Glucose-dependent insulin nutritive polypeptide (GIP)
Glucagon-like peptide-1 (GLP-1) receptor

Core Concept
¹³C and ¹⁵N are naturally occurring stable isotopes that are non-radioactive. Incorporating them into target molecules (e.g., drugs, metabolites) provides a unique "ID tag" or "tracer." This tag behaves almost identically to the original molecule in terms of chemical properties, but its slight mass difference can be precisely identified and distinguished by sensitive instruments like mass spectrometers. This enables researchers to accurately track the fate, transformation, and quantity of target molecules within complex biological systems.

Primary Applications
1. Pharmacokinetic Studies
This is one of the core applications. By administering the isotopically labeled drug to animals or humans, researchers can systematically and accurately study the entire "life cycle" of the drug in the body—its absorption, distribution, metabolism, and excretion (ADME). This helps determine the drug's targeting, concentration profiles in blood and tissues, half-life, and clearance pathways, providing critical data for new drug development and clinical dosing regimen design.
2. Metabolomics and Metabolic Pathway Research
In metabolic studies, the labeled compounds serve as tracers. By tracking the flow and transformation of ¹³C or ¹⁵N atoms within the body's complex metabolic networks (such as glucose, lipid, and amino acid metabolism), the metabolic fate of specific molecules and how drug interventions affect these pathways can be clearly mapped. This is crucial for understanding a drug's mechanism of action and potential side effects.
3. Quantitative Analysis in Biological Samples (as Internal Standards)
A major challenge in quantifying drugs or their metabolites in complex biological samples (e.g., plasma, urine, tissue) using mass spectrometry is sample matrix interference and analytical variability. Adding a known amount of a ¹³C/¹⁵N-labeled analog (as an internal standard) to the sample solves this. Because the labeled standard co-elutes and ionizes almost identically to the analyte but is distinguishable by mass, it undergoes the exact same processing steps. By comparing their signal intensities, the absolute concentration of the analyte can be calculated with high precision, making this the gold-standard quantitative method.
4. Proteomics Research
In protein quantitative analysis, ¹³C/¹⁵N-labeled peptides are a fundamental technical tool. For example, in the "Absolute Quantification" technique, synthesized labeled peptides serve as internal standards with precisely known concentrations. More broadly, in techniques like Stable Isotope Labeling by Amino acids in Cell culture (SILAC), comparing the mass spectrometry signal ratios of labeled versus unlabeled proteins/peptides allows for large-scale, high-throughput study of changes in protein expression under different physiological or pathological states. This is used to discover disease biomarkers or study drug targets.

Technical Summary
In summary, ¹³C and ¹⁵N stable isotope labeling is a powerful platform technology. Its core value lies in tracing and precise quantification. For a peptide drug like Tirzepatide, synthesizing a ¹³C,¹⁵N-labeled version is primarily intended to enable the clearest and most reliable elucidation of the drug molecule's behavior in cutting-edge metabolic, pharmacokinetic, and quantitative analytical research.

[1]. Efficacy and safety of LY3298176, a novel dual GIP and GLP-1 receptor agonist, in patients with type 2 diabetes: a randomised, placebo-controlled and active comparator-controlled phase 2 trial. Lancet. 2018 Nov 17;392(10160):2180-2193.
[2]. Tirzepatide is an imbalanced and biased dual GIP and GLP-1 receptor agonist. JCI Insight. 2020 Sep 3; 5(17): e140532.
[3]. LY3298176, a novel dual GIP and GLP-1 receptor agonist for the treatment of type 2 diabetes mellitus: From discovery to clinical proof of concept. Mol Metab. 2018 Dec:18:3-14.
[4]. Tirzepatide ameliorates spatial learning and memory impairment through modulation of aberrant insulin resistance and inflammation response in diabetic rats. Front Pharmacol. 2023 Aug 28;14:1146960.

Pharmacodynamics
Tilatide is a synthetic peptide with hypoglycemic effects. It exerts its glucose-dependent effects by stimulating insulin secretion in both the first and second phases and reducing glucagon levels. Tilatide has also been shown to delay gastric emptying, reduce fasting and postprandial blood glucose concentrations, decrease food intake, and reduce weight in patients with type 2 diabetes. Tilatide can improve insulin sensitivity. Because the peptide is coupled to the C20 fatty acid moiety at the 20th lysine residue via a hydrophilic linker, the drug is highly bound to albumin in plasma, thus prolonging its half-life. Background: LY3298176 is a novel dual glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist currently under development for the treatment of type 2 diabetes. This study aimed to investigate the efficacy and safety of LY3298176 in patients with poorly controlled type 2 diabetes, compared with placebo or dulaglutide, which selectively stimulates the GLP-1 receptor. Methods: In this double-blind, randomized, phase II study, patients with type 2 diabetes were randomized in a 1:1:1:1:1 ratio to receive once-weekly subcutaneous injections of LY3298176 (1 mg, 5 mg, 10 mg, or 15 mg), dulaglutide (1.5 mg), or placebo for 26 weeks. Grouping was stratified based on baseline glycated hemoglobin A1c (HbA1c), metformin use, and body mass index (BMI). Eligible participants (aged 18–75 years) with type 2 diabetes for at least 6 months (HbA1c 7.0–10.5%, inclusive 7.0% and 10.5%), whose glycemic control was not adequately achieved by diet and exercise or stable metformin therapy alone, and whose body mass index (BMI) was 23–50 kg/m2. The primary efficacy endpoint was the change in HbA1c from baseline to 26 weeks in the modified intention-to-treat (mITT) population (all patients who had received at least one treatment with the study drug and had at least one post-baseline measurement of any outcome measure). Secondary endpoints were measured in the mITT treatment dataset and included changes in HbA1c from baseline to week 12; changes in mean weight, fasting blood glucose, waist circumference, total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides; and changes in the proportion of patients achieving HbA1c targets (≤6.5% and <7.0%) from baseline to week 12 and week 26. The proportion of patients achieving at least 5% and 10% weight loss from baseline to week 26 were also included. This study was registered at ClinicalTrials.gov under accession number NCT03131687. Results: Eligibility for enrollment was assessed for 555 participants between May 24, 2017, and March 28, 2018, of whom 318 were randomized to one of the six treatment groups. Due to two untreated participants, the modified intention-to-treat and safety analysis populations comprised a total of 316 participants. 258 (81.7%) participants completed 26 weeks of treatment, and 283 (89.6%) completed the study. At baseline, the mean age was 57 years (standard deviation 9), the BMI was 32.6 kg/m² (5.9), the duration of diabetes diagnosis was 9 years (6), the HbA1c was 8.1% (1.0), 53% of patients were male and 47% were female. At week 26, the effect of LY3298176 on HbA1c changes was dose-dependent and did not reach a plateau. Compared with placebo, the mean changes in HbA1c from baseline after treatment with LY3298176 were: -1.06% in the 1 mg group, -1.73% in the 5 mg group, -1.89% in the 10 mg group, and -1.94% in the 15 mg group (compared to -0.06% in the placebo group) (posterior mean differences [80% confidence interval] compared with placebo: -1.00% [-1.22 to -0.79] in the 1 mg group, -1.67% [-1.88 to -1.46] in the 5 mg group, -1.83% [-2.04 to -1.61] in the 10 mg group, and -1.89% [-2.11 to -1.67] in the 15 mg group). Compared with dulaglutide (-1.21%), the posterior mean differences (80% confidence set) in HbA1c changes from baseline to 26 weeks with LY3298176 dose were: 0.15% (-0.08 to 0.38) in the 1 mg dose group, -0.52% (-0.72 to -0.31) in the 5 mg dose group, -0.67% (-0.89 to -0.46) in the 10 mg dose group, and -0.73% (-0.95 to -0.52) in the 15 mg dose group. At week 26, among patients treated with LY3298176, 33% to 90% achieved the target HbA1c level below 7.0% (52% in the dulaglutide group and 12% in the placebo group), and 15% to 82% achieved the target HbA1c level of at least 6.5% (39% in the dulaglutide group and 2% in the placebo group). Fasting blood glucose levels in the LY3298176 group ranged from -0.4 mmol/L to -3.4 mmol/L (0.9 mmol/L in the placebo group and -1.2 mmol/L in the dulaglutide group). Mean weight loss in the LY3298176 group ranged from -0.9 kg to -11.3 kg (0.4 kg in the placebo group and -2.7 kg in the dulaglutide group). At week 26, among patients treated with LY3298176, 14% to 71% achieved at least 5% of their weight loss target (22% in the dulaglutide group, 0% in the placebo group), and 6% to 39% achieved at least 10% of their weight loss target (9% in the dulaglutide group, 0% in the placebo group). Waist circumference changes in the LY3298176 group ranged from -2.1 cm to -10.2 cm (-1.3 cm in the placebo group, -2.5 cm in the dulaglutide group). Total cholesterol changes in the LY3298176 group ranged from 0.2 mmol/L to -0.3 mmol/L (0.3 mmol/L in the placebo group, -0.2 mmol/L in the dulaglutide group). There were no significant differences in HDL or LDL cholesterol changes between the LY3298176 and placebo groups. Triglyceride concentrations in the LY3298176 group ranged from 0 mmol/L to -0.8 mmol/L (0.3 mmol/L in the placebo group and -0.3 mmol/L in the dulaglutide group). The 12-week and 26-week results for all secondary endpoints were similar. Of the 316 subjects across the six treatment groups, 13 (4%) experienced 23 serious adverse events. Gastrointestinal events (nausea, diarrhea, and vomiting) were the most common adverse events occurring during treatment. The incidence of gastrointestinal adverse events was dose-related (23.1% in the 1 mg LY3298176 group, 32.7% in the 5 mg LY3298176 group, 51.0% in the 10 mg LY3298176 group, 66.0% in the 15 mg LY3298176 group, 42.6% in the dulaglutide group, and 9.8% in the placebo group); most adverse events were mild to moderate and transient. Decreased appetite was the second most common adverse event (3.8% in the 1 mg LY3298176 group, 20.0% in the 5 mg LY3298176 group, 25.5% in the 10 mg LY3298176 group, 18.9% in the 15 mg LY3298176 group, 5.6% in the dulaglutide group, and 2.0% in the placebo group). No serious hypoglycemic events were reported. One patient in the placebo group died of stage IV lung adenocarcinoma, unrelated to the study treatment. Conclusion: The dual GIP and GLP-1 receptor agonist LY3298176 showed more significant efficacy than dulaglutide in glycemic control and weight loss, with acceptable safety and tolerability. Combined stimulation of GIP and GLP-1 receptors may provide a new treatment option for type 2 diabetes. [1]

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Teratriide (LY3298176) is a dual GIP and GLP-1 receptor agonist being developed for the treatment of type 2 diabetes (T2DM), obesity, and non-alcoholic steatohepatitis. Early clinical trials of T2DM have shown that terazetide is superior to selective GLP-1 receptor agonists in improving clinical efficacy. Therefore, we hypothesize that the combined potency and signal transduction properties of terazetide give it unique pharmacological characteristics that can effectively improve a wide range of metabolic controls. This paper establishes a method for calculating the occupancy of each receptor at the clinically effective dose of this drug. Analysis results showed that tezepatide binds to the GIP receptor more strongly than to the GLP-1 receptor, confirming the imbalance of its mechanism of action. Pharmacological signal transduction studies showed that tezepatide mimics the action of natural GIP on the GIP receptor, but exhibits bias on the GLP-1 receptor, tending to promote cAMP production rather than β-arrestin recruitment, and its ability to drive GLP-1 receptor internalization is also weaker than that of GLP-1. Primary islet experiments showed that β-arrestin1 limited the insulin response of GLP-1 rather than GIP or tezepatide, suggesting that the biased agonist effect of tezepatide enhances insulin secretion. The GIP receptor imbalance, coupled with the unique signal transduction properties of the GLP-1 receptor, may jointly explain the good efficacy of the study drug. [2]

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Objective: To develop a novel dual GIP and GLP-1 receptor agonist, LY3298176, to determine whether the metabolic effects of GIP can enhance the established clinical benefits of selective GLP-1 receptor agonists in type 2 diabetes mellitus (T2DM). Methods: LY3298176 is a fatty acid-modified peptide with dual GIP and GLP-1 receptor agonist activity, designed for once-weekly subcutaneous injection. In vitro, LY3298176 was characterized using cell lines expressing recombinant or endogenous incretin receptors for signal transduction and functional analysis. In vivo, LY3298176 was characterized by mouse body weight, food intake, insulin secretion, and blood glucose profile. A phase I randomized, placebo-controlled, double-blind study was conducted in three parts: first, a single-dose escalation (SAD; dose 0.25–8 mg) study and a 4-week multiple-dose escalation (MAD; dose 0.5–10 mg) study in healthy subjects (HS); followed by a 4-week phase Ib multiple-dose proof-of-concept (POC; dose 0.5–15 mg) study in patients with type 2 diabetes mellitus (T2DM) (ClinicalTrials.gov registration number: NCT02759107). Doses higher than 5 mg were obtained by titration, with dulaglutide (DU) used as a positive control. The primary objective of this study was to investigate the safety and tolerability of LY3298176. Results: LY3298176 activated the GIP and GLP-1 receptor signaling pathways in vitro and, in mice, demonstrated glucose-dependent insulin secretion and improved glucose tolerance by acting on GIP and GLP-1 receptors. Long-term administration of LY3298176 significantly reduced body weight and food intake in mice; these effects were significantly stronger than those of GLP-1 receptor agonists. A total of 142 subjects received at least one dose of LY3298176, dulaglutide, or placebo. Pharmacokinetic studies of LY3298176 were conducted over a wide dose range (0.25–15 mg), and the results supported a once-weekly dosing regimen. In a phase 1b trial in diabetic patients, LY3298176 at doses of 10 mg and 15 mg significantly reduced fasting blood glucose compared to placebo (least square mean [LSM] difference [95% CI]: -49.12 mg/dL [-78.14, -20.12] and -43.15 mg/dL [-73.06, -13.21], respectively). In patients with MAD HS, the LY3298176 1.5 mg, 4.5 mg, and 10 mg dose groups showed significantly greater weight loss than the placebo group (least square mean difference [95% CI]: -1.75 kg [-3.38, -0.12], -5.09 kg [-6.72, -3.46], and -4.61 kg [-6.21, -3.01], respectively). The 10 mg and 15 mg dose groups also showed significant efficacy in patients with type 2 diabetes mellitus (least square mean difference [95% CI]: -2.62 kg [-3.79, -1.45] and -2.07 kg [-3.25, -0.88], respectively). The most common adverse reactions to LY3298176 were gastrointestinal reactions (vomiting, nausea, decreased appetite, diarrhea, and abdominal distension), which occurred in both patients with hepatitis B (HS) and type 2 diabetes mellitus. All adverse reactions were dose-dependent and mild to moderate in severity. Conclusion: Based on these results, the pharmacological properties of LY3298176 have been translated from preclinical studies to clinical studies. LY3298176 has the potential to provide clinically meaningful improvements in glycemic control and weight. These data support further clinical evaluation of LY3298176 for the treatment of type 2 diabetes mellitus and potential obesity. [3]

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Tilatin is a single peptide constructed by integrating GLP-1 activity into the GIP sequence. It is described as an “unbalanced and biased” dual agonist. Unbalanced refers to its higher affinity and potency for GIPR than for GLP-1R. Biased refers to its signal transduction properties on GLP-1R, which, unlike the natural ligand GLP-1, preferentially activates the cAMP pathway rather than recruiting β-arrestin. [2]
Biased agonist effect: Compared to GLP-1, the internalization effect of GLP-1R is weakened. In vitro perfusion experiments of Arrb1βcell-/- mouse islets showed that the absence of β-arrestin1 enhanced the insulin secretion response of GLP-1, but had no effect on GIP or tirzepatide, suggesting that the biased signaling of tirzepatide on GLP-1R may enhance its insulin secretion-promoting effect by avoiding β-arrestin-mediated restriction. [2] The good clinical efficacy of tirzepatide is thought to be due to the following: 1) complete and potent GLP-1R agonism; 2) unbalanced binding that favors GLP-1R, thus allowing for higher doses and potentially better tolerability; 3) biased signaling on GLP-1R may lead to enhanced insulin secretion. [2]
Tirzepatide is under development for the treatment of type 2 diabetes (T2DM), obesity, and non-alcoholic steatohepatitis (NASH). [2]

C219[13C]6H347N47[15N]1NAO68

4842.38 (Sodium salt)

4817.5420

2023788-19-2;13C,15N Tirzepatide;Tirzepatide hydrochloride (LY3298176 hydrochloride)

Solid powder

Tirzepatide internal standard; Labeled tirzepatide;isotope labeled tirzepatide;fluorescently labeled tirzepatide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)