GLP-1 receptor

Dulaglutide (50 nM and 100 nM; 24 h) protects human aortic endothelial cells (HAECs) against oxidative stress caused by low-density lipoprotein (LDL) and inhibits its effects on mitochondria.
Dulaglutide ameliorated ox-LDL-induced oxidative stress and mitochondrial dysfunction.[1]
Dulaglutide suppressed ox-LDL-induced secretion of IL-1β, IL-6, MCP-1, and HMG-1.[1]
Dulaglutide suppressed ox-LDL-induced reduction of cell viability and release of LDH.[1]
Dulaglutide suppressed attachment of THP-1 to HAECs by inhibiting VCAM-1, E-selectin.[1]
Dulaglutide promoted the expression of KLF2 through inhibiting the activation of p53.[1]

Dulaglutide (0, 0.05, 0.5, 1.5, or 5 mg/kg; s.c.; twice week, for 93 weeks) raises the incidence of thyroid C-cell hyperplasia and neoplasia in the rat carcinogenicity study[3]. The tumorigenic potential of dulaglutide was evaluated in rats and transgenic mice. Rats were injected sc twice weekly for 93 weeks with dulaglutide 0, 0.05, 0.5, 1.5, or 5 mg/kg corresponding to 0, 0.5, 7, 20, and 58 times, respectively, the maximum recommended human dose based on plasma area under the curve. Transgenic mice were dosed sc twice weekly with dulaglutide 0, 0.3, 1, or 3 mg/kg for 26 weeks. Dulaglutide effects were limited to the thyroid C-cells. In rats, diffuse C-cell hyperplasia and adenomas were statistically increased at 0.5 mg/kg or greater (P ≤ .01 at 5 mg/kg), and C-cell carcinomas were numerically increased at 5 mg/kg. Focal C-cell hyperplasia was higher compared with controls in females given 0.5, 1.5, and 5 mg/kg. In transgenic mice, no dulaglutide-related C-cell hyperplasia or neoplasia was observed at any dose; however, minimal cytoplasmic hypertrophy of C cells was observed in all dulaglutide groups. Systemic exposures decreased over time in mice, possibly due to an antidrug antibody response. In a 52-week study designed to quantitate C-cell mass and plasma calcitonin responses, rats received twice-weekly sc injections of dulaglutide 0 or 5 mg/kg. Dulaglutide increased focal C-cell hyperplasia; however, quantitative increases in C-cell mass did not occur. Consistent with the lack of morphometric changes in C-cell mass, dulaglutide did not affect the incidence of diffuse C-cell hyperplasia or basal or calcium-stimulated plasma calcitonin, suggesting that diffuse increases in C-cell mass did not occur during the initial 52 weeks of the rat carcinogenicity study [3].

Assessment of reactive oxygen species (ROS) [1]
Intracellular ROS in HAECs was measured using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. HAECs were stimulated with ox-LDL (100 μg/ml) in the presence or absence of dulaglutide at the concentrations of 50 and 100 nM for 24 h and washed 3 times with PBS. Cells were then loaded with 5 μM DCFH-DA for 15 min in darkness at 37 °C. Fluorescent signals were visualized using a Zeiss fluorescence microscope. Intracellular ROS was calculated using Image J software. Briefly, regions of interest (ROI) were defined in the fluorescent image, and the average number of cells present in the defined ROI was counted. The integrated density value (IDV) in the ROI was calculated and divided by the average number of cells. The results were used to represent the average level of intracellular ROS.

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Reduced glutathione (GSH) assay [1]
Intracellular levels of reduced glutathione (GSH) in HAECs were determined using a fluorometric assay. HAECs were stimulated with ox-LDL (100 μg/ml) in the presence or absence of dulaglutide at the concentrations of 50 and 100 nM for 24 h. Cells were then collected in ice cold 5% meta-phosphoric acid (MPA). Cells were then sonicated and centrifuged at 14,000 × g for 5 min. Supernatant was incubated with an equal volume of OPAME in methanol and borate buffer and incubated for 15 min at RT. Fluorescent signals were recorded at 350 nm excitation and 420 nm emission.

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Determination of mitochondrial membrane potential (MMP) [1]
Intracellular levels of MMP in HAECs were determined using tetramethylrhodamine methyl ester (TMRM) staining. HAECs were stimulated with ox-LDL (100 μg/ml) in the presence or absence of dulaglutide at the concentrations of 50 and 100 nM for 24 h. Cells were then washed 3 times with PBS and probed with 20 nmol/L TMRM. After incubation for 1 h at 37 °C, cells were washed 3 times and fluorescent signals were visualized using a Zeiss fluorescence microscope.

Cell Line: Human aortic endothelial cells (HAECs)
Concentration: 50 nM, 100 nM
Incubation Time: 24 hous
Result: Suppressed ox-LDL-induced reduction of cell viability and release of lactate dehydrogenase (LDH).

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Cellular adhesion assay [1]
HAECs were cultured to 80% confluence. Cells were stimulated with ox-LDL (100 μg/ml) in the presence or absence of dulaglutide at the concentrations of 50 and 100 nM for 24 h. A total of 2 × 105 THP-1 monocytes were stained with calcein acetoxymethyl ester for 30 min and incubated with HAECs for 2 h. Unattached THP-1 cells were washed away and attached THP-1 cells were visualized using a fluorescence microscope.

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Assessment of cell viability [1]
HAECs were seeded into 6-well plates and stimulated with ox-LDL (100 μg/ml) in the presence or absence of dulaglutide at the concentrations of 50 and 100 nM for 24 h. After 3 gentle washes, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) in phenol-free red medium at the final concentration of 5 mg/ml was added and incubated for 4 h at 37 °C in darkness, the product was dissolved with dimethyl sulfoxide (DMSO). OD value at 570 nm was measured to reflect the viability percentage.

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Measurement of lactate dehydrogenase (LDH) release [1]
HAECs were seeded into 6-well plates and stimulated with ox-LDL (100 μg/ml) in the presence or absence of dulaglutide at the concentrations of 50 and 100 nM for 24 h. 50 μl supernatant was collected and mixed with 50 μl of the LDH assay reagent in a fresh 96-well plate. After incubation for 30 min in darkness, the reaction was stopped with 50 μl stop buffer. OD value at 490 nm was recorded to assess LDH release.

Rats and Transgenic mice
0, 0.05, 0.5, 1.5, or 5 mg/kg; 0, 0.3, 1, or 3 mg/kg
SC, twice week, for 93 weeks; SC, twice week, for 26 weeks

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Plasma dulaglutide toxicokinetics [3]
Twenty-six-week mouse study [3]
Blood samples were collected on days 1, 85, and 176 of the dosing phase. Blood was collected from three animals per sex per group per time point. Blood was drawn before dosing and 4, 12, 24, 48, and 96 hours after dosing on days 1 and 176 and before dosing and 24 hours after dosing on day 85. Plasma samples were analyzed for dulaglutide concentrations using a validated ELISA method.

Microtiter plates were coated with mouse antihuman IgG (Fc) antibody. Dulaglutide standards, controls, and samples were prepared in mouse plasma. After the preparation, the samples were incubated on the coated plates for approximately 1.5 hours at room temperature. The dulaglutide complex on the plate was bound with a guinea pig anti-GLP-1 active antiserum and then detected using a goat anti-guinea pig IgG-horseradish peroxidase with tetramethyl benzidine substrate. The standard curve ranged from 0.25 to 125 ng/mL, with 2.0 and 30 ng/mL being the lower and upper limits of quantitation, respectively.

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Ninety-three-week carcinogenicity per 52-week rat thyroid C-cell studies [3]
For the 93-week carcinogenicity study, blood was drawn (three rats per sex per group per time point) before dosing and 4, 12, 24, 48, and 96 hours after dosing on days 1 and 24 and before dosing and 24 hours after dosing at weeks 13, 26, 78, and 93. For the 52-week morphometry study, blood samples were collected (three rats per sex per group per time point) approximately 6 days after dosing at the time the animals were killed on days 94, 185, 276, and 374. Plasma samples for both studies were analyzed for dulaglutide concentrations using a validated ELISA method.

Microtiter plates were coated with monoclonal anti-GLP-1 antibody. Dulaglutide standards, controls, and samples were prepared in rat plasma. After the preparation, the samples were incubated on the coated plates for approximately 1.5 hours at room temperature. The dulaglutide complex on the plate was detected using a mouse antihuman IgG4-horseradish peroxidase antibody (Southern Biotech) with tetramethyl benzidine substrate. The standard curve ranged from 0.39 to 50 ng/mL, with 0.80 and 40 ng/mL being the lower and upper limits of quantitation, respectively.

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Fifty-two-week rat thyroid C-cell study [3]
Microtiter plates were coated with mouse antihuman IgG (Fc) antibody. Dulaglutide standards, controls, and samples were prepared in rat plasma. After the preparation, the samples were incubated on the coated plates for approximately 1 hour at room temperature. The dulaglutide complex on the plate was bound with a mouse IgG2a kappa anti-GLP-1 antibody and then detected using a goat antimouse IgG2a-horseradish peroxidase with tetramethyl benzidine substrate. The standard curve ranged from 0.40 to 100 ng/mL, with 0.80 and 40 ng/mL being the lower and upper limits of quantitation, respectively.

26-week mouse study [3]
Life stages [3]
Dulaglutide administration had no effect on survival. No clinical symptoms associated with dulaglutide were observed. Compared with the control group, male mice in the dulaglutide treatment group generally had a lower mean food consumption, resulting in a corresponding decrease in mean body weight (Supplementary Figures 1-4). Similar but generally less significant changes in food consumption were also observed in female mice in the treatment group, and they did not lead to a slowdown in growth.
Plasma dulaglutide toxicokinetics [3]
Peak dulaglutide plasma concentrations were observed 4 to 12 hours after administration. Based on the area under the curve (AUC) and peak plasma concentration (Cmax) values, dulaglutide exposure increased with increasing dose, but at day 176, the proportion of the dose increase was generally lower than that of the dose increase. Systemic dulaglutide exposure was similar in males and females (Table 1). The plasma concentration of dulaglutide on day 85 (not shown) and the Cmax and AUC values on day 176 were generally 0.5 times lower or less than the corresponding values on day 1 (Table 1). The decrease in exposure during the study period may be due to the formation of dulaglutide anti-ADA; however, no specific assays for dulaglutide ADA were performed. Anatomical pathology [3] In both the control and treatment groups, no dulaglutide-related effects were detected in thyroid sections stained with routine hematoxylin and eosin, nor was there any evidence of thyroid C-cell hyperplasia or tumors. In calcitonin-stained thyroid sections, increased cytoplasmic volume of C cells was detected in all groups treated with the investigational drug and recorded as C-cell cytoplasmic hypertrophy/calcitonin staining enhancement (Table 2). The degree was mild, and no significant increase in the number of thyroid C cells was observed in mice treated with dulaglutide. Increased mortality and increased incidence of bronchioloalveolar adenomas and carcinomas, squamous cell papillomas and carcinomas, hemangiomas and angiosarcomas were observed in MNU-positive control animals, reflecting the typical response of this strain of mice after MNU administration.

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93-week rat carcinogenicity study [3]
In vivo phase [3]
Due to low survival rates in control animals (<20 animals per sex), the study was terminated at week 93 with the consent of the FDA Carcinogenicity Assessment Executive Committee. However, a sufficient number of animals survived to week 80 to adequately assess carcinogenicity. No specific cause of death associated with dulaglutide was identified. Survival was improved in all dulaglutide treatment groups, regardless of sex, and was statistically significant in the 0.5 mg/kg and 5 mg/kg male groups and the 0.05 mg/kg, 0.5 mg/kg and 1.5 mg/kg female groups (P ≤ 0.05) (Table 3). Compared with the control group, the decrease in mean body weight and mean food consumption induced by the drug was generally dose-dependent (Supplementary Figures 5-8). Plasma dulaglutide toxicokinetics [3] The mean time to peak plasma concentration of dulaglutide was observed 12 hours after administration on day 1 and ranged from 12 to 48 hours at week 52. Based on AUC and Cmax values, dulaglutide exposure increased with increasing dose from 0.05 mg/kg to 5 mg/kg at all evaluation dates (Table 1). Peak concentrations (Cmax) and AUC0-96h values at day 1 and week 52 were approximately dose-dependent. Drug exposure was similar in male and female rats at all dose groups and evaluation dates. Steady state appeared to be reached at week 13 and drug exposure was maintained until week 93. Cmax and AUC0-96h values at week 52 were higher than those at day 1, suggesting that dulaglutide may have accumulated in rat plasma after multiple administrations over week 92. Furthermore, at the end of week 93 of the study, the mean plasma dulaglutide concentrations in each toxicity group were substantially similar to the mean concentrations in the corresponding toxicokinetic groups.
Anatomical Pathology[3]
At dulaglutide doses of 0.5, 1.5, and 5 mg/kg, the incidence of thyroid C-cell adenomas in both males and females was significantly higher than in the control group (P ≤ 0.05) (Table 4). The incidence of thyroid C-cell carcinoma was numerically higher in the 5 mg/kg dose group, but did not reach statistical significance. The incidence of diffuse C-cell hyperplasia was significantly higher in males treated with dulaglutide 1.5 mg/kg and 5 mg/kg, and in females treated with dulaglutide 5 mg/kg, than in the control group (P ≤ 0.05). Compared with the control group, female rats treated with dulaglutide 0.5, 1.5, and 5 mg/kg had a higher incidence of focal C-cell hyperplasia. The incidence of focal C-cell hyperplasia was low in male rats treated with dulaglutide at 0.5, 1.5, or 5 mg/kg, and the incidence of focal C-cell hyperplasia also trended downward after an increase in the incidence of thyroid C-cell adenomas (Supplementary Figures 9 and 10).

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52-week rat thyroid C-cell study[3]
In vivo study phase[3]
No deaths or clinical symptoms associated with dulaglutide were observed. Three control rats died within minutes of receiving calcium chloride (CaCl2) on days 365 or 372 of the administration phase. In addition, three control rats and two rats treated with 5 mg/kg dulaglutide died at unplanned intervals. One rat receiving the 5 mg/kg dose died of a hematopoietic tumor; the cause of death of the remaining rats was unknown.
Plasma dulaglutide toxicokinetics[3]
At the midpoints of animal sacrifice (6 days after the last dose, i.e., days 94, 185, 276, and 374), plasma dulaglutide concentrations generally decreased as the study progressed, with considerable variability at day 185. At day 374 (13 days after the last dose), plasma dulaglutide concentrations were below the limit of quantitation in 18 of the 19 animals that received a dose of 5 mg/kg. One of these animals had a plasma concentration of 0.91 ng/mL. The decrease in dulaglutide exposure during the study period may be due to antibody formation; however, no specific determination of dulaglutide anti-antibody (ADA) was performed. Although dulaglutide plasma concentrations generally decreased during the study period, pharmacologically relevant effects [reduced food intake and reduced weight gain (Supplementary Figures 11 and 12)] were observed throughout the dosing phase, indicating that active dulaglutide remained present in the test animals throughout the study period. Anatomical Pathology [3]
In all autopsies, the mean final body weight of the test animals was reduced (to 83%–88% of the mean of the control group). The decrease in the mean absolute weight of the thyroid/parathyroid gland (to 81% of the mean of the control group) and the ratio of the thyroid/parathyroid gland to brain weight (to 84% of the mean of the control group) in the test animals was considered to be secondary to the decrease in body weight. The only microscopic findings considered to be associated with dulaglutide were an increased incidence and severity of focal or multifocal C-cell hypertrophy/hyperplasia in the thyroid gland of the treatment group rats after 52 weeks of administration (Table 5). At 26 or 39 weeks of administration, one or two animals in the treatment group developed focal or multifocal C-cell hypertrophy/hyperplasia, which was considered to be unrelated to dulaglutide; this lesion was a spontaneous background change in the rats, as evidenced by the lesion in a control animal after 39 weeks of administration. Focal and multifocal C-cell hypertrophy/proliferation is characterized by well-differentiated C-cell nodules of varying sizes, typically with an increased cytoplasmic volume (hypertrophy). The C-cells in these lesions are generally less calcitonin-positive than those in the surrounding C-cells. C-cell tumors were found only in animals examined after 52 weeks of drug administration; their incidence was similarly low in both control and treatment groups, and they were considered unrelated to dulaglutide.

[1]. Glucagon-like peptide-1 receptor agonist dulaglutide prevents ox-LDL-induced adhesion of monocytes to human endothelial cells: An implication in the treatment of atherosclerosis. Mol Immunol. 2019 Dec;116:73-79.

[2]. Dulaglutide and cardiovascular outcomes in type 2 diabetes (REWIND): a double-blind, randomised placebo-controlled trial. Lancet . 2019 Jul 13;394(10193):121-130.

[3]. Chronic Toxicity and Carcinogenicity Studies of the Long-Acting GLP-1 Receptor AgonistDulaglutide in Rodents. Endocrinology. 2015 Jul;156(7):2417-28.

Atherosclerosis is a common complication of type 2 diabetes and one of the leading causes of death worldwide. Oxidized low-density lipoprotein (ox-LDL) promotes the development of atherosclerosis by inducing oxidative stress, mitochondrial dysfunction, and the expression of pro-inflammatory cytokines and chemokines (including interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein 1 (MCP-1)), adhesion molecules (including vascular cell adhesion molecule 1 (VCAM-1) and E-selectin), and downregulating the expression of the Krüppel-like factor 2 (KLF2) transcription factor. Notably, ox-LDL can induce THP-1 monocytes to adhere to endothelial cells. In this study, we demonstrated for the first time that the specific glucagon-like peptide-1 receptor (GLP-1R) agonist dulaglutide can prevent the atherosclerotic effects of ox-LDL by blocking the inhibition of KLF2 in human aortic endothelial cells by p53 protein. KLF2 has been shown to play an important role in protecting vascular endothelial cells from ox-LDL and oscillatory shear stress damage. Therefore, therapies that can modulate the KLF2 signaling pathway may be an attractive and effective treatment option for the prevention of atherosclerosis. [1]

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Background: Three different glucagon-like peptide-1 (GLP-1) receptor agonists can reduce the incidence of cardiovascular events in patients with type 2 diabetes who have high glycated hemoglobin A1c (HbA1c) concentrations and high cardiovascular risk. We evaluated the effect of adding the GLP-1 receptor agonist dulaglutide to existing glucose-lowering regimens on major adverse cardiovascular events in patients with type 2 diabetes (regardless of prior history of cardiovascular disease and with varying glycemic control levels). Methods: This multicenter, randomized, double-blind, placebo-controlled trial was conducted at 371 research centers in 24 countries. Men and women aged at least 50 years with type 2 diabetes and a history of cardiovascular events or cardiovascular risk factors were randomly assigned in a 1:1 ratio to either dulaglutide (1.5 mg) subcutaneous injection (once weekly) or placebo. Randomization was performed using a computer-generated randomization code and stratified by research center. All investigators and participants were unaware of their treatment allocation. Participants were followed up at least every 6 months to assess for new cardiovascular events and other serious clinical outcomes. The primary endpoint was a composite endpoint of first nonfatal myocardial infarction, nonfatal stroke, or death from cardiovascular causes (including unexplained death), assessed in the intention-to-treat population. This study was registered at ClinicalTrials.gov under registration number NCT01394952. Results: Between August 18, 2011 and August 14, 2013, a total of 9,901 participants (mean age 66.2 years [standard deviation 6.5], median glycated hemoglobin 7.2% [interquartile range 6.6-8.1], of whom 4,589 were female [46.3%]) were enrolled and randomly assigned to the dulaglutide group (n=4,949) or the placebo group (n=4,952). The median follow-up time was 5.4 years (interquartile range 5.1–5.9 years). In the dulaglutide group, 594 subjects (12.0%) experienced the primary composite endpoint event, with an incidence of 2.4 per 100 person-years; in the placebo group, 663 subjects (13.4%) experienced the primary composite endpoint event, with an incidence of 2.7 per 100 person-years (hazard ratio [HR] 0.88, 95% confidence interval [CI] 0.79–0.99; p=0.026). There was no significant difference in all-cause mortality between the two groups (dulaglutide group 536 cases [10.8%] vs placebo group 592 cases [12.0%]; HR 0.90, 95% CI 0.80–1.01; p=0.067). During the follow-up period, 2347 (47.4%) of the subjects receiving dulaglutide treatment reported gastrointestinal adverse events, compared with 1687 (34.1%) of the subjects receiving placebo treatment (p<0.0001). Conclusion: Dulaglutide can be used to treat glycemic control in middle-aged and elderly patients with type 2 diabetes mellitus who have a history of cardiovascular disease or cardiovascular risk factors. [2]

3313.597

923950-08-7

GLP-1 moiety from Dulaglutide; 1197810-60-8

171042928

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG

Typically exists as solid at room temperature

1.4±0.1 g/cm3

1.706

3.81

48

53

109

235

7740

29

CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)NCC(=O)NCC(=O)O)NC(=O)[C@H](CC3=CC=CC=C3)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC4=CC=C(C=C4)O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC5=CC=CC=C5)NC(=O)[C@H]([C@@H](C)O)NC(=O)CNC(=O)[C@H](CCC(=O)O)NC(=O)CNC(=O)[C@H](CC6=CNC=N6)N

HPNPLWNTQBSMAJ-FBXRENMFSA-N

InChI=1S/C149H221N37O49/c1-16-76(10)121(147(233)164-79(13)126(212)172-103(58-85-61-155-90-34-24-23-33-88(85)90)137(223)174-99(54-73(4)5)138(224)183-119(74(6)7)145(231)171-91(35-25-27-51-150)128(214)159-64-110(195)156-63-109(194)157-67-118(208)209)185-139(225)101(55-82-29-19-17-20-30-82)175-134(220)97(45-50-116(204)205)168-131(217)92(36-26-28-52-151)166-125(211)78(12)162-124(210)77(11)163-130(216)94(41-46-108(153)193)167-132(218)95(43-48-114(200)201)169-133(219)96(44-49-115(202)203)170-135(221)98(53-72(2)3)173-136(222)100(57-84-37-39-87(192)40-38-84)176-142(228)105(68-187)179-144(230)107(70-189)180-146(232)120(75(8)9)184-141(227)104(60-117(206)207)177-143(229)106(69-188)181-149(235)123(81(15)191)186-140(226)102(56-83-31-21-18-22-32-83)178-148(234)122(80(14)190)182-112(197)66-160-129(215)93(42-47-113(198)199)165-111(196)65-158-127(213)89(152)59-86-62-154-71-161-86/h17-24,29-34,37-40,61-62,71-81,89,91-107,119-123,155,187-192H,16,25-28,35-36,41-60,63-70,150-152H2,1-15H3,(H2,153,193)(H,154,161)(H,156,195)(H,157,194)(H,158,213)(H,159,214)(H,160,215)(H,162,210)(H,163,216)(H,164,233)(H,165,196)(H,166,211)(H,167,218)(H,168,217)(H,169,219)(H,170,221)(H,171,231)(H,172,212)(H,173,222)(H,174,223)(H,175,220)(H,176,228)(H,177,229)(H,178,234)(H,179,230)(H,180,232)(H,181,235)(H,182,197)(H,183,224)(H,184,227)(H,185,225)(H,186,226)(H,198,199)(H,200,201)(H,202,203)(H,204,205)(H,206,207)(H,208,209)/t76-,77-,78-,79-,80+,81+,89-,91-,92-,93-,94-,95-,96-,97-,98-,99-,100-,101-,102-,103-,104-,105-,106-,107-,119-,120-,121-,122-,123-/m0/s1

(4S)-5-[[2-[[(2S,3R)-1-[[(2S)-1-[[(2S,3R)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[2-[[2-(carboxymethylamino)-2-oxoethyl]amino]-2-oxoethyl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-carboxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-2-oxoethyl]amino]-4-[[2-[[(2S)-2-amino-3-(1H-imidazol-4-yl)propanoyl]amino]acetyl]amino]-5-oxopentanoic acid

Dulaglutide; GLP-1 moiety from Dulaglutide; 923950-08-7; Dulaglutide; 1197810-60-8; HPNPLWNTQBSMAJ-FBXRENMFSA-N; LY2189265

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: Please refer to the "Guidelines for Dissolving Peptides" section in the 4th page of the "Instructions for use" file (upper-right section of this webpage) for how to dissolve peptides.

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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.)