EC50 (for human): 5.79 (GCGR), 0.0643 (GIPR), 0.775 nM (GLP-1R) [1]. EC50 (for mouse): 2.32 (GCGR), 0.191 (GIPR), 0.794 nM (GLP-1R) [1]. Ki (for human): 5.6 (GCGR), 0.057 (GIPR), 7.2 nM (GLP-1R) [1]. Ki (for mouse): 73 (GCGR), 2.8 (GIPR), 1.3 nM (GLP-1R)[1].

Retatrutide (LY3437943) acetate has EC50 values of 5.79, 0.0643, and 0.775 nM, respectively, making it active against human GCGR, GIPR, and GLP-1R[1]. Retatrutide acetate has EC50 values of 2.32, 0.191, and 0.794 nM, respectively, against mouse GCGR, GIPR, and GLP-1R[1]. Retatrutide acetate exhibits binding affinities of 5.6, 0.057, and 7.2 nM for human GCGR, GIPR, and GLP-1R, respectively [1]. Retatrutide acetate exhibits binding affinities of 73, 2.8, and 1.3 nM for mouse GCGR, GIPR, and GLP-1R, respectively [1].

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Retatrutide (LY3437943)/LY activates the human GCGR, GIPR, and GLP-1R in vitro [1]
LY was engineered to have potent agonist activity at the human GCGR, GIPR, and GLP-1R. To assess the intrinsic potency of LY, HEK-293 clonal cell lines expressing either the human GCGR, human GIPR, or human GLP-1R were used to measure 3′,5′-cyclic adenosine monophosphate (cAMP) accumulation as the second messenger downstream of receptor activation. LY is 2.9-fold less potent than human glucagon at the human GCGR (LY 50% effective concentration [EC50] = 5.79 nM, SEM = 0.28 nM versus human glucagon EC50 = 1.97 nM, SEM = 0.04 nM) (Figure 1B; Table S1). LY shows 8.9-fold greater potency at human GIPR than GIP(1-42)NH2 (LY EC50 = 0.0643 nM, SEM = 0.0037 nM versus human GIP(1-42)NH2 EC50 = 0.574 nM, SEM = 0.026 nM) (Figure 1C; Table S1). At the human GLP-1R, LY is 2.5-fold less potent than GLP-1(7-36)NH2 (LY EC50 = 0.775 nM, SEM = 0.041 nM versus human GLP-1(7-36)NH2 EC50 = 0.312 nM, SEM = 0.007 nM) (Figure 1D; Table S1). LY is a full agonist at the GCGR, GIPR, and GLP-1R in the functional assays (Table S1). All cAMP assays were performed using clonal cell lines with low receptor densities. This affords minimal signaling pathway amplification and allows optimal measurement of the intrinsic potency and efficacy of molecules (Willard et al., 2020). Target engagement was independently confirmed using radioreceptor binding assays (Table S1). Comparatively, a range of potencies for activating these receptors was observed for several comparator mono, dual, and triple receptor agonists, including SAR441255 (Table S2). To further explore in vitro activity of LY in human cell-based models with endogenous GIPR or GCGR expression, we investigated the functional endpoints in differentiated human adipocytes preferentially expressing GIPR and human induced pluripotent stem cell-derived hepatocytes preferentially expressing GCGR. In the hepatocytes, LY demonstrated potency similar to native glucagon in stimulating glucose output (Figure 1E). In the adipocytes, LY was more potent than native GIP for stimulation of lipolysis (Figure 1F). Taken together, these in vitro findings demonstrate that LY is a potent agonist at the human GCGR, GIPR, and GLP-1R.

Retatrutide (LY3437943) acetate (subcutaneous injection; 0.47 mg/kg; single dose) acts on GIP or GLP-1 receptors to enhance glucose tolerance in ipGTT and to engage in GCGR in vivo [1]. Retatrutide acetate (subcutaneous injection; 10 mL/kg; every 3 days; for 21 days) activates the glucagon receptor, which results in a substantial decrease in body weight and an increase in energy expenditure [1]. It is acceptable and safe to use retirutide acetate [1].

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Retatrutide (LY3437943)/LY is a triple agonist at the GCGR, GIPR, and GLP-1R in vivo [1]
To further characterize LY in vivo, multiple genetically modified mouse models were employed to assess target receptor engagement and functional contribution to nutrient and energy metabolism. First, we confirmed that LY is a potent agonist at the mouse GCGR, GIPR, and GLP-1R in receptor binding affinity and functional potency assays in vitro (Table S1). We characterized the pharmacokinetic properties of LY in mice to assess the timing of dosing (Table S3; Figure S1). To assess GIPR and GLP-1R activity of LY, glycemic control in vivo was assessed using the intraperitoneal glucose tolerance test (ipGTT) in normal (wild type), GIPR-null (GIPr−/− mice), and GLP-1R-null mice (GLP-1r−/− mice). Similar to semaglutide and long-acting GIP, treatment with LY improved glucose excursions in all 3 genotypes (Figures 1G–1I). Together, these results indicate that LY can improve glucose tolerance in an ipGTT through either the GIP or GLP-1 receptors. To assess glucagon activity, we blocked both GIPR and GLP-1R activity by treating GLP-1R-null mice with a GIPR antibody antagonist (Killion et al., 2018) 24 h prior to LY dosing. With lack of GIPR and GLP-1R activity in the mice, LY increased plasma glucose 1 h after dosing (Figure 1J). However, this increase in glucose was blocked by pretreatment with a GCGR antibody antagonist (Jun et al., 2015), demonstrating LY functionally engages GCGR in vivo, increasing plasma glucose when tested in the absence of GIPR and GLP-1R agonism (Figure 1J). Importantly, while GCGR activity is present in LY, the insulinotropic effect from activity on both the GIP and GLP-1 receptors supersedes the GCGR effect on glucose tolerance, as LY (0.1 nmol/kg) maintained a similar ED50 to that of tirzepatide (0.2 nmol/kg) on glucose reduction in wild-type mice during an ipGTT (Coskun et al., 2018).

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Retatrutide (LY3437943)/LY promotes body weight loss in obese mice [1]
To investigate the effects on body weight, energy metabolism, body composition, and hepatic steatosis, LY was administered to C57/Bl6 diet-induced obese (DIO) mice. LY dose-dependently reduced body weight (ED50: 4.73 nmol/kg) (Figures 2A and 2C) and calorie intake (Figure 2B). The reduction in body weight was primarily due to reduced fat mass (Figure 2D) with minimal effect on lean mass (Figure 2E), as illustrated by the fat mass/lean mass ratio (Figure 2F). LY reduced blood glucose and plasma insulin (Figures 2G and 2H), indicating improved glycemic control and suggesting potential improvements in insulin sensitivity, consistent with what has been demonstrated with dual GIPR/GLP-1R agonism (Samms et al., 2021). LY improved liver health as demonstrated by a decrease in plasma alanine aminotransferase and liver triglycerides (Figures 2I and 2J). In an additional experiment, we tested the efficacy of LY versus tirzepatide on body weight loss in obese mice. LY promoted more body weight loss than tirzepatide in obese mice when treated daily at 10 nmol/kg (Figure 2K), and this was associated with greater reduction in calorie intake (Figure 2L). This might partially explain additional weight loss with LY. To elucidate this, we performed additional studies with LY to assess the contribution of energy expenditure on weight loss.

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Retatrutide (LY3437943)/LY increases energy expenditure through glucagon receptor engagement in obese mice [1]
We previously demonstrated that treatment with tirzepatide results in a small but significant effect to increase energy expenditure (Coskun et al., 2018), but a key driver for body weight loss with tirzepatide in mice is reduction in overall calorie intake. To assess the effect of LY on calorie intake and energy expenditure, we conducted a series of experiments at thermoneutrality (27°C). We studied the effect of LY at 10 nmol/kg compared with a calorie-intake matched (CIM) group. LY led to ∼35% body weight loss during the first 10 days of the treatment period, while CIM resulted in 20% body weight loss (Figure 3A). During this 10-day period, the calorie intake was substantially suppressed (Figure 3B). The reduction of caloric intake in the CIM group was accompanied by a significant reduction in energy expenditure. As the initial inhibitory treatment effect on calorie intake waned (Figure 3B), the CIM group gradually gained weight with slow recovery from declined energy expenditure (Figure 3C). In comparison, mice treated with LY continued to maintain initial body weight loss (Figure 3B) as a result of maintaining the before-treatment energy expenditure levels (Figure 3C). In comparison with the CIM group, LY increased energy expenditure (Figure 3C), which resulted in a significant negative energy balance (Figure 3D) and lipid oxidation as illustrated with decline in respiratory exchange ratio (Figure 3E). Treatment with LY did not change the locomotor activity compared to other groups (Figure 3F). Both CIM- and LY-treated groups lost fat mass and lean mass (Figures 3G and 3H) as a result of body weight loss. While changes in lean mass were similar between groups, LY produced a significantly greater reduction in fat mass than seen in the CIM group. The GCGR antibody antagonist minimized the body weight loss with LY (Figure 3I), essentially matching the level observed in the CIM group (Figure 3B). Administration of the GCGR antibody antagonist did not alter the overall effect of LY on calorie intake reduction (Figure 3J), but blocked the effect of LY on energy expenditure (Figure 3K), providing evidence that GCGR agonism is a key contributor of energy expenditure.

In vitro binding affinity and functional potency of Retatrutide (LY3437943) for recombinant human or mouse glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon-like peptide-1 receptor (GLP-1R) Retatrutide (LY3437943) (LY), glucagon (GCG), glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1), and the [125I]-radiolabeled GCG, GIP, and GLP-1 were prepared at Lilly or elsewhere as described in the key resources table.

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Receptor binding assays [1]
Receptor binding assays were performed using a Scintillation Proximity Assay format with wheat germ agglutinin beads as previously described (Willard et al., 2020) using membranes from human embryonic kidney (HEK-293) clonal cell lines expressing the human or mouse GIPR, GLP-1R, or GCGR (Samms et al., 2021; Willard et al., 2020). Unlabeled GCG/GIP/GLP-1 controls for non-specific binding (NSB) were used at 1 μM final for GCG, 0.25 μM final for GIP, or 0.25 μM final for GLP-1 for each respective receptor binding assay. Radioligand concentrations were as follows for each respective receptor binding assay: [125I]-GCG (0.15 nM final), [125I]-GIP (0.075–0.15 nM final), and [125I]-GLP-1 (0.15 nM final). GCGR membranes (1.5 μg/well human or 6.5 μg/well mouse), GIPR membranes (3.0 μg/well human or 7.0 μg/well mouse), or GLP-1R membranes (0.5 μg/well human or 0.12 μg/well mouse) were added for each respective receptor binding assay. Ki values were determined by nonlinear regression analysis using the amount of radiolabel bound versus the concentration of peptide added. GIP radiotracer is purified by HPLC and described to be a 1:1 mixture of [125I]-Tyr1-GIP(1-42) and [125I]-Tyr10-GIP(1-42). Homologous and heterologous competition experiments were performed with non-radioactive peptide analogues[127I]-Tyr1-GIP(1-42) and [127]-Tyr10-GIP(1-42) to ensure quantification of the high-affinity binding site of the GIPR. Peptide analogues were generated using synthetic [127I]-Tyr amino acid building blocks.

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Human receptor functional in vitro assay [1]
Functional activity of LY was determined using 3′,5′-cyclic adenosine monophosphate (cAMP) formation in HEK-293 clonal cell lines with a low-expression density of human GCGR (1,300 receptors/cell), human GIPR (1,700 receptors/cell), or human GLP-1R (1,400 receptors/cell) (Willard et al., 2020). A generation of the low-expressing human GCGR clonal cell line was performed as described previously for the human GLP-1R and human GIPR low-expressing clonal cell lines (Willard et al., 2020). The cAMP formation assays were performed in the presence and absence of fatty acid-free, globulin-free human serum albumin as previously described (Willard et al., 2020), and the fluorescent signal was detected using a PerkinElmer Envision instrument with excitation at 320 nm and emission at 665 nm and 620 nm.

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Mouse receptor functional in vitro assay [1]
Functional activity of LY was determined using cAMP formation in HEK-293 clonal cell lines expressing murine GCGR, murine GIPR, or murine GLP-1R as previously described (Samms et al., 2021). The cAMP formation assays were performed in the absence of serum albumin as described above, and the fluorescent signal was detected using a PerkinElmer Envision instrument with excitation at 320 nm and emission at 665 and 620 nm.

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Human GLP-1R β-Arrestin recruitment assay [1]
ARRB2 recruitment was performed as described using GLP-1R CHO-K1 PathHunter cells (Willard et al., 2020). The assays were performed in the absence of albumin and after addition of the PathHunter detection reagent, the chemiluminescent signal was read using an Envision plate reader.

Human hepatocyte assay [1]
Human induced pluripotent stem hepatocytes were cultured in RPMI 1640 medium supplemented with Oncostatin M, gentamicin, and bovine serum albumin. To acclimatize cells to glucose-free conditions, cells were washed and incubated for 1 h prior to treatment with a glucose-free HGO buffer containing MgSO4 (0.82 mM), NaHCO3 (9 mM), HEPES (0.02 mM), fatty acid-free BSA (0.1%), CaCl2 (2.25 mM), NaCl (117.6 mM), KCl (5.4 mM), and KH2PO4 (1.5 mM). Following incubation, HGO buffer was removed and cells were treated with test peptides in concentration response in HGO buffer for 2 h. After treatment, conditioned media was collected and analyzed for glucose content to assess cellular glucose output into the HGO buffer. Glucose analysis was performed using the Amplex Red Glucose Oxidase Assay kit per manufacturer protocol. Concentration response curves for peptides tested were plotted as glucose in conditioned media (y-axis) versus log concentration of molecule (x-axis). EC50 was determined using 4 parameter variable slope non-linear regression fit analysis (GraphPad Prism 7.0). To assess potency, EC50 was reported as a mean of 3 experiments with 3 biologic replicates per dose in each experiment.

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Human adipocyte assay [1]
Isolated and cultured human preadipocytes were differentiated into mature adipocytes, incubated for 1 day in preadipocyte media followed by 6 days in adipocyte differentiation media, and an additional 6 days in adipocyte maintenance media. Lipolysis assay on differentiated adipocytes was performed per Cultured Human Adipocyte Lipolysis Assay Kit. Briefly, cells were washed twice followed by treatment with test peptides in concentration response for 3 h in assay buffer. After incubation, conditioned media was transferred to a separate plate and assessed for glycerol content per kit protocol. Concentration response curves for peptides tested were plotted as glycerol in conditioned media (y-axis) versus log concentration of peptide (x-axis). EC50 was determined using 4 parameter variable slope nonlinear regression fit (GraphPad Prism 7.0).

Animal/Disease Models: Male CD-1 mice[1]
Doses: 0.47 mg/kg
Route of Administration: subcutaneous (sc) administration, single
Experimental Results: AUClast, ng*h/mL AUC0-∞, ng*h/mL Cmax, ng/mL Tmax, h t1 /2, h CLF, mL/h/kg 41135 41905 1680 12 21 11.22.

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Animal/Disease Models: Diet-induced obese (DIO) male C57/BL6 black mouse (24-25 weeks, 40-51 g)[1]
Doses: 10 mL /kg
Route of Administration: subcutaneous (sc) injection, cycle every 3 days, for 21 days
Experimental Results: diminished body weight and improved glycemic control.

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In vivo efficacy studies in obese mice [1]
Diet-induced obese (DIO) male C57/Bl6 mice 24 weeks to 25 weeks old, maintained on a calorie-rich diet since arrival, were used in the following studies. Animals were individually housed in a temperature-controlled (24°C to 27°C) facility with a 12-h light/dark cycle (lights on 22:00) and free access to food and water.
After a minimum of 2 weeks acclimation to the facility, the mice were randomized according to their body weight, so each experimental group of animals would have similar body weight. Mice body weight ranged from 40 g to 51 g. All groups contained 6 mice. Vehicle (20 mM Tris-HCl at pH 8.0) or LY (dose range 0.3 nmol/kg to 30 nmol/kg) dissolved in vehicle was administered by subcutaneous (SC) injection (10 mL/kg) to ad libitum-fed DIO mice 30 min–90 min prior to the onset of the dark cycle every 3 days for 21 days. SC injections were administered on Days 1, 4, 7, 10, 13, 16 and 19. Body weight and food intake were measured daily throughout the study. General animal health and welfare was monitored by veterinary staff in all studies. No adverse effects were reported.
Absolute changes in body weight were calculated by subtracting the body weight of the same animal prior to the first injection of molecule. On Days 0 and 21, total fat mass was measured by quantitative nuclear magnetic resonance. On Day 21, animals were sacrificed prior to dark photoperiod, blood was collected by cardiac stick and plasma was analyzed by a clinical chemistry analyzer. Liver triglycerides were determined from homogenates of livers collected at sacrifice. Insulin was measured via Meso Scale Discovery enzyme-linked immunosorbent assay kit.
For indirect calorimetry studies, animals were placed in TSE PhenoMaster/LabMaster calorimeter for 3 days of acclimation and all the experiments were performed at thermoneutrality (27°C). On Days 0 and 22, total fat mass was measured by nuclear magnetic resonance (NMR) using an Echo Medical System instrument. Vehicle or Retatrutide (LY3437943) (10 nmol/kg) were subcutaneously administered to ad libitum fed DIO mice 30 to 90 min prior to the onset of the dark cycle daily for 22 days. To determine the extent of the effect of LY34379343 treatment on body weight, body metabolism or energy metabolism occurred independently of changes of calorie intake, a group of mice was matched to that consumed by Retatrutide (LY3437943) group. In another experiment, mice were treated weekly by either control or GCGR antibody antagonist at 10 mg/kg dose. Daily body weight and food intake were measured throughout the study. Absolute changes in body weight were calculated by subtracting the body weight of the same animal prior to the first injection of molecule. Heat and respiratory exchange ratio (RER) were measured by indirect calorimetry using an open-circuit calorimetry system. RER is the ratio of the volume of CO2 produced (VCO2) to the volume of O2 consumed (VO2).

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Non-clinical safety CV study in non-human primates [1]
Single dose monkey CV study with Retatrutide (LY3437943) [1]
Eighteen male cynomolgus monkeys (six monkeys/group) were administered a single subcutaneous dose of vehicle control article or 0.05 or 0.5 mg/kg Retatrutide (LY3437943) on Day 1 in a parallel dosing design. Assessment of cardiovascular function was based on qualitative electrocardiography (ECG) evaluation and quantitative analyses of ECG (QT interval and corrected QT [QTc]) and hemodynamic (heart rate and dP/dtmax derived from the left ventricular pressure waveform; systolic, diastolic, and mean arterial pressures; and arterial pulse pressure) parameters. The ECG and hemodynamic data were recorded by telemetry for at least 90 min prior to dosing and continuously through at least 169 h postdose.

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6-Month repeat dose toxicity study in monkeys with Retatrutide (LY3437943). [1]
Male and female cynomolgus monkeys were assigned to four groups, and subcutaneous doses were administered at 0.05, 0.15, 0.5 mg/kg. Telemetry measurements were collected and analyzed once for each sex during the predose phase; on Days 8 (Groups 1 through 3 only), 36 (Groups 1 and 4 only; due to the need for dosing suspensions in Group 4 during the first weeks of study), 99, and 169 of the dosing phase; and on Day 108 (males) or 106 (females) of the recovery phase.

[1]. LY3437943, a novel triple glucagon, GIP, and GLP-1 receptor agonist for glycemic control and weight loss: From discovery to clinical proof of concept. Cell Metab. 2022 Sep 6;34(9):1234-1247.e9.

With an increasing prevalence of obesity, there is a need for new therapies to improve body weight management and metabolic health. Multireceptor agonists in development may provide approaches to fulfill this unmet medical need. LY3437943 is a novel triple agonist peptide at the glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon-like peptide-1 receptor (GLP-1R). In vitro, LY3437943 shows balanced GCGR and GLP-1R activity but more GIPR activity. In obese mice, administration of LY3437943 decreased body weight and improved glycemic control. Body weight loss was augmented by the addition of GCGR-mediated increases in energy expenditure to GIPR- and GLP-1R-driven calorie intake reduction. In a phase 1 single ascending dose study, LY3437943 showed a safety and tolerability profile similar to other incretins. Its pharmacokinetic profile supported once-weekly dosing, and a reduction in body weight persisted up to day 43 after a single dose. These findings warrant further clinical assessment of LY3437943.[1]

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Conclusions and future directions In conclusion, Retatrutide (LY3437943)/LY possesses an imbalanced activity at GIPR (in favor of GIPR activity) and balanced activity at GCGR and GLP-1R. LY achieved body weight reductions of up to 45% in obese mice with glucose-lowering efficacy and improvement in insulin resistance. Our data demonstrate that the GCGR activity in the triagonist accounts for 30%–35% of the observed body weight loss in mice as a result of increased energy expenditure. Healthy participants in the single ascending dose study showed that LY was well tolerated. The body weight reduction after a single administration of LY was maintained up to day 43 post-dose with an initial decrease in appetite. The observed safety, tolerability, and efficacy profile of LY in this study support evaluation of multiple ascending doses of LY in participants with obesity (NCT04881760) and T2D (NCT04867785).[1]

C221H342N46O68.C2H4O2

4791.38

Retatrutide;2381089-83-2;Retatrutide TFA

Tyr-{Aib}-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{α-Me-Leu}-Leu-Asp-Lys-{diacid-C20-gamma-Glu-(AEEA)-Lys}-Ala-Gln-{Aib}-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

Typically exists as solid at room temperature

LY3437943 acetate ; Retatrutide acetate ; LQ42M82ZU6; LY-3437943; LY-3437943 acetate

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.)