GPR119

AR231453 is highly selective for GPR119, as evidenced by its inactivity at all other GPCRs tested (more than 230, including all known pancreatic islet receptors)[1]. AR231453 exhibits a maximal efficacy akin to that of forskolin, potently stimulating cAMP accumulation (EC50 = 4.7 nM). With an EC50 of 3.5 nM, AR 231453 dramatically increases insulin release in HIT-T15 cells[1]. In isolated mouse islets, AR 231453 also induces the release of insulin at glucose concentrations between 8 and 17 mM[1].

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GPR119 agonist stimulates insulin release in vitro [1]
The observation that GPR119 couples to Gαs and is expressed in β-cells prompted us to test the hypothesis that GPR119 is an insulinotropic receptor. In this regard, AR231453 significantly enhanced insulin release in HIT-T15 cells, with an EC50 of 3.5 nM (Fig. 4A), similar to its effect on cAMP (EC50 of 4.7 nM; see Fig. 3D), and similar in efficacy to forskolin. To assess the GPR119 dependence of the insulinotropic effects seen with AR231453, we tested its activity in RIN-5F cells lacking GPR119 (RIN-5F/vector) and in RIN-5F cells stably transfected with human GPR119 (RIN-5F/hGPR119). AR231453 stimulated insulin release in RIN-5F/hGPR119 cells but not in RIN-5F/vector cells (Fig. 4B). These studies indicate that AR231453 is insulinotropic specifically in GPR119-expressing β-cell lines. We next examined the effect of GPR119 agonist on glucose-stimulated insulin release in isolated rat and mouse islets. At 15 mM glucose, 300 nM AR231453 enhanced insulin release in rat islets similarly to GLP-1 (Fig. 4C). However, this compound had no effect on islets incubated in 5 mM glucose. The insulinotropic effect of GPR119 agonists in islets is therefore glucose dependent. AR231453 also stimulated insulin release in isolated mouse islets at glucose concentrations ranging from 8–17 mM (Fig. 4D). This indicates that the insulinotropic effect of GPR119 agonists requires only modest elevations of blood glucose.

AR231453 (20 mg/kg, Oral) improves oral glucose tolerance in a dose-dependent manner that is comparable to the sulfonylurea glyburide's maximally effective doses[1]. GPR119 agonist improves glucose tolerance and enhances glucose-dependent insulin release in mice [1]
AR231453 exhibited good oral bioavailability in C57BL/6 mice, attaining micromolar plasma levels for more than 2 h at the doses used here. AR231453 (20 mg/kg, orally) markedly improved oral glucose tolerance in a dose-dependent fashion, with efficacy similar to maximally effective doses of the sulfonylurea glyburide (Fig. 5A). On average, AR231453 maximally inhibited the glycemic excursion by approximately 42% (21 independent experiments). When glucose was administered ip, AR231453 improved glucose tolerance somewhat less effectively (23%; three independent experiments) (Fig. 5B).

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AR231453 is inactive in GPR119-deficient mice [1]
To demonstrate that the in vivo effects of AR231453 occur via GPR119, we generated GPR119-deficient mice (Fig. 7). As in human, the gene for mouse GPR119 is located on the X chromosome (Unigene Cluster Mm.34953) (Fig. 7A). Deletion of GPR119 was confirmed by Southern blot analysis (Fig. 7B), RT-PCR analysis of pancreatic GPR119 mRNA (Fig. 7C), and immunofluorescent analysis of GPR119 receptor on pancreatic sections from the knockout mice (Fig. 7D). Islets from GPR119-deficient mice retained normal morphology (Fig. 7D) and a normal response to glucose and GLP-1 (Fig. 7E). These mice also had normal size, body weight, and fed/fasted blood glucose levels (not shown) and a typical response to glyburide in glucose tolerance tests (Fig. 7, F and G). Thus, deletion of GPR119 does not grossly impact glucose homeostasis. However, isolated islets from these animals no longer responded to AR231453 (Fig. 7E). AR231453 also had no effect on glucose tolerance in GPR119-deficient mice (Fig. 7G), indicating that its observed in vivo activities were indeed mediated via GPR119.

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Methods: A total of 100 syngenic C57BL/6 mouse islets were transplanted under the left kidney of each chemically induced diabetic C57BL/6 mouse. Starting from the day of transplantation, these recipients were given bromodeoxyuridine (BrdU) daily with or without AR231453 at 10 mg/kg/d. Islet graft function was monitored by measuring blood glucose levels. At 4 weeks, left nephrectomy was performed to remove the kidney bearing the islet grafts to determine β-cell replication in the islet grafts. Insulin and BrdU immunofluorescence staining was performed to detect replicated β cells. Insulin(+) and BrdU(+) β cells in islet grafts were counted using a confocal microscope. To determine whether AR231453 increases plasma GLP-1 levels, we collected plasma from AR231453 treated mice at 30 minutes after treatment and measured plasma active GLP-1 by enzyme-linked immunosorbent assay.
Results: Although all recipient mice achieved normoglycemia at 28 days with or without treatment, normoglycemia was achieved in significantly fewer days in AR231453-treated mice. The vehicle-treated mice achieved normoglycemia in 16 ± 6 days, while AR231453-treated mice only required only 8 ± 3 days (P < .01). The percentage of insulin(+) and BrdU(+) β cells in islet grafts was significantly higher in AR231453-treated mice than in vehicle-treated mice. The mean percentage of insulin(+) and BrdU(+) β cells in islet grafts was 21.5% ± 6.9% in AR231453-treated mice and 5.6% ± 3.7% in vehicle-treated mice (P < .01). The plasma active GLP-1 levels were also significantly higher in AR231453-treated mice than in vehicle-treated mice (P < .05).
Conclusion: Our data demonstrate that AR231453, a GPR119 agonist, can stimulate β-cell replication and improve islet graft function [2].

In vitro assays [1]
cAMP measurements were done with a Flash Plate adenylyl cyclase kit. Briefly, HEK293 cells were transfected with either empty vector DNA or GPR119 expression plasmid DNA (described above) using Lipofectamine. After 24 h, transfected cells were harvested in GIBCO cell dissociation buffer (catalog item 13151-014) and resuspended in assay buffer (50% 1× PBS/50% stimulation buffer). Compounds were incubated with 105 cells per well for 60 min at room temperature. After another 2-h incubation with tracer in detection buffer, plates were counted in a Wallac MicroBeta scintillation counter. Values of cAMP per well were extrapolated from a standard cAMP curve that was included on each assay plate. cAMP assays in hamster insulinoma-derived HIT-T15 cells and GPR119-transfected RIN-5F stable lines were performed essentially in the same way but without transient transfection. In experiments designed to selectively lower endogenous GPR119 expression in HIT-T15 cells, cells were transfected with either a control RNA oligo with sequence derived from hamster GPR119 in sense orientation (5′-CUAUGCUGCUAUCAAUCUA-3′) (control) or in antisense orientation (5′-UAGAUUGAUAGCAGCAUAG-3′) [small interfering RNA (siRNA)]. Forty-eight hours after transfection, cells were assayed for GPR119 expression and for agonist-stimulated cAMP production as described above.

For assays measuring inositol phosphate accumulation, HEK293 cells in 96-well plates were transfected with the indicated G protein receptor expression plasmid. Additionally, cells were cotransfected with either an empty cytomegalovirus (CMV) vector or a Gαq/Gαi expression plasmid in which the terminal six residues of Gαq were replaced by the corresponding residues of Gαi. The receptor and G protein chimera were transfected at a molar ratio of 4:1, respectively. The following day, cells were incubated in 100 μl inositol-free/serum-free DMEM containing 0.4 μCi [3H]myoinositol. Cells were incubated overnight, after which the medium was replaced with 100 μl inositol-free/serum-free DMEM containing 10 μM pargyline and 10 mM lithium chloride. One hour later, the cells were lysed and inositol phosphates were isolated using chromatography on AG1-X8 formate resin. After additional purification through binding, four washes, and elution from a multiscreen filter plate, eluted counts were quantified using a Wallac scintillation counter.

For in vitro insulin release, HIT-T15 insulinoma cells were plated in 24-well plates (2.5 × 105 cells per well) for insulin release assays. One day before the assay, culture media were changed to DMEM (3 mM glucose) with 10% dialyzed horse serum and 2.5% fetal bovine serum. On the next day, cells were washed twice with PBS and incubated for 1 h with test compounds in DMEM in the presence of 15 mM glucose in 0.25 ml HEPES-buffered Krebs-Ringer buffer. Supernatant insulin levels were determined using an Ultra Sensitive Insulin ELISA kit. Insulin assays with RIN-5F stable lines were performed in essentially the same manner.

Insulin release assays with pancreatic islets were done using islets isolated from female Sprague Dawley rats (body weight, 175–185 g) or male C57BL/6 mice (body weight, ∼25 g). Insulin release was determined in static islet incubation as described previously. Briefly, groups of five islets each were placed in incubation wells. After a 30-min preincubation with HEPES-buffered Krebs-Ringer buffer (pH 7.4) containing 5 mM glucose, islets were transferred to wells containing 2 ml HEPES-buffered Krebs-Ringer buffer and different concentrations of glucose and test compounds. The studies were performed at 37 C in a water bath shaker with an atmosphere of 95% O2/5% CO2. Samples of incubation buffer were collected at 60 min for insulin determination using ELISA methodology.

In vivo experiments [1]
C57BL/6 male mice were used. For the oral glucose tolerance test, overnight fasted mice (n = 6 per treatment) were given either vehicle (80% polyethylene glycol 400/10% Tween 80/10% ethanol) or test compounds at desired doses via oral gavage. A glucose bolus was then delivered (3 g/kg orally or 2 g/kg ip). Plasma glucose levels were determined at desired time points over a 2-h period using blood (∼5 μl) collected from tail nick and a glucose meter. For insulin pharmacodynamic studies, vehicle or AR231453 was administered orally to fasted animals (n = 6 per treatment group and time point). After 30 min, a glucose bolus of 3 g/kg was administered orally. Blood was collected in heparinized blood collection tubes at desired time points. Plasma samples were obtained via centrifugation at 500 × g for 20 min and assayed for insulin as described above.

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Islet Transplantation and Treatment [2]
The islets were isolated and transplanted according to a protocol that is similar to a previously published isolation protocol.6 Islets free of acinar cells, vessels, lymph nodes, and ducts were used for culture and transplantation. A total of 100 islets was transplanted into each recipient mouse. The recipient mice were randomly divided into two groups and treatments started from the day of transplantation. Mice in group 1 were orally treated with vehicle daily. Mice in group 2 were orally treated with AR231453 at 10 mg/kg/d. All recipient mice were also intraperitoneally treated with bromodeoxyuridine (BrdU) at 100 mg/kg/d for 4 weeks. After 4 weeks, nephrectomy was performed and all of the left kidneys bearing primary islet grafts were collected.

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Plasma GLP-1 Active Immunoassay [2]
Mice were orally treated with vehicle or AR231453 at 10 mg/kg. At 30 minutes after treatment, blood samples were collected. Active GLP-1 in the plasma was measured using a mouse GLP-1 enzyme-linked immunosorbent assay kit.

[1]. A role for beta-cell-expressed G protein-coupled receptor 119 in glycemic control by enhancing glucose-dependent insulin release. Endocrinology. 2007 Jun;148(6):2601-9.

[2]. Stimulating β-cell replication and improving islet graft function by AR231453, A GPR119 agonist. Transplant Proc. 2011 Nov;43(9):3217-20.

Pancreatic beta-cell dysfunction is a hallmark event in the pathogenesis of type 2 diabetes. Injectable peptide agonists of the glucagon-like peptide 1 (GLP-1) receptor have shown significant promise as antidiabetic agents by virtue of their ability to amplify glucose-dependent insulin release and preserve pancreatic beta-cell mass. These effects are mediated via stimulation of cAMP through beta-cell GLP-1 receptors. We report that the Galpha(s)-coupled receptor GPR119 is largely restricted to insulin-producing beta-cells of pancreatic islets. Additionally, we show here that GPR119 functions as a glucose-dependent insulinotropic receptor. Unlike receptors for GLP-1 and other peptides that mediate enhanced glucose-dependent insulin release, GPR119 was suitable for the development of potent, orally active, small-molecule agonists. The GPR119-specific agonist AR231453 significantly increased cAMP accumulation and insulin release in both HIT-T15 cells and rodent islets. In both cases, loss of GPR119 rendered AR231453 inactive. AR231453 also enhanced glucose-dependent insulin release in vivo and improved oral glucose tolerance in wild-type mice but not in GPR119-deficient mice. Diabetic KK/A(y) mice were also highly responsive to AR231453. Orally active GPR119 agonists may offer significant promise as novel antihyperglycemic agents acting in a glucose-dependent fashion. [1]

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Objective: G protein-coupled receptor 119 (GPR119) is predominantly expressed in β cells and intestinal L cells. AR231453 is a selective small-molecular GPR119 agonist that enhances glucose-dependent insulin secretion and glucagon-like peptide 1 (GLP-1) release. We investigated whether AR231453 can directly stimulate β-cell replication and improve islet graft function in diabetic mice.[2]

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These data provide strong evidence that GPR119 is a significant modulator of glucose-stimulated insulin release in pancreatic β-cells. The GPR119-selective agonist AR231453 stimulated cAMP accumulation and insulin release in 1) transfected RIN-5F insulinoma cells, 2) HIT-T15 insulinoma cells that express the receptor endogenously, and 3) isolated rat and mouse islets. These effects were substantially the same as those elicited by forskolin or GLP-1, suggesting significant physiological relevance. In vivo, AR231453 stimulated insulin release and improved glucose tolerance with comparable efficacy to glyburide, a very robust stimulator of insulin release in rodents and humans. Importantly, the activity of AR231453 was particularly impressive in diabetic KK/Ay mice. All these effects are very likely occurring via GPR119. RIN-5F control cells lacking GPR119 are unresponsive to AR231453. In HIT-T15 cells, siRNA-mediated reduction in GPR119 levels was associated with virtually complete loss of responsiveness to AR231453. Finally, AR231453 does not enhance glucose-stimulated insulin release in islets derived from GPR119-deficient mice, nor does it improve glucose tolerance in GPR119-deficient mice. GPR119-deficient mice had no obvious defect in homeostasis, but this perhaps is not surprising because mice lacking either GIP receptor or GLP-1 receptor have very minor alterations in this regard. Because we did not measure AR231453 exposures in GPR119-deficient mice, its inactivity in glucose tolerance tests performed in these mice could simply be due to strain-dependent differences in plasma exposure of the compound. However, this seems unlikely. In C57BL/6 mice, AR231453 achieves exposures more than 100-fold greater than its in vitro EC50. Additionally, the compound is effective in all other strains of mice used here. Numerous, structurally distinct compounds have been assessed in wild-type littermates and GPR119-deficient mice, with similar outcomes to those seen for AR231453 (not shown). Taken together, these data strongly suggest that the actions of AR231453 are mediated via GPR119 and firmly establish that GPR119 is a physiologically significant mediator of glucose homeostasis. Although these studies support the hypothesis that GPR119 regulates glucose homeostasis by direct enhancement of pancreatic β-cell function, it is important to recognize that GPR119 may work via additional mechanisms. The effectiveness of AR231453 is reduced by almost 50% when glucose is administered ip, suggesting that its actions may also involve modulation of incretin signaling. This hypothesis warrants further investigation. Our studies in islets and mice indicate that GPR119 stimulates insulin release in a glucose-dependent manner. Even when administering doses of AR231453 (100 mg/kg in C57BL/6 mice) that greatly exceed the maximal in vivo efficacy of this compound, there is no observed stimulation of insulin release or lowering of glucose under fasted conditions. By contrast, glyburide elicited significant insulin release and marked hypoglycemia in fasted mice. These data are entirely consistent with the well-established mechanism by which GIP and GLP-1 stimulate insulin release.[1]

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In this study, we found that AR231453 treatment significantly enhanced the reversal of diabetes in mice that received a marginally therapeutic dose of islets. It confirmed our earlier studies of using oleoylethanolamide (OEA), an endogenous GPR119 ligand, and PSN632408, a selective small-molecular GPR119 agonist, to enhance islet graft function.8 Reversal of diabetes depended on the function of the islet grafts since hyperglycemia reoccurred after removal of the left kidney bearing the islet grafts. We also found that AR231453 treatment significantly increased β-cell replication in islet grafts. In our previous study, we found that exendin-4 can stimulate β-cell replication in mouse islet grafts from both young and old donors.4 Therefore, we used islets from old donors for transplantation. Although it has been shown that the capacity for β-cell replication in aged mice is declined,our data showed that AR231453 could stimulate β-cell replication in islets isolated from old donors. AR231453 directly stimulates insulin secretion from β-cells in vitro and improves glucose tolerance and enhances glucose-dependent insulin release in vivo.1 Recently we also found that OEA and PSN632408 can directly stimulate β-cell replication in cultured islets (manuscript submitted for publication). Thus, AR231453 may improve islet graft function and stimulate β-cell replication in islet grafts by direct activation of GPR119 on islets. However, AR231453 can also stimulate GLP-1 secretion from intestinal enteroendocine L-cells through activation of GPR119. We found that AR231453 treatment increased the plasma GLP-1 levels in mice. Therefore, it is possible that AR231453-stimulated β-cell replication in islet grafts is also due to increased plasma GLP-1 concentrations. To address the direct and/or indirect effect of AR231453 through activating GRP119 on improving islet graft function and stimulating β-cell replication in islet grafts, GLP-1 receptor knockout mice and GPR119 knockout mice will have to be used as islet donors and/or transplant recipients. Targeting GPR119 is a novel therapeutic approach to increase β-cell replication and to improve islet graft function. It can potentially be used for human islet transplantation. Single-donor islet transplantation is a very attractive goal for clinical islet transplantation. The islet mass from single donors is often not adequate to restore normoglycemia, although diabetes could be reversed with islets isolated from a single-donor pancreas.11 Therefore, developing GPR119 agonist-based strategies to increase β-cell mass and to improve islet grafts function can potentially facilitate successful single-donor islet transplantation. Since human patients with still have some β-cell mass remaining at the onset of type 1 diabetes, GPR119 agonists may also be used to restore normoglycemia by stimulating β-cell replication and increasing β-cell mass.[2]

C₂₁H₂₄FN₇O₅S

505.52

505.154

C, 49.89; H, 4.79; F, 3.76; N, 19.40; O, 15.82; S, 6.34

733750-99-7

24939268

Light yellow to yellow solid powder

5.303

1

12

6

35

833

0

CC(C)C1=NOC(C2CCN(C3=NC=NC(NC4=CC=C(S(=O)(C)=O)C=C4F)=C3[N+]([O-])=O)CC2)=N1

DGBKNTVAKIFYNU-UHFFFAOYSA-N

InChI=1S/C21H24FN7O5S/c1-12(2)18-26-21(34-27-18)13-6-8-28(9-7-13)20-17(29(30)31)19(23-11-24-20)25-16-5-4-14(10-15(16)22)35(3,32)33/h4-5,10-13H,6-9H2,1-3H3,(H,23,24,25)

N-(2-fluoro-4-methylsulfonylphenyl)-5-nitro-6-[4-(3-propan-2-yl-1,2,4-oxadiazol-5-yl)piperidin-1-yl]pyrimidin-4-amine

AR-231453; AR 231453; AR231,453; 733750-99-7; AR 231,453; AR231,453; N-(2-Fluoro-4-(methylsulfonyl)phenyl)-6-(4-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)-5-nitropyrimidin-4-amine; AR-231453; N-(2-fluoro-4-methylsulfonylphenyl)-5-nitro-6-[4-(3-propan-2-yl-1,2,4-oxadiazol-5-yl)piperidin-1-yl]pyrimidin-4-amine; CHEMBL461384; 07Z1P4981I; AR231,453; AR-231453; AR231,453

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)

DMSO: ~25 mg/mL (~49.5 mM)

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.)