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.

Twenty-six-week mouse study [3]
In-life phase [3]
Administration of dulaglutide had no effect on survival. No dulaglutide-related clinical signs were observed. Mean food consumption for males generally decreased for dulaglutide-treated mice compared with controls, resulting in correlative decreases in mean body weight (Supplemental Figures 1–4). Similar, but generally less prominent effects on food consumption were observed for the treated females but did not produce reductions in growth.

Plasma dulaglutide toxicokinetics [3]
Times to peak plasma concentration of dulaglutide were observed between 4 and 12 hours after dosing. Exposure to dulaglutide, as assessed by area under the curve (AUC) concentration and peak plasma concentration (Cmax) values, increased with increasing doses but was generally less than proportional with an increasing dose on day 176. Systemic exposure of dulaglutide was similar between males and females (Table 1). Plasma concentrations of dulaglutide on day 85 (not shown) and Cmax and AUC values on day 176 were generally 0.5-fold or less than the corresponding day 1 values (Table 1). The decreases in exposure over the duration of the study are likely due to dulaglutide antidrug antibody (ADA) formation; however, the specific determination of dulaglutide ADA was not conducted.

Anatomic pathology [3]
There were no detectable dulaglutide-related effects and no evidence of thyroid C-cell hyperplasia or neoplasia in the control or treated groups using routine hematoxylin and eosin-stained sections of the thyroid. In sections of thyroid stained with calcitonin, an increased C-cell cytoplasmic volume was detected in all treated groups given the test article and was recorded as C cells, cytoplasmic hypertrophy/increased calcitonin staining (Table 2). The severity was mild, and there was no qualitative increase in the number of thyroid C cells in dulaglutide-treated mice. Increased mortality and increased incidences of bronchiolar alveolar adenoma and carcinoma, squamous cell papilloma and carcinoma, and hemangioma and hemangiosarcoma were observed in the MNU-positive control animals, reflecting a typical response in this strain of mouse after MNU administration.

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Ninety-three-week rat carcinogenicity study [3]
In-life phase [3]
Due to the low survival in the control animals (<20 animals/sex), the study was terminated during week 93 with the agreement of the Executive Carcinogenesis Assessment Committee of the FDA. However, sufficient numbers of animals survived to week 80 to adequately evaluate carcinogenicity. No specific dulaglutide-related cause of death was identified. Survival was numerically increased for both genders in all dulaglutide treatment groups and reached statistical significance (P ≤ .05) in the 0.5- and 5-mg/kg males and in the 0.05-, 0.5-, and 1.5-mg/kg females (Table 3). Compound-related decreases in mean body weight and mean food consumption compared with controls were generally dose dependent (Supplemental Figures 5–8).

Plasma dulaglutide toxicokinetics [3]
Mean times to peak plasma concentration values of dulaglutide were observed at 12 hours after dosing on day 1 and ranged between 12 and 48 hours at week 52. Exposure to dulaglutide, as assessed by AUC and Cmax values, increased with increasing dose from 0.05 to 5 mg/kg on all days evaluated (Table 1). Peak concentration (Cmax) and AUC0–96h values on day 1 and at week 52 were approximately dose proportional. The exposures were similar in male and female rats at all dose groups and days evaluated. Steady state appeared to have been achieved by week 13, and exposure was maintained through week 93. Values for Cmax and AUC0–96h were higher at week 52 than at day 1, indicating possible accumulation of dulaglutide in rat plasma after multiple dosing for 92 weeks. In addition, mean plasma dulaglutide concentrations for the toxicity groups at the time of week 93 study termination were generally similar to the mean concentrations observed for the respective toxicokinetic groups.

Anatomic pathology [3]
The incidence of thyroid C-cell adenoma was significantly (P ≤ .05) increased compared with controls in males and females at the dulaglutide 0.5-, 1.5-, and 5-mg/kg doses (Table 4). Animals given 5 mg/kg had a numerically higher dulaglutide-related incidence of thyroid C-cell carcinoma that did not reach statistical significance. The incidence of diffuse C-cell hyperplasia was significantly (P ≤ .05) higher compared with controls in males administered dulaglutide 1.5 mg/kg and 5 mg/kg and in females administered dulaglutide 5 mg/kg. The incidence of focal C-cell hyperplasia was higher compared with controls in females treated with dulaglutide 0.5, 1.5, and 5 mg/kg. The incidence of focal C-cell hyperplasia was lower in males treated with dulaglutide 0.5, 1.5, or 5 mg/kg, with a negative trend after the positive trend for an increase in the incidence of thyroid C-cell adenomas (Supplemental Figures 9 and 10).

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Fifty-two-week rat thyroid C-cell study [3]
In-life phase [3]
No dulaglutide-related deaths or clinical signs were observed. Three control rats died within several minutes after receiving the CaCl2 dose on day 365 or 372 of the dosing phase. In addition, three control rats and two rats given 5 mg/kg died at unscheduled intervals. The cause of death was a hematopoietic neoplasm in one rat given 5 mg/kg; the cause of death in the other rats was undetermined.

Plasma dulaglutide toxicokinetics [3]
Plasma dulaglutide concentrations at interim times the animals were killed (6 d after the last dose on d 94, 185, 276, and 374) generally decreased as the study progressed, and there was considerable variability in the concentrations of dulaglutide on day 185. Plasma dulaglutide concentrations 13 days after the final dose (day 374) were below the quantifiable limit for 18 of the 19 animals given 5 mg/kg. One animal had a plasma concentration of 0.91 ng/mL. The decreases in exposure to dulaglutide over the duration of the study were likely due to the formation of antibodies; however, specific determination of dulaglutide ADA was not conducted. Although dulaglutide plasma concentrations generally decreased over the study, effects related to the pharmacology of dulaglutide [decreased food consumption and decreased body weight gain (Supplemental Figures 11 and 12)] were observed throughout the dosing phase, indicating that active dulaglutide was present in the treated animals throughout the study.

Anatomical pathology [3]
Mean terminal body weights were decreased in treated animals (83%–88% of control means) at all necropsies. Decreased mean absolute thyroid/parathyroid weight (81% of control mean) and thyroid/parathyroid to brain weight ratio (84% of control mean) in treated animals were considered secondary to the decreased body weights. The only microscopic finding considered to be dulaglutide related was an increased incidence and severity of focal or multifocal C-cell hypertrophy/hyperplasia in the thyroids of treated rats after 52 weeks of dosing (Table 5). Focal or multifocal C-cell hypertrophy/hyperplasia in one or two treated animals after 26 or 39 weeks of dosing was not considered dulaglutide related; this lesion is a spontaneous background change in rats, as evidenced by the occurrence in one control animal after 39 weeks of dosing. Focal and multifocal C-cell hypertrophy/hyperplasias were characterized by variably sized nodules of well-differentiated C cells that often had increased cytoplasmic volume (hypertrophy). C cells in these foci were usually less intensely calcitonin immunopositive than surrounding C cells. C-cell neoplasms were identified only in the animals examined after 52 weeks of dosing, occurred at a similarly low incidence in control and treated animals, and 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 comorbidity of type II diabetes and a leading cause of death worldwide. The presence of oxidized low-density lipoprotein (ox-LDL) drives atherogenesis by inducing oxidative stress, mitochondrial dysfunction, expression of proinflammatory cytokines and chemokines including interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein 1 (MCP-1), adhesion molecules including vascular cellular adhesion molecule 1 (VCAM-1) and E-selectin, and downregulating expression of the Krüppel-like factor 2 (KLF2) transcription factor. Importantly, ox-LDL induced the attachment of THP-1 monocytes to endothelial cells. In the present study, we demonstrate for the first time that the specific glucagon-like peptide 1 receptor (GLP-1R) agonist dulaglutide may prevent these atherosclerotic effects of ox-LDL by preventing suppression of KLF2 by p53 protein in human aortic endothelial cells. KLF2 has been shown to play a major role in protecting vascular endothelial cells from damage induced by ox-LDL and oscillatory shear, and therefore, therapies capable of mediating KLF2 signaling may be an attractive treatment option for preventing the development and progression of atherosclerosis. [1]

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Background: Three different glucagon-like peptide-1 (GLP-1) receptor agonists reduce cardiovascular outcomes in people with type 2 diabetes at high cardiovascular risk with high glycated haemoglobin A1c (HbA1c) concentrations. We assessed the effect of the GLP-1 receptor agonist dulaglutide on major adverse cardiovascular events when added to the existing antihyperglycaemic regimens of individuals with type 2 diabetes with and without previous cardiovascular disease and a wide range of glycaemic control. Methods: This multicentre, randomised, double-blind, placebo-controlled trial was done at 371 sites in 24 countries. Men and women aged at least 50 years with type 2 diabetes who had either a previous cardiovascular event or cardiovascular risk factors were randomly assigned (1:1) to either weekly subcutaneous injection of dulaglutide (1·5 mg) or placebo. Randomisation was done by a computer-generated random code with stratification by site. All investigators and participants were masked to treatment assignment. Participants were followed up at least every 6 months for incident cardiovascular and other serious clinical outcomes. The primary outcome was the first occurrence of the composite endpoint of non-fatal myocardial infarction, non-fatal stroke, or death from cardiovascular causes (including unknown causes), which was assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01394952. Findings: Between Aug 18, 2011, and Aug 14, 2013, 9901 participants (mean age 66·2 years [SD 6·5], median HbA1c 7·2% [IQR 6·6-8·1], 4589 [46·3%] women) were enrolled and randomly assigned to receive dulaglutide (n=4949) or placebo (n=4952). During a median follow-up of 5·4 years (IQR 5·1-5·9), the primary composite outcome occurred in 594 (12·0%) participants at an incidence rate of 2·4 per 100 person-years in the dulaglutide group and in 663 (13·4%) participants at an incidence rate of 2·7 per 100 person-years in the placebo group (hazard ratio [HR] 0·88, 95% CI 0·79-0·99; p=0·026). All-cause mortality did not differ between groups (536 [10·8%] in the dulaglutide group vs 592 [12·0%] in the placebo group; HR 0·90, 95% CI 0·80-1·01; p=0·067). 2347 (47·4%) participants assigned to dulaglutide reported a gastrointestinal adverse event during follow-up compared with 1687 (34·1%) participants assigned to placebo (p<0·0001). Interpretation: Dulaglutide could be considered for the management of glycaemic control in middle-aged and older people with type 2 diabetes with either previous 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.)