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 β-cell dysfunction is a hallmark event in the pathogenesis of type 2 diabetes. Injectable glucagon-like peptide-1 (GLP-1) receptor agonists have shown great potential as antidiabetic drugs due to their ability to enhance glucose-dependent insulin release and maintain pancreatic β-cell numbers. These effects are mediated by stimulating the release of cAMP from GLP-1 receptors on β-cells. We found that the Gα(s)-coupled receptor GPR119 is primarily localized to insulin-secreting β-cells in the pancreas. Furthermore, we discovered that GPR119 possesses glucose-dependent insulinotropic function. Unlike GLP-1 receptors and other peptide receptors that mediate enhanced glucose-dependent insulin release, GPR119 is suitable for developing highly potent, orally effective small-molecule agonists. The GPR119-specific agonist AR231453 significantly increased cAMP accumulation and insulin release in HIT-T15 cells and rodent islets. In both cases, the absence of GPR119 led to the inactivation of AR231453. AR231453 also enhances glucose-dependent insulin release in vivo and improves oral glucose tolerance in wild-type mice, but has no such effect in GPR119-deficient mice. Diabetic KK/A(y) mice also showed a high response to AR231453. Orally administered active GPR119 agonists are expected to become novel glucose-dependent hypoglycemic drugs. [1]

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Objective: G protein-coupled receptor 119 (GPR119) is mainly expressed in β cells and intestinal L cells. AR231453 is a selective small molecule 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 proliferation in diabetic mice and improve islet transplantation function. [2]

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These data strongly demonstrate that GPR119 is an important regulator of glucose-stimulated insulin release in pancreatic β cells. The GPR119 selective agonist AR231453 stimulates cAMP accumulation and insulin release in the following cells: 1) transfected RIN-5F islet tumor cells; 2) HIT-T15 islet tumor cells endogenously expressing this receptor; and 3) isolated rat and mouse islets. These effects are essentially the same as those of fossolin or GLP-1, indicating significant physiological implications. In vivo, AR231453 stimulates insulin release and improves glucose tolerance, with efficacy comparable to glibenclamide (a potent insulin-stimulating drug in rodents and humans). Notably, AR231453 exhibits particularly pronounced activity in diabetic KK/Ay mice. All these effects are likely mediated by GPR119. RIN-5F control cells lacking GPR119 do not respond to AR231453. In HIT-T15 cells, siRNA-mediated decreased GPR119 levels were associated with almost complete loss of response to AR231453. Finally, AR231453 neither enhanced glucose-stimulated insulin release from the pancreas in GPR119-deficient mice nor improved glucose tolerance in these mice. No significant homeostasis was observed in GPR119-deficient mice, which is perhaps unsurprising given the slight alterations in this regard in mice lacking either the GIP or GLP-1 receptors. Since we did not detect AR231453 exposure in GPR119-deficient mice, the ineffectiveness of this compound in glucose tolerance tests in these mice could simply be due to differences in plasma exposure across different mouse strains. However, this possibility seems unlikely. In C57BL/6 mice, AR231453 exposure was more than 100-fold higher than its in vitro EC50 value. Furthermore, the compound was effective in all other mouse strains used in this study. Many structurally different compounds have been evaluated in wild-type littermates and GPR119-deficient mice, with results similar to AR231453 (data not shown). In summary, these data strongly suggest that the action of AR231453 is mediated by GPR119, clearly demonstrating that GPR119 is a physiologically significant regulator of glucose homeostasis. While these studies support the hypothesis that GPR119 regulates glucose homeostasis by directly enhancing pancreatic β-cell function, it must be recognized that GPR119 may act through other mechanisms. The efficacy of AR231453 decreased by nearly 50% after intraperitoneal glucose injection, suggesting that its action may also involve the regulation of incretin signaling. This hypothesis warrants further investigation. Our studies in islets and mice show that GPR119 stimulates insulin release in a glucose-dependent manner. Even when administered well above the maximum effective dose of AR231453 in vivo (100 mg/kg in C57BL/6 mice), no stimulation of insulin release or reduction in blood glucose was observed under fasting conditions. In contrast, glibenclamide induced significant insulin release and marked hypoglycemia in fasted mice. These data are consistent with the known mechanisms by which GIP and GLP-1 stimulate insulin release. [1] In this study, we found that AR231453 treatment significantly enhanced diabetes reversal in mice receiving microdose of islet transplants. This confirms our previous study of using the endogenous GPR119 ligand oleoylethanolamine (OEA) and the selective small molecule GPR119 agonist PSN632408 to enhance islet transplant function. [8] Diabetes reversal depends on the function of the islet transplant, as hyperglycemia relapses after removal of the left kidney carrying the islet transplant. We also found that AR231453 treatment significantly increased β-cell proliferation in islet transplants. In our previous study, we found that exendin-4 stimulated β-cell proliferation in islet transplants from both young and old donor mice. Therefore, we used islets from older donors for transplantation. Although previous studies have shown that β-cell proliferation is reduced in aged mice, our data indicate that AR231453 can stimulate β-cell proliferation in islets isolated from aged 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 proliferation in cultured islets (paper submitted). Therefore, AR231453 may improve islet transplantation function and stimulate β-cell proliferation in islet transplantation by directly activating GPR119 on islets. However, AR231453 can also stimulate GLP-1 secretion from intestinal endocrine L cells by activating GPR119. We found that AR231453 treatment increased plasma GLP-1 levels in mice. Therefore, the stimulation of β-cell proliferation in islet transplantation by AR231453 may also be related to increased plasma GLP-1 concentration. To investigate the direct and/or indirect effects of AR231453 on improving islet transplant function and stimulating β-cell proliferation during islet transplantation through GRP119 activation, GLP-1 receptor knockout mice and GPR119 knockout mice were needed as islet donors and/or transplant recipients. Targeting GPR119 is a novel therapeutic approach that increases β-cell proliferation and improves islet transplant function, with potential applications in human islet transplantation. Single-donor islet transplantation is a highly attractive target in clinical islet transplantation. The number of cells from a single donor islet is often insufficient to restore normal blood glucose levels, although islets isolated from a single donor pancreas can reverse diabetes. Therefore, developing strategies based on GPR119 agonists to increase β-cell numbers and improve islet transplant function may help improve the success rate of single-donor islet transplantation. Since patients with type 1 diabetes retain a certain number of β-cells at the onset of the disease, GPR119 agonists could also be used to restore normal blood glucose levels by stimulating β-cell replication and increasing β-cell numbers. ^[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.)