Glucagon-like peptide-1 receptor (GLP-1R)

Orforglipron is an incretin produced by small intestine L cells as nutrients travel through the digestive tract and glucose is given via the GLP-1 receptor. Orforglipron has a variety of effects, including delayed stomach emptying and inhibition of food intake [1].

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 In this study, researchers report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over β-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein–coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule[2].

Orforglipron hemicalcium hydrate (plasma concentration 0.94-4.8 nM, intravenous injection for 30 minutes; or 0.05-0.1 mg/mL, oral gavage, 5 days) dose-dependently inhibited food intake, promoted insulin secretion and lowered blood glucose in cynomolgus monkeys in the animal model [1]. Orforglipron hemicalcium hydrate (0.05-1.35 mg/kg, oral gavage) reached Cmax 2 hours after administration, and the increase in plasma drug exposure was roughly proportional to the increase in dose, indicating that Orforglipron hemicalcium hydrate was absorbed in the gastrointestinal tract in a dose-dependent manner [1]. Pharmacokinetic analysis of Orforglipron hemicalcium hydrate in cynomolgus monkeys[1] route Dose (mg/kg) Tmax (h) Cmax (ng/mL) AUC0-24h (ng·h/mL) ig 0.05 2.0 4.78 23.7 ig 0.15 2.0 20.7 135 ig 0.45 2.0 32.0 208 ig 1.35 2.0 148 1040.

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In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.[2]

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Pharmacokinetics and Function in Cynomolgus Monkeys. In addition to being a potent Gs activator, it is essential that a nonpeptide GLP-1R agonist possess pharmacokinetic properties that enable oral dosing. Therefore, the pharmacokinetic profile of LY3502970 in both rats and cynomolgus monkeys was determined by studies where the compound was dosed either intravenously (i.v.) or orally. The elimination half-life following oral administration (T1/2) was 10.4 to 12.4 h in rats (n = 4) and 3.4 to 4.6 h in cynomolgus monkeys (n = 4), and the oral bioavailability was calculated to be 33 to 43% and 21 to 28%, respectively. This contrasts with the 0.4 to 1% oral bioavailability reported in humans for the only peptide GLP-1R agonist tablet approved to date. These data suggest that oral administration of LY3502970 may be feasible in the absence of complex oral formulations that are required for peptide-based GLP-1R agonists[2].

Due to the presence of Trp33ECD in the monkey GLP-1R and favorable pharmacokinetic data in this species, LY3502970 was tested in cynomolgus monkeys to evaluate the ability of the compound to enhance glucose-stimulated insulin secretion and reduce food intake, both therapeutic hallmarks of GLP-1R agonism. Intravenous glucose tolerance tests (IVGTTs) were conducted to assess the ability of LY3502970 to enhance insulin secretion. The compound or exenatide was i.v. administered, followed by continuous infusion to maintain steady-state drug concentrations during the test. Glucose was administered 40 min after the infusion of LY3502970 or exenatide (Fig. 5A). Prior to the glucose administration, neither LY3502970 nor exenatide stimulated insulin secretion. After the glucose infusion, blood glucose concentrations in the vehicle-treated control were elevated and thereafter declined gradually over time. Serum insulin levels were slightly increased and remained elevated for 40 min. Treatment with LY3502970 or exenatide significantly increased the insulin concentrations and lowered blood glucose during the experiment (Fig. 5 B–E). Insulin secretion effected by the high dose of LY3502970 (steady-state concentration: 9.1 ± 0.8 nmol/L; mean ± SEM, n = 7) was comparable to that stimulated by high-dose exenatide (43.0 ± 4.1 pmol/L; mean ± SEM, n = 7). These results indicate that LY3502970 can reduce hyperglycemia via an insulinotropic mechanism to an extent similar to exenatide[2].

In Vitro Pharmacology. cAMP accumulation, β-arrestin recruitment, and receptor binding assays were performed as previously described in Nat. Commun. 7, 13384 (2016) and Nat. Chem. Biol. 16, 1105–1110 (2020).

Animal/Disease Models:cynomolgus monkey model[1]
Doses: 0.9-4.8 nM; or 0.05-0.1 mg/mL
Route of Administration: continuous i.v. administration for 30 minutes until a plasma concentration of 0.9-4.8 nM at steady state; i.g. for 5 days with dose of 0.05-0.1 mg/mL
Experimental Results: Increased insulin secretion and decreased plasma-glucose. Suppressed food intake in a dose-dependent manner.

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Compound Formulation. LY3502970 was prepared in 10% polyethylene glycol 400 (PEG400)/10% propylene glycol (PG)/80% glycine buffer (100 mM glycine, 64 mM NaOH, pH 10) buffer. Exenatide was prepared in phosphate-buffered saline (PBS) containing 0.05 wt/vol% Tween80 buffer. The vehicle solutions without the test articles were used as controls.[2]

Pharmacokinetics. [2]
LY3502970 was administered orally at doses of 0.05, 0.15, or 0.45 mg/kg or i.v. at 0.15 mg/kg to 8-wk-old male rats (n = 4 rats/group) or oral doses of 0.04, 0.12, or 0.36 mg/kg or i.v. at 0.12 mg/kg to 3-y-old male cynomolgus monkeys (n = 4 monkeys/group). Blood was collected predose and 30 min and 1, 2, 3, 4, 6, 8, 12, 16, and 24 h after administration in orally dosing group. Blood samples were also collected predose and 2, 10, and 30 min and 1, 2, 4, 8, 12, 16, and 24 h after i.v. administration. Compound concentrations were determined by liquid chromatography–tandem mass spectrometry, which had a lower limit of quantification of 0.1 ng/mL. Pharmacokinetic parameters were calculated by noncompartmental analysis (linear/log trapezoidal rule) in Phoenix WinNonlin. Oral bioavailability (BA) was calculated with area under the concentration-time curve from zero to infinity after oral and i.v. administration by BA (%) = AUCinf, by mouth, orally (p.o.)/AUCinf, i.v. × 100.[2]

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Glucose Tolerance Tests. [2]
Mice fasted overnight were orally dosed with vehicle or LY3502970, followed 5 h later by an intraperitoneal injection of glucose (2 g/kg). Blood glucose concentrations were measured over time up to 120 min after glucose administration using glucometers. Data were used to calculate the area under the curve (AUC) (n = 5 mice/group). Male cynomolgus monkeys (3.9 to 7.5 kg) were administered atropine sulfate i.v. (0.5 mg Tanabe, 0.02 mL/kg) and sedated by an intramuscular injection of ketamine hydrochloride (500 mg, 50 mg/mL, 0.2 mL/kg). Animals were then anesthetized by inhalation of isoflurane (Isoflu, 0.5 to 2.0%) using a ventilator. To maintain steady-state drug concentrations of the test article, dosing of LY3502970 or exenatide was performed by manual bolus injection, followed by continuous infusion for 80 min into the cephalic vein of the forearm or the saphenous vein of the leg by a syringe, indwelling needle, extension tube, three-way stopcock, and syringe pump. Low and high doses were 1,800 and 5,400 ng/kg, respectively, for LY3502970 and 4.2 and 13.4 ng/kg for exenatide. Dosing volumes were 2 mL/kg for the bolus administration, and the infusion rates for low- and high-dose LY3502970 were 1,280 and 3,840 ng⋅kg−1⋅h−1 and were 6.5 and 21.8 ng⋅kg−1⋅h−1 for low- and high-dose exenatide. Infusion volume was 2.7 mL/kg at a speed of 2 mL⋅kg−1⋅h−1. Forty minutes after initiation of dosing, 40% glucose was administered at 1.25 mL⋅kg−1⋅min−1 via the cephalic or saphenous vein. Blood was collected from the femoral vein 5 min before and after dosing and then at 5, 10, 15, 20, 30, and 40 min following administration of 40% glucose. The studies were conducted at intervals of 7 or 24 d (days 8, 15, 22, 29, 36, and 60) using a 7 × 6 cross-over design.[2]

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Food Consumption Studies. [2]
Eight male cynomolgus monkeys (7.5 to 9.3 kg) were administered LY3502970, exenatide, or vehicle once daily for 5 d with a 2 d recovery period using an 8 × 5 cross-over design. Food consumption during the 90 min period following presentation of food was measured in animals previously administered LY3502970, exenatide, or vehicle as follows: 1) LY3502970 at 0.05 or 0.1 mg/kg by oral administration 180 min before feeding, 2) exenatide at 0.3 or 0.6 µg/kg by s.c. injection 30 min before feeding, or 3) the matched vehicle administered at the appropriate time.

[1]. Pyrazolopyridine derivative having glp-1 receptor agonist effect. WO2018056453A1.

[2]. Structural basis for GLP-1 receptor activation by LY3502970, an orally active nonpeptide agonist. Proc Natl Acad Sci U S A. 2020;117(47):29959-29967.

Since peptide-based GLP-1R agonists exhibit an anorexigenic effect as part of their overall ability to improve metabolic control, LY3502970 was orally administered to monkeys to examine the ability of the compound to reduce feeding. Following LY3502970 or exenatide treatment, food consumption was measured for 90 min. For these studies, LY3502970 was administered orally 180 min before feeding, and exenatide was s.c. injected 30 min prior to food availability, in line with the time of maximum concentration observed (Tmax) in monkey pharmacokinetic studies. Dosing was conducted once daily for 5 d with a 2 d recovery period. LY3502970 at 0.05 and 0.1 mg/kg decreased food consumption from the first to the fifth days of dosing in a dose-dependent manner (Fig. 5F), similar to that observed for 0.3 and 0.6 µg/kg of exenatide (Fig. 5G). The mean LY3502970 and exenatide concentrations which decreased food consumption were 8.3 ± 0.8 nmol/L and 83.1 ± 4.5 pmol/L, respectively (mean ± SEM, n = 8). These results indicate that orally dosed LY3502970 can achieve a reduction of food intake similar to the injectable GLP-1R agonist, exenatide. Taken together, LY3502970 displays a preclinical pharmacodynamic profile similar to marketed peptide-based GLP-1R agonists and possesses pharmacokinetic properties compatible with oral dosing in humans. Consequently, LY3502970 is currently being evaluated in early stage clinical trials for its potential as an antidiabetic agent (identifier, NCT04426474).[2]

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For the treatment-regimen estimand,4 each dose of orforglipron led to statistically significant A1C reductions. In the key secondary endpoint for body weight, 12 mg and 36 mg doses led to statistically significant reductions.

A1C reduction: 1.2% (3 mg), 1.5% (12 mg), 1.5% (36 mg), 0.4% (placebo)
Percent weight reduction: 4.5% (3 mg), 5.8% (12 mg), 7.6% (36 mg), 1.7% (placebo)
Weight reduction: 4.2 kg (9.3 lbs; 3 mg), 5.2 kg (11.5 lbs; 12 mg), 7.2 kg (15.8 lbs; 36 mg), 1.5 kg (3.4 lbs; placebo)
The overall safety profile of orforglipron in ACHIEVE-1 was consistent with the established GLP-1 class. The most commonly reported adverse events were gastrointestinal-related and generally mild to moderate in severity. The most common adverse events for participants treated with orforglipron (3 mg, 12 mg and 36 mg, respectively) were diarrhea (19%, 21% and 26%) vs. 9% with placebo, nausea (13%, 18% and 16%) vs. 2% with placebo, dyspepsia (10%, 20% and 15%) vs. 7% with placebo, constipation (8%, 17% and 14%) vs. 4% with placebo, and vomiting (5%, 7% and 14%) vs. 1% with placebo. Overall treatment discontinuation rates due to adverse events were 6% (3 mg), 4% (12 mg) and 8% (36 mg) for orforglipron vs. 1% with placebo. No hepatic safety signal was observed.

The ACHIEVE-1 results will be presented at ADA's 85th Scientific Sessions and published in a peer-reviewed journal. More results from the ACHIEVE Phase 3 clinical trial program will be shared later this year, along with findings from the ATTAIN Phase 3 clinical trial program evaluating orforglipron for weight management. Lilly expects to submit orforglipron for weight management to global regulatory agencies by the end of this year, with the submission for the treatment of type 2 diabetes anticipated in 2026.

About orforglipron
Orforglipron (or-for-GLIP-ron) is an investigational, once-daily small molecule (non-peptide) oral glucagon-like peptide-1 receptor agonist that can be taken any time of the day without restrictions on food and water intake.5 Orforglipron was discovered by Chugai Pharmaceutical Co., Ltd. and licensed by Lilly in 2018. Chugai and Lilly published the preclinical pharmacology data of this molecule together.6 Lilly is running Phase 3 studies on orforglipron for the treatment of type 2 diabetes and for weight management in adults with obesity or overweight with at least one weight-related medical problem. It is also being studied as a potential treatment for obstructive sleep apnea and hypertension in adults with obesity.

About ACHIEVE-1 and ACHIEVE clinical trial program
ACHIEVE-1 (NCT05971940) is a Phase 3, 40-week, randomized, double-blind, placebo-controlled trial comparing the efficacy and safety of orforglipron 3 mg, 12 mg and 36 mg as monotherapy to placebo in adults with type 2 diabetes and inadequate glycemic control with diet and exercise alone. The trial randomized 559 participants across the U.S., China, India, Japan and Mexico in 1:1:1:1 ratio to receive either 3 mg, 12 mg or 36 mg orforglipron or placebo. The objective of the study was to demonstrate that orforglipron (3 mg, 12 mg, 36 mg) is superior in A1C reduction from baseline after 40 weeks, compared to placebo, in people with type 2 diabetes who have not taken any anti-diabetic medications for at least 90 days prior to visit 1, and are naïve to insulin therapy. Study participants had a HbA1c between ≥7.0% and ≤9.5% and a BMI of ≥23 kg/m2. All participants in the orforglipron treatment arms started the study at a dose of orforglipron 1 mg once-daily and then increased the dose in a step-wise approach at four-week intervals to their final randomized maintenance dose of 3 mg (via a 1 mg step), 12 mg (via steps at 1 mg, 3 mg and 6 mg) or 36 mg (via steps at 1 mg, 3 mg, 6 mg, 12 mg and 24 mg). Flexible dosing was not permitted.

The ACHIEVE Phase 3 global clinical development program for orforglipron has enrolled more than 6,000 people with type 2 diabetes across five global registrational trials. The program began in 2023 with results anticipated later this year and into 2026.https://investor.lilly.com/news-releases/news-release-details/lillys-oral-glp-1-orforglipron-demonstrated-statistically

C48H50CAF2N10O6

941.05

940.35087

3008544-96-2

2212020-52-3 (free);2415797-61-2 (calcium); 3008544-96-2; 2212021-26-4 (calcium hydrate);

171390963

White to off-white solid powder

2

11

7

67

1950

4

[Ca].FC1C(C)=CC(=CC=1C)N1C(=C2C(CCN([C@H]2C)C(C2=CC3C=C([C@H]4CCOC(C)(C)C4)C=CC=3N2[C@@]2(C3=NOC(N3)=O)C[C@@H]2C)=O)=N1)N1C=CN(C2C=CC3=C(C=NN3C)C=2F)C1=O.O

XMVXKSTUSYLMQM-BJPQXFNBSA-N

InChI=1S/C48H48F2N10O5.Ca.H2O/c1-25-18-32(19-26(2)40(25)49)60-42(58-16-15-57(46(58)63)37-11-10-36-33(41(37)50)24-51-55(36)7)39-28(4)56(14-12-34(39)53-60)43(61)38-21-31-20-29(30-13-17-64-47(5,6)23-30)8-9-35(31)59(38)48(22-27(48)3)44-52-45(62)65-54-44;;/h8-11,15-16,18-21,24,27-28,30H,12-14,17,22-23H2,1-7H3,(H,52,54,62);;1H2/t27-,28-,30-,48-;;/m0../s1

LY3502970 hemicalcium hydrate; GLP-1 receptor agonist 1 hemicalcium hydrate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 25 mg/mL (27.14 mM; with sonication)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)