm 1.

[0760] The key secondary objective of this study is:

[0761] ● Compare the effects of CT-868 titrated to 4.1 mg (arm 3) versus placebo (arm 1) on the change (%) of HbA1c from baseline (day 1) to week 16; and

[0762] ● Compare the effects of CT-868 treatment titrated to 1.8 mg (arm 2) versus placebo (arm 1) on the change (%) of HbA1c from baseline (day 1) to week 16.

[0763] The key secondary endpoint of this study was:

[0764] ● Mean HbA1c change (%) from baseline day 1 to week 16 in arm 3 compared to arm 1; and

[0765] ● The mean change (%) of HbA1c from baseline day 1 to week 16 in arm 2 compared to arm 1.

[0766] Other secondary objectives of this study are:

[0767] ●Evaluate the effect of CT-868 relative to placebo on the following parameters in each treatment arm:

[0768] ○CGM indicators (TIR, duration of hypoglycemia and hyperglycemia, glycemic risk index);

[0769] ○ Percentage of participants achieving HbA1c <7.0%, ≤6.5%, and <5.7%;

[0770] ○ Weight changes;

[0771] ○ Percentage of participants who achieved weight loss of ≥5%, ≥10%, ≥15%, and ≥20%;

[0772] Number of Grade 3 hypoglycemic events;

[0773] ○Incidence of DKA; and / or

[0774] ○ Absolute and relative changes in basal, pulse, and total daily insulin use (units / day and units / kg / day).

[0775] ● The safety and tolerability of CT-868 administered via an injection pen over 16 weeks were assessed in each treatment arm of overweight or obese participants with T1DM compared to placebo.

[0776] ●Assess CT-868 plasma concentration.

[0777] Other secondary endpoints of this study were:

[0778] ● Changes in the following CGM parameters in each treatment arm from baseline day 1 to week 8 and week 16:

[0779] ○ Average sensor glucose (mg / dL) and its standard deviation and %CV [intra-day (i.e., within 24 hours) and inter-day (i.e., over multiple days)];

[0780] ○ Percentage of CGM values ​​within the range (TIR 70–180 mg / dL);

[0781] ○ Percentage of participants who showed improvement of ≥5 percentage points and ≥10 percentage points after taking TIR 70-180 mg / dL;

[0782] ○ Percentage of CGM values ​​<70-54 mg / dL (Grade 1 hypoglycemia) and <54 mg / dL (Grade 2 hypoglycemia);

[0783] ○ Percentage of CGM values ​​>180–250 mg / dL (Grade 1 hyperglycemia) and >250 mg / dL (Grade 2 hyperglycemia);

[0784] ○Time (hours / minutes) consumed per 24 hours within the range (70–180 mg / dL) during the CGM wearing period;

[0785] ○Time consumed per 24 hours (hours / minutes) in hypoglycemia grade 1 (<70-54 mg / dL) and grade 2 (<54 mg / dL) during CGM wearing period;

[0786] ○Time consumed per 24 hours (hours / minutes) in hyperglycemia grade 1 (>180–250 mg / dL) and grade 2 (>250 mg / dL) during CGM wearing period;

[0787] ○ The number of events in which the sensor glucose level is <70 mg / dL for at least 120 minutes (the event ends when glucose recovers to ≥70 mg / dL and lasts for ≥15 minutes).

[0788] ○ The number of events in which the sensor glucose level is >250 mg / dL for at least 120 minutes (the event ends when glucose recovers to ≤180 mg / dL and lasts for ≥15 minutes).

[0789] ○ The proportion of participants with a daily TIR dose of 70–180 mg / dL >70%;

[0790] ○ The proportion of participants with daily TBR <70 mg / dL <4%;

[0791] ○ The proportion of participants with daily TBR <54 mg / dL <1%; and / or

[0792] ○ Blood sugar risk index.

[0793] ●HbA1c

[0794] ○ Changes in HbA1c (%) from baseline day 1 to weeks 4, 8, and 12; and / or

[0795] ○ Percentage of participants who achieved HbA1c <7.0%, ≤6.5%, and <5.7% at week 16.

[0796] ●Weight

[0797] ○ Percentage change (%) in body weight from baseline day 1 to weeks 4, 8, 12 and 16;

[0798] ○ Absolute change in body weight (kg) from baseline day 1 to weeks 4, 8, 12, and 16; and / or

[0799] ○ Percentage of participants who achieved weight loss of ≥5%, ≥10%, ≥15%, and ≥20% by week 16.

[0800] ● The number of grade 3 hypoglycemic events in each treatment arm over a 16-week period.

[0801] ● The number of “affirmative” DKA events (listed below) (Kitabchi et al., Diabetes Care (2009) 32(7): 1335-43).

[0802] ● The number of DKA events that resulted in hospitalization or emergency room visits.

[0803] ● Changes (absolute and percentage change) in basal, pulse, and total daily insulin use (units / day and units / kg / day) over a 16-week period in each treatment arm.

[0804] ● AE / ADE, physical examination, vital signs (temperature, blood pressure, and heart rate), ECG, and safety laboratory measurements (urinalysis, hematology, chemistry, BHB, amylase, lipase, and calcitonin) for each treatment arm during a 16-week period.

[0805] ●Plasma drug concentrations at week 2, 4, 6, 8, 12 and 16 visits.

[0806] Discontinuation of the study drug may be considered if a study participant meets any of the following criteria:

[0807] ● Any event that strongly suggests serious drug-induced liver injury. Examples may include injury with any of the following outcomes:

[0808] ○ Clinical manifestations (e.g., liver-related hospitalization, diagnosis of acute liver failure).

[0809] ○ Consider symptoms consistent with the diagnosis of acute drug-induced liver injury (e.g., abdominal pain, vomiting, jaundice).

[0810] ○ Biochemical findings, such as those described below, are based on the FDA guidance (CDER 2009) regarding criteria for discontinuing drug use due to drug-induced liver injury in individual participants:

[0811] ○ALT or AST >8× ULN

[0812] ○ALT or AST >5×ULN, lasting >2 weeks

[0813] ○ALT or AST >3×ULN and total bilirubin >2×ULN or international normalized ratio >1.5×ULN

[0814] ○ALT or AST >3×ULN, accompanied by fatigue, nausea, vomiting, pain or tenderness in the right upper quadrant, fever, rash, and / or eosinophilia (>5%).

[0815] ○ALP >3×ULN

[0816] ○ALP >2.5×ULN and total bilirubin >×ULN, or

[0817] ○ALP >2.5×ULN, accompanied by fatigue, nausea, vomiting, right quadrant pain or tenderness, fever, rash and / or eosinophilia (>5%).

[0818] ● CT-868 was not tolerated at least 1.8, 3.3, or 4.1 mg / day in arm 2, arm 3, or arm 4, respectively.

[0819] ● Do not administer any prescription or over-the-counter medication for weight loss for more than one week.

[0820] ●Any anti-hyperglycemic medications that have been in use for more than 1 week are prohibited.

[0821] ● Calcitonin ≥100 ng / L.

[0822] ●eGFR <15 mL / min.

[0823] ●Suspected pancreatitis.

[0824] ● Experiencing any of the following clinically significant hypoglycemic events in the absence of any confounding conditions that lower blood glucose:

[0825] ○ At any point during the study, participants experienced grade 3 hypoglycemia, defined as a severe event characterized by impaired mental (cognitive) and / or physical functioning requiring external assistance to recover; or

[0826] ○ At any time during the study, a CGM value <54 mg / dL (3.0 mmol / L), with or without symptoms, lasting for ≥120 minutes (prolonged grade 2 hypoglycemia).

[0827] ●Experiencing a “definite” DKA episode [defined as the presence of acidosis (blood pH <7.3, serum bicarbonate level <18 mEq / L) with symptoms / signs according to the ADA consensus statement on the diagnosis of DKA, or if the DKA episode results in hospitalization or emergency room stay].

[0828] The exploratory goal of this study is:

[0829] ●Assess the effect of CT-868 relative to placebo on composite remission parameters in each treatment arm at week 16.

[0830] ●Assess the effect of CT-868 on gastric emptying (via paracetamol absorption) in each treatment arm during the MMTT.

[0831] ●Assess the effects of CT-868 relative to placebo on metabolic, inflammatory, and endothelial health blood biomarkers in each treatment arm.

[0832] ● The effect of CT-868 relative to placebo on total lean body mass and total fat mass in each treatment arm was assessed by DEXA.

[0833] ● The PRO questionnaire was used to assess the impact of CT-868 relative to placebo on quality of life and treatment satisfaction in each treatment arm.

[0834] ●Assess the effect of CT-868 relative to placebo on VAS ratings of hunger, satiety, fullness, and expected food consumption in each treatment arm.

[0835] ●Assess the percentage of participants at the end of week 16 who were on the 4.1 mg, 5.2 mg, and 6.6 mg doses, and the time to reach their final dose (arm 4 only).

[0836] ● Determine the correlation between the time to reach the highest CT-868 dose and the change in HbA1c and percentage change in body weight from baseline to week 16 (arm 4 only).

[0837] ●Assess the percentage of participants who permanently discontinued the study drug from day 1 randomization to week 16.

[0838] ● The time (in weeks) for evaluating the permanent discontinuation of research drugs.

[0839] The exploratory endpoint of this study is:

[0840] ●Percentage of participants in each treatment arm who had the following conditions at week 16 compared to their corresponding day 1 baseline:

[0841] ○HbA1c improvement ≥0.5 percentage points and <1% TBR <54 mg / dL;

[0842] ○≥70% TIR 70-180 mg / dL and <4% TBR <70 mg / dL;

[0843] ○ ≥70% TIR 70-180 mg / dL and <1% TBR <54 mg / dL; and / or

[0844] ○ HbA1c improvement ≥0.5 percentage points or ≥70% TIR 70-180 mg / dL, and <1% TBR <54 mg / dL, without the need for a significant increase in daily insulin use [i.e., an increase of >10% in total (pulse + basal) insulin (units / kg / day)].

[0845] ● Changes in plasma glucose, insulin, C-peptide, glucagon, GLP-1 (total activity), GIP, and FFA in each treatment arm from baseline day 1 to week 16.

[0846] ● Changes in the following MMTT parameters from baseline day 1 to week 16 in each treatment arm:

[0847] ○ Glucose iAUC 0-240min ;

[0848] ○Insulin iAUC 0-240min ;

[0849] ○C peptide iAUC 0-240min ;

[0850] ○glucagon iAUC 0-240min ;

[0851] ○GLP-1 (Total Activity) iAUC 0-240min ;

[0852] ○GIP iAUC 0-240min ;

[0853] ○FFA iAUC 0-240min ;

[0854] ○HOMA-IR, QUICKI, Matsuda Index, HOMA-B, Disposal Index; and / or

[0855] Paracetamol C max T max AUC 0-240min TWA 0-240min .

[0856] ● Changes in the following fasting blood biomarkers from baseline day 1 to week 16 in each treatment arm: lipid profile (triglycerides, total cholesterol, LDL-C, HDL-C, VLDL, apoB), adiponectin, leptin, hsCRP, PAI-1, CTX-1, MCP-1, IL-6, endothelin-1, E-selectin, VCAM-1, 3-nitrotyrosine, and UACR.

[0857] ● Changes in total lean body mass and total fat mass, determined by DEXA, in each treatment arm from baseline day 1 to week 16.

[0858] ● Changes in diabetes-related quality of life assessed using ADDQoL in each treatment arm. The baseline for these assessments was at the end of the screening period and before they experienced the CGM device provided in the study (i.e., at the start of a self-determined insulin adjustment period or before the placebo induction period).

[0859] ● Changes in diabetes treatment satisfaction assessed using DTSQ and DTSQc in each treatment arm. The baseline for DTSQ was at the end of the screening period and before they experienced the CGM device provided in the study (i.e., at the start of a self-determined insulin adjustment period or before the placebo induction period).

[0860] ● Changes in the overall appetite score, assessed by VAS ratings of hunger, satiety, fullness, and expected food consumption, from baseline day 1 to week 8 and week 16 in each treatment arm.

[0861] ● The number of participants who maintained doses of 4.1 mg, 5.2 mg, and 6.6 mg at the end of week 16 (arm 4 only).

[0862] ● Time to reach the final dose (weeks) (arm 4 only).

[0863] ● Correlation between time to reach peak CT-868 dose and HbA1c change and percentage change in body weight from baseline day 1 to week 16 (arm 4 only).

[0864] ●Percentage of participants who permanently discontinued the study drug from day 1 randomization to week 16.

[0865] ● The duration (in weeks) of permanent interruption of research drug use.

[0866] The objectives related to pharmacokinetics and immunogenicity are:

[0867] ● Evaluate the CT-868 time-concentration curve over 24 hours (in a subset of the total study population).

[0868] ●Evaluate the correlation between CT-868 plasma exposure and relevant efficacy parameters.

[0869] ●Assess for anti-drug antibodies against CT-868 that appear during treatment (arms 2, 3, and 4 only).

[0870] The endpoints related to pharmacokinetics and immunogenicity are:

[0871] ●CT-868 PK parameters (C max T max C trough C last T last AUC 0-24 AUC 0-last and t ½ );

[0872] ●CT-868 Plasma Exposure (C trough C max The correlation between HbA1c (AUC) and changes in body weight; and / or

[0873] ● Anti-drug antibodies against CT-868 appearing on day 1 and week 16 of treatment (arms 2, 3, and 4 only).

[0874] Inclusion criteria

[0875] Participants must meet all of the following criteria to be eligible to participate in this study:

[0876] 1. The person signing the informed consent form must be a male or female aged 18 or older.

[0877] 2. Screening for T1DM diagnostic records ≥1 year prior to the visit.

[0878] 3. Body mass index ≥ 25.0 kg / m²2

[0879] 4. At the screening visit, the glycated hemoglobin A1c (HbA1c) level was between 7.0% and 9.5% (inclusive).

[0880] 5. Prior to the screening visit, the patient has been treated with insulin for ≥6 months using continuous subcutaneous insulin infusion via an insulin pump (CSII) or multiple daily injections (MDI).

[0881] 6. Based on the investigator's judgment and records, patients must have received stable insulin preparations, dosages (i.e., within 10% of the total daily dose), and devices (i.e., not switching from MDI to a pump, or vice versa) for ≥2 months prior to the screening visit.

[0882] Exclusion criteria

[0883] Participants who meet any of the following criteria will be excluded from the study:

[0884] 1. Diagnosed with type 2 diabetes mellitus (T2DM) or any other type of diabetes, except for T1DM.

[0885] 2. Screening patients who have experienced diabetic ketoacidosis (DKA) within 3 months prior to the screening visit.

[0886] 3. Screening patients who have experienced severe hypoglycemia (Grade 3 as defined in the ADA Diabetes Care Standards (ADA 2022)) within the 3 months prior to the screening visit.

[0887] 4. Impaired perception of hypoglycemia (i.e., a Clarke Questionnaire score ≥4 at the screening visit [Clarke et al., Diabetes Care (1995) 18 (4): 517-22])

[0888] 5. Use any insulin formulation that is not delivered via CSII or MDI (e.g., inhaled insulin).

[0889] 6. Screen for significant changes in diet or exercise programs within the previous 3 months. This includes any of the following activities that were performed or planned during the study period (e.g., very low-calorie, low-carbohydrate, very high-protein, ketogenic diet, time-restricted diet / intermittent fasting or other nutritional interventions, or initiating a new exercise program).

[0890] 7. Previous use (i.e., within 6 months prior to the screening visit) or current use of any of the following:

[0891] a) Any medication that may interfere with glycemic control (except insulin) (e.g., monoamine oxidase inhibitors, growth hormone);

[0892] b) Any adjunctive treatment for diabetes (e.g., pramlintide, metformin, glucagon-like peptide-1 [GLP-1] analogues [e.g., exenatide], GLP-1 receptor agonists (GLP-1RAs) [e.g., cemaglutide, liraglutide, or dulaglutide], GLP-1 / GIP RA (Mounjaro™), sodium-glucose cotransporter-2 inhibitors, sulfonylureas, or dipeptidyl peptidase-4 inhibitors).

[0893] c) Any prescription drugs and / or any nonprescription products, including herbal and / or dietary supplements for weight loss (e.g., orlistat, locacerin, phentermine topiramate, naltrexone-bupropion, zemagranate, liraglutide, Garcinia Cambogia extract, Hydroxycut®, or glucomannan) or for weight gain (e.g., testosterone, growth hormone, anabolic steroids, or amino acid supplements); or

[0894] d) Received or has received long-term (>14 consecutive days) systemic glucocorticoid therapy (excluding topical, intraocular, intranasal, intra-articular, or inhaled formulations) within 3 months prior to the screening visit, or there is evidence of a significant active autoimmune disorder (e.g., lupus or rheumatoid arthritis) that the investigator deems necessary (within the past 3 months) or likely to require concurrent treatment with systemic glucocorticoids (excluding topical, intraocular, intranasal, intra-articular, or inhaled formulations) during study participation.

[0895] e) Any prescription or over-the-counter medications known to interfere with bowel motility, including but not limited to chronic opioids, anticholinergics, antispasmodics, linaclotide, and dopamine antagonists.

[0896] Note: Lipid-lowering and / or antihypertensive medications are permitted, provided that the dosage / regimen of these medications has remained stable over the last two months prior to the screening visit.

[0897] 8. History of intolerance, allergic reaction, or lack of efficacy (i.e., no response) to any previous treatment with any GLP-1 analog, GLP-1RA, or GLP-1 / GIP RA.

[0898] 9. Screen for any self-reported weight gain or loss of ≥5% within the 3 months prior to the visit.

[0899] 10. Have undergone or plan to undergo any surgical treatment or use of weight loss devices or any other weight loss procedures (e.g., LAP-Band®, gastric balloon, duodenal cannula, skin resurfacing) for obesity (any type) during the study period.

[0900] Note: If liposuction and / or abdominal fat reduction were performed more than 1 year prior to the screening visit, the investigator may decide whether to allow it, provided all other eligibility criteria are met.

[0901] 11. Obesity induced by other endocrine disorders (e.g., Cushing's syndrome, acromegaly, or undertreated hypothyroidism) or diagnosed as a monogenic or syndromic form of obesity (e.g., melanocortin 4 receptor deficiency or Prader-Willi syndrome).

[0902] 12. Clinically significant diagnosis and / or history of gastroparesis, abnormal gastric motility, abnormal gastric emptying, malabsorption, chronic constipation, chronic diarrhea, inflammatory bowel disease, bowel resection, irritable bowel syndrome, or severe gastroesophageal reflux disease.

[0903] 13. Screening participants with any of the following conditions within 6 months prior to the visit: myocardial infarction, unstable angina, coronary artery bypass grafting, percutaneous coronary intervention (including participants who may plan and / or anticipate undergoing percutaneous endovascular coronary angioplasty [PTCA] during the study period, such as participants with prior angiographic evidence that may require PTCA); transient ischemic attack, cerebrovascular accident, or hospitalization due to congestive heart failure.

[0904] 14. Current New York Heart Association Class III or IV heart failure.

[0905] 15. Clinically significant electrocardiogram (ECG) results at screening visits (e.g., QT interval [QTcF] >450 msec (male), QTcF >470 msec (female), left bundle branch block, corrected using the Fridericia formula), or any other ECG result considered indicative of active heart disease that the investigator believes would interfere with the interpretation of ECG changes during the study or could pose a safety concern to the participant.

[0906] 16. Screen for uncontrolled hypertension at the time of visit (i.e., mean sitting systolic blood pressure ≥160 mmHg and / or mean sitting diastolic blood pressure ≥100 mmHg).

[0907] 17. Screen for any clinically significant changes in ophthalmology within the 12 months prior to the visit, including the presence of clinically significant proliferative retinopathy, macular edema, or other diabetic retinopathy.

[0908] 18. History of any blood conditions that may interfere with HbA1c measurement results (e.g., hemolytic anemia, sickle cell disease, other hemoglobinopathies).

[0909] 19. Screening visits for uncontrolled thyroid disease defined as the presence of active symptoms (e.g., palpitations, somnolence, weight gain or loss) and / or thyroid-stimulating hormone (TSH) levels exceeding the central laboratory's normal reference range.

[0910] (This may include participants who are adequately treated for hypothyroidism, provided they are clinically asymptomatic, have TSH levels within the normal reference range, and have maintained a stable thyroid hormone replacement dose for ≥2 months prior to the screening visit.)

[0911] 20. History of acute or chronic pancreatitis or current diagnosis of acute or chronic pancreatitis or risk factors for pancreatitis, such as alcoholism, gallstones (without cholecystectomy), hypercalcemia, or severe hypertriglyceridemia.

[0912] 24. The central laboratory determines that any of the following results exist during the screening period:

[0913] a) Alanine aminotransferase (ALT), aspartate aminotransferase (AST), or gamma-glutamyl transferase (GGT) > 3.0 × 10⁻⁶ (Upper limit of normal, reference range).

[0914] Note: Participants with non-alcoholic fatty liver disease were eligible to participate in this study if their ALT level was ≤3.0 × ULN of the reference range.

[0915] b) Alkaline phosphatase (ALP) >1.5 × ULN of the reference range

[0916] c) Total bilirubin > ULN of the reference range

[0917] d) Amylase or lipase levels > 2 × ULN of the reference range

[0918] e) Fasting triglyceride level >500 mg / dL

[0919] f) Estimated glomerular filtration rate (eGFR) calculated using the dietary correction equation for kidney disease <45 mL / min / 1.73 m 2

[0920] g) If eGFR ≥ 60 mL / min / 1.73 m 2 If the calcitonin level is ≥20 ng / L, then the calcitonin concentration is ≥60 mL / min / 1.73 m. 2 If calcitonin is ≥35 mg / L

[0921] h) History of hepatitis screening (including hepatitis B surface antigen [HBsAg] and hepatitis C antivirus) or positive hepatitis screening results

[0922] i) Positive for human immunodeficiency virus (HIV) antibodies at screening

[0923] j) Hemoglobin level <11 g / dL (male participants) or <10 g / dL (female participants)

[0924] 26. Based on the safety information provided in the study regarding the CGM device (e.g., Dexcom G6), it is unavoidable to use >1000 mg of acetaminophen / paracetamol every 6 hours.

[0925] Research Design

[0926] This is a phase 2, double-blind, randomized, placebo-controlled study evaluating the efficacy, safety, tolerability, and pharmacokinetics of CT-868 in overweight or obese adult participants with type 1 diabetes mellitus (T1DM).

[0927] Although participants’ T1DM can be managed with an insulin pump or with the aid of an insulin MDIs, all participants must have used a CGM device for ≥2 months prior to the screening visit and must be willing to wear and maintain the study-provided CGM device (e.g., Dexcom G6) during the study.

[0928] All enrolled participants are expected to maintain their daily routines during the study (e.g., not to start a novel diet or exercise program that differs from their daily routine) and follow the standard care for T1DM, including maintaining all other standard care therapies for other stable chronic conditions (where appropriate) throughout the study.

[0929] Following a screening period of up to 3 weeks (weeks -7 to -4), eligible participants will be administered a diabetes-related quality of life and treatment satisfaction questionnaire at the start of Visit 2 (or Visit 3 if no self-adjustment period has been initiated) to establish a baseline of their perception and satisfaction with their current diabetes treatment. After this assessment, participants may enter a self-adjustment period of up to 2 weeks (weeks -4 to -2), during which investigators will have the opportunity to assess the adequacy of their current insulin regimen and determine whether participants are within their target treatment goals, defined as ≥70% of the time within the range of 70–180 mg / dL (3.9–10 mmol / L) and <4% of the time within the range of <70 mg / dL (<3.9 mmol / L) (Battelino et al., Lancet Diabetes Endocrinol. (2023) 11:42–57). During the period prior to the mandatory 2-week single-blind placebo induction period (week -2 to day 1), the researchers may adjust the participants' insulin regimen at their discretion if necessary.

[0930] On day 1, participants will enter a 16-week double-blind treatment period and will be randomly assigned to one of the four treatment arms in a 1:1:1:1 ratio.

[0931] The combined placebo arm 1 will consist of 3 groups of approximately 8 participants each, receiving a placebo dose matched to the volume and dose of CT-868. The total number of placebo participants in arm 1 (n~24) will be similar to the number of participants in each of the CT-868 treatment arms (i.e., n~24 in each of arms 2, 3, and 4).

[0932] During randomization, participants were stratified according to their HbA1c level (<8.2% or ≥8.2%) at the screening visit and their insulin administration method (pump or MDI). Stratification by these two factors allows for a better understanding of the impact of CT-868 treatment on glycemic control for both insulin pump and MDI users across different HbA1c levels.

[0933] Insulin dosage will be adjusted based on initial HbA1c values ​​and CGM data to minimize the risk of hypoglycemia (see Table 25).

[0934] To mitigate the risk of DKA, especially during specific insulin dose reduction periods, it is recommended to perform systematic and frequent ketone monitoring and take appropriate mitigation measures (Table 26).

[0935] Just as insufficient insulin therapy can lead to diabetic ketoacidosis (DKA), excessive insulin therapy can also lead to hypoglycemia. Appropriate mitigation measures will be taken to further minimize the risk of hypoglycemia.

[0936] Following randomization on Day 1 and after all necessary baseline assessments have been completed, participants will be observed administering the first dose of the study drug at the clinic. Participants will again be instructed to administer the study drug via SC injection into the abdomen at approximately the same time each morning.

[0937] Dosage form and route of administration

[0938] CT-868 injection 15 mg / mL and CT-868 injection placebo solution will be provided in a needle-based injection system (CT-868 pen injector (pen)) with an integrated, non-replaceable 3-mL Type 1 glass cartridge. The CT-868 pen is a multi-dose, single-participant, disposable pen designed to deliver five fixed doses of 1.1 mg (0.07 mL), 1.8 mg (0.12 mL), 2.6 mg (0.17 mL), 3.3 mg (0.22 mL), and 4.1 mg (0.27 mL) via SC injection in the abdomen. The CT-868 pen contains 3 mL of a clear, colorless, sterile aqueous solution of 15 mg / mL CT-868 (active ingredient only) in 20 mM disodium hydrogen phosphate heptahydrate / sodium dihydrogen phosphate buffer, propylene glycol, and phenol at pH 7.0. The placebo pen contains the same ingredients except for CT-868. Each active ingredient pen contains approximately 45 mg / 3 mL of CT868. Each pen is individually packaged in a cardboard box including instructions for use (IFU). In each dose group, the CT 868 and placebo pens will appear identical.

[0939] CT-868 and placebo pens must be stored refrigerated and brought to room temperature before administration.

[0940] CT-868 and placebo will be titrated according to the protocol and in arm 4 based on individual participant tolerability up to a maximum dose of 6.6 mg CT-868. Detailed dosage and titration protocols for each treatment arm are provided in Table 25.

[0941] Participants randomly assigned to arm 2, arm 3, or arm 4 were required to tolerate the lowest dose of 1.8 mg, 3.3 mg, or 4.1 mg, respectively, to continue participating in the study and have their data used for the primary efficacy analysis.

[0942] Evaluation criteria

[0943] Efficacy will be assessed using HbA1c, various CGM indicators, weight / body composition measurements, MMTT, and PRO.

[0944] The security assessment will include the following:

[0945] ●AE / ADE, including injection site reactions

[0946] ● Particularly interesting AEs / ADEs (hypoglycemia, DKA, pancreatitis, diabetic retinopathy, acute kidney injury, pen malfunction, pen misuse, intentional pen abuse, and poor adherence to IFU).

[0947] (Note: Participants who report specific symptoms of hypoglycemia will also be asked to provide a description of such events.)

[0948] ● Vital signs

[0949] ●Physical examination

[0950] ●ECG

[0951] ● Clinical laboratory tests (hematology, chemistry, including β-hydroxybutyrate, amylase, lipase, calcitonin, and urinalysis)

[0952] Table 25. Insulin Adjustment Guidelines

[0953]

[0954] Table 26. Recommended Action Plan for DKA Management

[0955]

[0956] Table 27. Dosage / Titration Adjustment Protocol

[0957]

[0958] Example 7: Phase 2 trial of CT-868, a novel dual GLP-1 and GIP receptor modulator, in overweight and obese adults with type 2 diabetes mellitus (T2DM).

[0959] method

[0960] This example describes data from a phase 2, 26-week, randomized, placebo-controlled, double-blind, multicenter study evaluating the efficacy and safety of CT-868 in adults with T2D and overweight / obesity who are under-optimally controlled by diet and exercise (with or without metformin).

[0961] Goals and End Points

[0962] The primary objective of this study was to assess changes in HbA1c and characterize the safety and tolerability profile of CT-868 administered via daily subcutaneous injection.

[0963] The primary endpoint of this study was to assess changes in HbA1c levels at week 26.

[0964] Secondary endpoints of the study included assessment of changes in body weight, fasting plasma glucose and lipids at week 26, and safety / tolerance relative to baseline.

[0965] Research Design

[0966] One hundred and three participants (male / female: 39 / 64), aged 18 to 75 years (median age: 50 years), diagnosed with type 2 diabetes mellitus (T2D) >6 months and with a BMI ≥27 kg / m², were included in the study. 2 And HbA1c is 7%–10% (average BW / BMI / HbA1c: 92 kg / 35.3 kg / m²). 2 (8.1%), and at screening, participants received either diet / exercise alone or metformin monotherapy, and were randomly assigned in a 1:1:2 ratio to one of the three treatment arms:

[0967] Arm 1: Volume-matched placebo (placebo; n~24);

[0968] Arm 2: CT-868 1.75 mg (n~24); and

[0969] Arm 3: CT-868 dose is administered at a maximum of 4 mg (n~48) based on individual tolerance.

[0970] The titration protocol is shown in Figure 51. In arm 3, 32 participants received a 4 mg dose, and Eighteen participants received a 3.25 mg dose. Therefore, three doses of CT-868 (1.75, 3.25, and 4 mg) were administered along with a placebo. To account for this variability, the final dose (1.75 mg, 3.25 mg, or 4 mg) received by each participant, rather than their initial randomization group, was analyzed.

[0971] result

[0972] The demographics of the participants are shown in Table 28 below.

[0973] Table 28. Demographic and Baseline Characteristics

[0974]

[0975] Blood sugar control

[0976] At week 26, HbA1c and fasting plasma glucose (FPG) levels were decreased in each CT-868 arm (1.75, 3.25, 4 mg), with placebo-corrected mean LS differences of -1.92%, -1.67%, and -2.30% (p<0.001) and -52.9, -56.7, and -60.1 mg / dL (p<0.001), respectively. Compared with placebo, CT-868 had a higher proportion of patients achieving HbA1c ≤6.5% at week 26 (p<0.01): 52.2% (1.75 mg), 72.2% (3.25 mg), 69.0% (4 mg), and 18.2% (placebo).

[0977] Compared with placebo, HbA1c in all three CT-868 arms was significantly improved from baseline at week 26. The mean treatment difference in HbA1c change from baseline between CT-868 4.0 mg and placebo was -2.3% (95% CI -3.03 to -1.58, p<0.001). Compared with placebo, significantly more participants in all three CT-868 arms achieved HbA1c ≤6.5% and HbA1c <7%. Compared with placebo, CT-868 significantly improved FPG (treatment difference -51.8 to -60.5 mg / dL, p<0.001, for all arms). See Figure 52.

[0978] weight

[0979] The mean percentage change in body width (BW) in the CT-868 4 mg arm was -5.1% (p=0.025), compared to -2.5% in placebo. 14% of the CT-868 4 mg treatment group achieved a weight loss of >10%, compared to 0% in placebo.

[0980] At week 26, a 4.0 mg dose of CT-868 was associated with a weight loss of (5.7 ± 4.4)% relative to baseline. The weight loss in the placebo arm was (2.3 ± 3.9)%. At week 26, 53.3% of participants receiving a 4.0 mg dose of CT-868 had a weight loss (WL) ≥5%, compared to 22.7% in the placebo arm (p=0.027).

[0981] lipid parameters

[0982] In the CT-868 4mg arm, the mean placebo-adjusted changes in T-Chol, TG, LDL-C, and ApoB were -26, -139, -15, and -20 mg / dL, respectively (p < 0.01).

[0983] Compared with placebo, the CT-868 treatment arm showed lower levels of total cholesterol, triglycerides, LDL-C, VLDL, and apolipoprotein B at week 26. See Figure 53.

[0984] vital signs

[0985] At week 26, CT-868, administered at 3.25 mg and 4.0 mg, reduced both systolic and diastolic blood pressure (SBP). The SBP / DBP ratio for the CT-868 4-mg arm was -7.6 / -3.6 mmHg, compared to (+1 / +1.4 mmHg) for placebo. See Figure 54.

[0986] The mean heart rate on the CT-868 treatment arm increased by 1.7 ± 8.8 to 2.3 ± 8.7 bpm, compared to a decrease of 4.5 ± 8.2 bpm in the placebo.

[0987] Liver test

[0988] Compared with placebo, liver enzymes were reduced by 20%–25% in the CT-868 treatment arms. In all CT-868 treatment arms, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) at week 26 were reduced by approximately 15%–25% relative to baseline, while no significant changes were observed with placebo. No clinically significant changes in bilirubin were observed. See Figure 54.

[0989] Safety and tolerability

[0990] CT-868 was safe and well-tolerated, with no treatment-related interruptions in the CT-868 arm. The most common adverse events (AEs) were gastrointestinal (GI) related, observed in both the CT-868 (60%) and placebo (56%) arms [nausea (16-28% vs. 26%), constipation (8-20% vs. 19%), vomiting (8-10% vs. 4%), diarrhea (37-44% vs. 22%), without dose dependence, and most were mild (Grade 1). No clinically significant hypoglycemia was reported.

[0991] Table 29. Overall Summary of Treatment-Acting Adverse Events (TEAEs)

[0992]

[0993] Table 30. Gastrointestinal TEAEs

[0994]

[0995] in conclusion

[0996] CT-868 demonstrated robust glycemic control (a -2.3% reduction in HbA1c compared to placebo) in 67%–72% of patients, achieving ≤6.5% HbA1c at week 26 (within the non-diabetic range). Improvements in cardiovascular risk factors were also demonstrated. CT-868, administered at doses up to 4.0 mg, improved key cardiovascular risk factors (LDL-C, apoB, VLDL, TG, blood pressure) and liver enzymes. CT-868 was well tolerated at 4.0 mg. This favorable tolerability profile supports higher CT-868 dosing in subsequent studies to maximize its weight-loss benefits.

[0997] Example 8: Phase 2 trial of CT-868, a novel dual GLP-1 and GIP receptor modulator, in overweight and obese adults with type 1 diabetes mellitus (T1D).

[0998] The primary objective of this study was to measure patients' glycemic control and weight loss. Treatment lasted 48 weeks or less. Titrations were performed over two to four weeks. Titration doses were 5, 10, and 15 mg or 3, 6, 9, 12, and 15 mg. Figure 55 shows the overall trial design, including arms and doses. The screening period lasted less than three weeks, the self-induction period lasted less than two weeks, and the placebo diet and exercise period lasted two weeks. The self-induction period included instructions on CGM management, insulin dose optimization, and safety aspects of the AID pump function.

[0999] The mandatory placebo, diet, and exercise induction period included baseline assessments of all endpoints and safety, as well as CGM + insulin dose collection.

[1000] Inclusion criteria

[1001] Inclusion criteria include:

[1002] ○ Male or female, aged ≥18 [≤70] years;

[1003] ○ Clinical diagnosis records of T1DM prior to screening must be at least 1 year, and fasting C-peptide levels must be <0.6 ng / mL;

[1004] ○ Body mass index ≥ 27.0 kg / m² 2 ;

[1005] ○HbA1c: Between ≥7.0% and ≤10.5%;

[1006] ○ Use insulin formulations / delivery methods stably for at least 3 months;

[1007] ○ Use an insulin pump (manual or AID) or MDI;

[1008] ○ Have CGM experience and be willing to use the sensors provided by the research several times, each time at 2-week intervals;

[1009] ○ Willing to comply with all prescribed diabetes self-administration tasks, including insulin dose adjustment, blood ketone monitoring, and maintaining current glucagon products;

[1010] ○ Must be a non-smoker;

[1011] ○ Willing to abide by contraceptive rules; and

[1012] ○ Willing to inject sq daily to study the drug.

[1013] assessment

[1014] The evaluation criteria include:

[1015] ○AE and ConMed;

[1016] ○ CGM download and PI discussion;

[1017] ○ Blood and urine collection;

[1018] ○ Questionnaires: DTSQ, ADDQoL, CoEQ, TLFB;

[1019] ○ Weight and body measurements

[1020] ○ Vital signs, ECG, physical examination

[1021] Patient education: diet and exercise

[1022] Figure 56 shows the main study activities before and after randomization of the Phase 2 trial of CT-868 in overweight and obese adults with T1D, including 2-week CGM and insulin dose data collection and patient education on diet and exercise.

[1023] Primary study endpoint:

[1024] Common primary endpoints include:

[1025] ● Change in HbA1c (%) from week FB to week 48; and

[1026] ● Percentage change in body weight from week 48 to week 48

[1027] Key secondary endpoints include:

[1028] ● Changes in HbA1c (%) from FB to weeks 12, 24, and 36;

[1029] ● Achieve a state where HbA1c < 7.0 or 6.5%;

[1030] ● Percentage change in body weight from week 12 to week 36;

[1031] ●Absolute weight change (kg) at weeks 12, 24, 36, and 48;

[1032] ● The percentage of participants reaching >5%, >10%, and >15% in weeks 24 and 48, respectively; and

[1033] ●Percentage change in insulin dose from week 12 to week 24, 36, and 48.

[1034] Other secondary endpoints include:

[1035] ●Changes in daily insulin requirements from week 24 to week 48;

[1036] ● Body composition as assessed by Dexa at weeks 24 and 48;

[1037] ● Changes in the following CGM parameters for FB at weeks 12, 24, 36, and 48:

[1038] ○ Average sensor glucose (mg / dL) and its standard deviation (SD) and %CV;

[1039] ○ Percentage and amount of time spent within the TIR, TBR, and TAR ranges;

[1040] ○ Blood sugar risk index; and

[1041] ○ Composite endpoint;

[1042] ●FB to weeks 12, 24, 36, and 48: LDLC, HDL-C, VLDL, FFA, and ApoB;

[1043] ● Changes in SBP and DBP in FB at weeks 12, 24, 36, and 48; and

[1044] ● Changes in the following glucose metabolism parameters during FB at weeks 12, 24, 36, and 48: FPG, insulin, C-peptide, glucagon, GLP1, GIP, HOMA-IR, and QUICKI.

[1045] Exploratory endpoints include:

[1046] ● Changes in adiponectin, leptin, hsCRP, PAI-1 activity, CTX-1, MCP-1, IL-6, endothelin-1, E-selectin, VCAM1, and ALT at weeks 12, 24, 36, and 48 of FB;

[1047] ● Changes in UACR from FB to weeks 12, 24, 36, and 48;

[1048] ●Changes in the eGFR slope from FB to weeks 24 and 48; and

[1049] ●Changes to the following PRO tools for FB to weeks 12, 24, 36, and 48: DTSQ, ADDQoL and CoEQ, TLFB.

[1050] Figure 57 shows the main study activities before and after randomization in the Phase 2 trial of CT-868 in overweight and obese adults with T1D, including HbA1c, PRO, CRU visit, blood samples, study drug concentration (approximately 24 hours after administration), and extended subgroup PK sampling.

[1051] Lessons learned from CT-868-004 regarding improvements to follow-up studies include:

[1052] ● Includes the full range of insulin pumps, including the AID pump;

[1053] ● When developing inclusion criteria for I / E, clarity and accuracy are essential to eliminate ambiguity and confusion;

[1054] ●Establish a mechanism to notify sponsors and allow for intervention in participant selection prior to randomization;

[1055] ● The education and research center helps patients understand common GI side effects and how to alleviate and treat them;

[1056] ●Integrate patient education courses on diet and exercise, CGM, and insulin pump function;

[1057] ● Limit CGM data collection to a two-week period at baseline, mid-study, and end of the study;

[1058] ●Reduce the burden of data collection on subjects by using systems that automatically input data (such as blood glucose meters, CGMs, insulin pumps); and

[1059] ● We used the monitoring digital platform capacity from Roche Diabetes Care for our study's data acquisition requirements.

[1060] Example 9: Stability of CT-868 Injection

[1061] CT-868 Injection 15 mg / mL and CT-868 Injection Placebo Cartridge

[1062] Table 31 provides the release and stability specifications for the CT-868 Injection 15 mg / mL Pharmaceutical Product (DP) cartridges to ensure quality, strength, identity, purity, and microbiological properties. Except for purity testing, the release and stability testing for the CT-868 Injection placebo DP is the same as for the CT-868 Injection 15 mg / mL DP. The CT-868 placebo DP identity and assay acceptance criteria have been replaced with the absence of active ingredient and 0%, respectively.

[1063] Table 31. Specifications of CT-868 Injection 15 mg / mL and Placebo Capsules

[1064]

[1065] Stability Summary and Conclusions [CT-868 Injection 15 mg / mL]

[1066] The CT-868 drug substance is formulated as a sterile aqueous solution for use in the following three drug products (DP) for administration to SCs: 2-mL CT-868 injection (5 mg / mL, in a glass vial), 1-mL CT-868 injection (5 mg / mL / matched placebo, in a disposable prefilled syringe (PFS), or 3-mL CT-868 injection (15 mg / mL / matched placebo, designed to be assembled into a disposable, multi-dose, single-patient pen syringe (integrated non-replaceable cartridge)).

[1067] As part of the design validation, a stability study under simulated use was completed in a cold storage (5 ± 3°C) to support the use of the CT-868 pen injector for up to 31 days.

[1068] The stability program included one engineered batch of CT-868 active ingredient DP and five GMP batches (three CT-868 active ingredient DPs and two CT-868 placebo DPs) to support shelf-life dates and use during clinical trials. Additional stability data for up to 18 months at long-term storage at 5 ± 3°C were submitted for engineered batch ENG-161-001-001, and additional stability data for up to 18 months, 12 months, and 18 months at long-term storage at 5 ± 3°C were submitted for GMP batches 161-002-001, 161-002-002, and 161-001-001, respectively, as listed in Table 32. Another batch of CT-868 active ingredient DP, 161-002-003, has been included in the stability program, with data available for 6 months at 5 ± 3°C long-term storage and 25 ± 2°C / 60% RH (25°C). Additional data for up to 12 months at 5 ± 3°C long-term storage and 25°C were submitted for CT-868 placebo DP. Based on the 18-month stability data for batches 161-001-001 and 161-002-001 at 5 ± 3°C long-term storage, and the 12-month stability data for batch 161-002-002 at 5 ± 3°C long-term storage, the recommended shelf life for CT-868 injection DP cartridges is 24 months.

[1069] Information regarding batch stability is provided in Table 32. Table 33 provides the stability storage conditions, time points, and tests performed. Subsequently, phenol was added to the planned stability tests, and the results were reported. Acceptance criteria used to evalu...

Claims

1. A method of treating Type 1 diabetes mellitus (T1DM) in a patient in need thereof, the method comprising administering to the patient in need thereof by subcutaneous injection a compound comprising the following structure: (Formula VII) (SEQ ID NO: 1), or a pharmaceutically acceptable salt or ester thereof, wherein the compound is administered at a dose of 1 mg to 20 mg that is independent of body weight.

2. A method of treating Type 2 diabetes mellitus (T2DM) in a patient in need thereof, the method comprising administering to the patient in need thereof by subcutaneous injection a compound comprising the following structure: (Formula VII) (SEQ ID NO: 1), or a pharmaceutically acceptable salt or ester thereof, wherein the compound is administered at a dose of 1 mg to 20 mg that is independent of body weight.

3. The method of claim 1 or 2, wherein the method is used to: improve glycemic control in the patient, increase insulin sensitivity in the patient, increase insulin-independent glucose disposal in the patient, reduce the patient’s need for therapeutic insulin, relieve hypertension in the patient, and / or reduce atherogenic lipids in the patient.

4. The method of any one of claims 1 to 3, wherein the patient is overweight or obese and the method reduces the patient’s body weight.

5. A method of weight management in a patient in need thereof, the method comprising administering to the patient in need thereof by subcutaneous injection a compound comprising the following structure: (Formula VII) (SEQ ID NO: 1), or a pharmaceutically acceptable salt or ester thereof, wherein the compound is administered at a dose of 1 mg to 20 mg that is independent of body weight.

6. The method of claim 5, wherein the patient has Type 1 or Type 2 diabetes mellitus.

7. The method of any one of claims 1 to 6, wherein the administration of the compound is adjunctive to one or more additional therapies.

8. The method of claim 7, wherein the one or more additional therapies comprise insulin therapy, dietary therapy, exercise therapy, hypertension therapy, and / or lipid-lowering therapy.

9. The method of any one of claims 1 to 8, wherein the step of administering is repeated at intervals of one to seven days.

10. The method of any one of claims 1 to 8, wherein the step of administering is repeated once per day.

11. The method of any one of the preceding claims, wherein the compound is administered at a dose that agonizes both GLP-1 receptors and GIP receptors. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 12. The method of any one of the preceding claims, wherein the dose is about 1.5 mg, 1.8 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

13. The method of any one of the preceding claims, wherein the dose is 1.77 mg, 3.25 mg, or 4 mg.

14. The method of any one of the preceding claims, wherein the dose is 1.1 mg, 1.8 mg, 2.6 mg, 3.3 mg, 4.1 mg, 5.2 mg, or 6.6 mg.

15. The method of any one of the preceding claims, wherein the patient has a BMI of 25 kg / m 2 or higher.

16. The method of any one of the preceding claims, wherein the compound is administered to the patient in a pharmaceutical composition comprising: the compound at a concentration of 15 mg / mL, disodium phosphate heptahydrate / sodium phosphate monobasic buffer, optionally at a concentration of 20 mM, propylene glycol, and phenol, further optionally wherein the pharmaceutical composition is at pH 7.

0.

17. A pharmaceutical composition comprising: (i) the compound comprising the following structure or a pharmaceutically acceptable salt or ester thereof at a concentration of 15 mg / mL: (Formula VII) (SEQ ID NO: 1), (ii) disodium phosphate heptahydrate / sodium phosphate monobasic buffer, optionally at a concentration of 20 mM, (iii) propylene glycol, and (iv) phenol, further optionally wherein the pharmaceutical composition is at pH 7.

0.

18. A compound comprising the following structure or a pharmaceutically acceptable salt or ester thereof or a pharmaceutical composition according to claim 17 for use in a method according to any one of claims 1 to 16: (Formula VII) (SEQ ID NO: 1).

19. Use of a compound comprising the following structure or a pharmaceutically acceptable salt or ester thereof or a pharmaceutical composition according to claim 17 in the manufacture of a medicament for use in a method according to any one of claims 1 to 16: (Formula VII) (SEQ ID NO: 1).

20. An article of manufacture for use in a method according to any one of claims 1 to 16, wherein the article of manufacture comprises one or more units of the dose, optionally a single dose unit.

21. An article of manufacture for use in a method according to any one of claims 1 to 16, wherein the article of manufacture comprises five units of the dose.

22. The article of manufacture for use according to claim 20 or 21, wherein the article of manufacture is a syringe, a pen, or a syringe, optionally wherein the article of manufacture is for single use.

23. The article for use according to any one of claims 20 to 22, wherein the article comprises a needle-based injection system having an integrated non-replaceable 3-mL type 1 glass cartridge and a pharmaceutical composition comprising 3 mL of the agonist at a concentration of 15 mg / mL.

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