HIF-PH/hypoxia-inducible factor prolyl hydroxylase
HIF prolyl hydroxylases (PHDs), specifically PHD2
Inhibition of PHDs leads to stabilization of HIF-1α under normoxic conditions.[4]

Vadadustat improves iron mobilization and stimulates the body's natural production of erythropoietin. Vadadustat raises reticulocytes, plasma EPO, and Hb levels in a dose-dependent manner and is well tolerated in both healthy volunteers and patients with chronic renal disease. The vadadustat-induced increases in plasma EPO levels followed a typical diurnal pattern, peaking before the subsequent dose and being similar in size to physiological increases at moderate elevations. By raising transferrin levels and decreasing hepcidin, vadadustat promotes iron homeostasis. When compared to conventional ESAs, once-daily oral vadadustat that has been titrated to raise and maintain Hb within the target range may offer a number of benefits [1]. It has been noted that Vadadustat has a half-life of roughly 4.5 hours. The mean hemoglobin levels of the patients rose from 9.91 g/dL at baseline to 10.54 g/dL on day 29. From 334.1 ng/mL at baseline to 271.7 ng/mL on day 29, ferritin levels dropped [2].

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Vadadustat preconditioning (40 μM, 6 h) downregulated expression of immune-related genes (IL24, IL1B, CXCL8, PDCD1LG1, PDCD1LG2, HIF1A, CCL2, IL6) and upregulated IL17RD, CCL28, and LEP in human bone marrow-derived mesenchymal stromal cells (BM-MSCs).
Secretome analysis showed decreased secretion of IL6 (by 51%), HGF (by 47%), CCL7 (by 42%), and CXCL8 (by 40%) after 24 h treatment with 40 μM Vadadustat.
In mixed lymphocyte reaction (MLR), Vadadustat enhanced the inhibitory effect of MSCs on proliferation of allostimulated human PBMCs.
Transwell migration assay demonstrated that secretome from Vadadustat-preconditioned MSCs reduced chemotaxis of monocyte-enriched PBMCs by 46% compared to control MSCs secretome.[4]

Current treatment of anemia in chronic kidney disease (CKD) with erythropoiesis-stimulating agents can lead to substantial hemoglobin oscillations above target range and high levels of circulating erythropoietin. Vadadustat (AKB-6548), a novel, titratable, oral hypoxia-inducible factor prolyl hydroxylase inhibitor induces endogenous erythropoietin synthesis and enhances iron mobilization. In this 20-week, double-blind, randomized, placebo-controlled, phase 2b study, we evaluated the efficacy and safety of once-daily vadadustat in patients with stages 3a to 5 non-dialysis-dependent CKD. The primary endpoint was the percentage of patients who, during the last 2 weeks of treatment, achieved or maintained either a mean hemoglobin level of 11.0 g/dl or more or a mean increase in hemoglobin of 1.2 g/dl or more over the predose average. Significantly, the primary endpoint was met in 54.9% of patients on vadadustat and 10.3% of patients on placebo. Significant increases in both reticulocytes and total iron-binding capacity and significant decreases in both serum hepcidin and ferritin levels were observed in patients on vadadustat compared with placebo. The overall incidence of adverse events was comparable between the 2 groups. Serious adverse events occurred in 23.9% and 15.3% of the vadadustat- and placebo-treated patients, respectively. Three deaths occurred in the vadadustat arm. Thus, this phase 2b study demonstrated that vadadustat raised and maintained hemoglobin levels in a predictable and controlled manner while enhancing iron mobilization in patients with nondialysis-dependent CKD[1].

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Wild type and ERFE knockout mice with and without CKD were treated with vadadustat or vehicle. In both wild type and ERFE knockout CKD models, vadadustat was similarly effective, as evidenced by normalized hemoglobin concentrations, increased expression of duodenal iron transporters, lower serum hepcidin levels, and decreased tissue iron concentrations. This is consistent with ERFE-independent increased iron mobilization. Vadadustat treatment also lowered serum urea nitrogen and creatinine concentrations and decreased expression of kidney fibrosis markers. Lastly, vadadustat affected fibroblast growth factor 23 (FGF23) profiles: in non-CKD mice, vadadustat increased plasma total FGF23 out of proportion to intact FGF23, consistent with the known effects of hypoxia-inducible factor-1α and erythropoietin on FGF23 production and metabolism. However, in the mice with CKD, vadadustat markedly decreased both total and intact FGF23, effects likely contributed to by the reduced loss of kidney function. Thus, in this CKD model, vadadustat ameliorated anemia independently of ERFE, improved kidney parameters, and decreased FGF23. How vadadustat affects CKD progression in humans warrants future studies.[3]

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In a Phase 1a single-dose study involving 8 healthy men (6 receiving Vadadustat, 2 placebo), Vadadustat was observed to increase endogenous erythropoietin (EPO) levels in a manner comparable to the expected physiologic diurnal variation.[2]
In a Phase 2a open-label dose escalation study, 10 patients with non-dialysis dependent (NDD) chronic kidney disease (CKD) stages 3–4 received Vadadustat once daily for 28 days (starting dose: 400 mg for stage 3, 300 mg for stage 4). Hemoglobin levels increased from 9.91 g/dL at baseline to 10.54 g/dL by day 29, and serum ferritin levels decreased from 334.1 ng/mL to 271.7 ng/mL.[2]
In a Phase 2b, multicenter, double-blind, randomized, placebo-controlled study of 210 NDD-CKD patients, Vadadustat treatment resulted in a significantly greater percentage of patients achieving a successful hemoglobin response (≥11.0 g/dL or ≥1.2 g/dL increase from baseline) compared to placebo (54.9% vs 10.3%, p<0.0001).[2]
In a trial of 94 hemodialysis patients switched from ESA to Vadadustat (300 mg once daily, 450 mg once daily, or 450 mg thrice weekly), mean hemoglobin levels remained stable from baseline to week 16 (change ranged from -0.02 to -0.04 g/dL).[2]

Analysis of BM-MSCs Cytokine Secretion by Antibody Array Proteome Profiler[4]
The relative changes in secretory activity of Vadadustat treated BM-MSCs compared to control cells were examined using the Proteome Profiler Human XL Cytokine Array. The Proteome Profiler membrane-based antibody array enables to simultaneously measure the relative level of 102 human cytokines in a single sample. For the purpose of this assay, BM-MSCs from 6 donors were grown in a standard growth medium on a 6-well plates until approximately 80% confluency was achieved. 24 h before the start of the experiment, all cells were primed with IFNγ (25 ng/mL). Next day, cells were washed and culture medium was replaced with OptiMEM Medium, no phenol red with reduced FBS content to 4% and supplemented with 1.0% penicillin–streptomycin with/without Vadadustat 40 μM. Cells of each population were treated in triplicate. After 24 h treatment cells supernatants were collected in an Eppendorf tube (1.5 mL), centrifuged at 4500 rpm for 5 min, transferred to new tubes, mixed and divided into 200 µL aliquots and frozen in −80 °C. Prior to the analysis, cell supernatants from 6 donors were thawed on ice and pooled. The analysis was performed according to the manufacturer’s instruction. Chemiluminescence of membranes was detected with ChemiDoc MP Imaging System and the integrated optical density of each spot was measured and quantified using Image Lab software.

Preconditioning of Human BM-MSCs with Vadadustat[4]
\nIn the presented study “pharmacological” hypoxia was achieved by culturing cells with the selective PHDs inhibitor, Vadadustat (AKB-6548). Based on preliminary data (Western blot analysis of HIF-1α stabilization and the MTT test) we decided to select the Vadadustat concentration of 40 μM for further studies. Vadadustat was dissolved and stored in −80 °C as 5 mM stock solution in DMSO according to the manufacturer instruction. Notably, no more than 0.8% (v/v) of DMSO was finally present in the culture medium, which did not cause any noticeable cytotoxic effect (MTT analysis presented in Supplementary Figure S1). The control group of MSCs was incubated with the same dose (0.8% v/v) of DMSO alone.\n

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\n\nMixed Lymphocyte Reaction (MLR) Assay[4]
\nFor the purpose of MLR assay human PBMCs were isolated from buffy coats from 6 healthy blood donors. The assay was performed in three independent sets of experiments on two donors each. Supernatants from 6 populations of IFNγ (25 ng/mL) primed BM-MSCs treated for 24 h with/without Vadadustat 40 μM were used to determine the effect of Vadadustat pretreatment on immunomodulatory activity of MSCs secretome. In this study, half of the isolated PBMCs were inactivated for 90 min with γ-irradiation. Next, 1 × 105 both responder (active) and irradiated (stimulatory) PBMCs were seeded into wells of 96-well plates in a combination of auto- (AAir, BBir) and allo- (ABir, BAir) stimulation. Cells were maintained in RPMI-1640 supplemented with 10% FBS and antibiotic–antimycotic solution (1% penicillin-streptomycin; 0.5% amphotericin B). The MLR assay were performed using 96-well plates. In the part of the wells where the direct effect of Vadadustat on auto- and allostimulated PBMCs as well as its effect on the interaction between MSCs and PBMCs were studied, 40 μM Vadadustat was added to the experimental wells daily as a stock solution. Control wells were treated daily with equivalent volumes of DMSO. In the remaining wells, in which the indirect effect of Vadadustat pre-conditioning on the interaction between MSCs and PBMCs was studied, a 1:1 mixture of RPMI-1640 growth medium and supernatants from 24 h cell culture of control or Vadadustat preconditioned MSCs was added once at the beginning of the experiment. Plates were then cultured for 5 days at 37 °C in a humidified atmosphere with 5% CO2. After 5 days of cell culture, PBMCs were pulsed with 1 μCi/well of 3H-thymidine (113 Ci/nmol, NEN) for the last 18 h of incubation and then harvested with an automated cell harvester. The 3H-thymidine incorporation into cells was measured based on the level of radioactivity reported as ‘Corrected Counts per Minute’ (CCPM) using a scintillation counter. All treatments were performed in triplicate.\n

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\nTranswell Migration Assay[4]
\nThe effect of 40 μM Vadadustat preconditioning on the chemotactic properties of the BM-MSCs secretome was investigated using a “96 Well Cell Migration Assay” reagent kit from Cultrex®, which utilize a simplified design of a Boyden chamber with polyethylene terephthalate (PET) membrane with pores of 8 μm size. For the migration test, we used monocyte-enriched PBMCs (n = 4) suspended in RPMI containing 0.5% human serum at a density of 4 × 106/mL. 50 μL of cell suspension from each donor were applied to the upper chambers of the plate (2 × 104 cells per well), each in duplicate. Quantities of 150 µL per well of growth medium (RPMI with 0.5% human serum) or freshly thawed, pooled supernatants from cultures of 7 MSCs populations were applied to the bottom chambers of the plate. Each of three treatments: growth medium alone, supernatants from control MSCs and MSCs preconditioned with Vadadustat was applied in duplicate. Plates were then incubated under standard conditions (37 °C, 5% CO2) for 48 h. After incubation, the upper chambers were carefully aspirated and the cells that migrated to the bottom compartments of the plate were detached using a cell dissociation solution with calcein acetomethylester (calcein-AM). Afterwards, plates were incubated at 37 °C for 30 min. During this time, cells internalized calcein-AM, and cellular esterases then cleaved it into free calcein. Released calcein possess strong fluorescence, that was used to estimate the number of migrated cells. After incubation, plates were disassembled and bottom chambers were fluorescently read at 485 nm excitation and 520 nm emission on Perkin Elmer Victor X4 plate reader. The degree of cell migration was assessed by comparing fluorescence in the wells with MSCs culture supernatants to fluorescence in wells with growth medium alone, and expressed as the ratio of migrating cells.\n\n

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Isolation and culture of BM-MSCs: Human BM-MSCs were isolated from bone marrow aspirates, cultured in low glucose DMEM supplemented with 10% FBS and antibiotics. Cells were grown at 37°C, 5% CO₂, and medium was replaced every other day. Cells between passages 4–6 were used for experiments.
Preconditioning with Vadadustat: BM-MSCs were treated with 40 μM Vadadustat for 6 h (for gene expression) or 24 h (for secretome analysis) under normoxic conditions. Control cells were treated with equivalent DMSO (0.8% v/v).
RNA isolation and real-time PCR: Total RNA was extracted, reverse transcribed, and real-time PCR was performed using SYBR Green. Gene expression was normalized to B2M.
Secretome analysis: Cytokine secretion was analyzed using Proteome Profiler Human XL Cytokine Array and Luminex multiplex immunoassay for IL6, CXCL8, IL4, IL10, and HGF.
Mixed lymphocyte reaction (MLR): PBMCs were isolated from buffy coats, irradiated for inactivation, and co-cultured with MSCs or their secretome. Proliferation was assessed by ³H-thymidine incorporation after 5 days.
Transwell migration assay: Monocyte-enriched PBMCs were placed in upper chambers, and MSCs secretome was added to lower chambers. Migration was assessed after 48 h using calcein-AM fluorescence.[4]

Full methods are detailed in the Supplementary Material. Six-week-old male EKO mice and WT littermates were placed on 8-week diets that did or did not contain 0.2% adenine, which induces CKD. To assess the treatment effects of vadadustat in the setting of CKD, for the last 3 weeks of the diets, the mice were treated daily with vadadustat solution, dosed at 75 mg/kg/d via oral gavage, or vehicle solution. Mice were killed at 14 weeks of age. We obtained whole blood, serum, plasma, liver, spleen,...[3]

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Effects of vadadustat on erythropoietic parameters in WT and EKO mice with and without CKD...[3]
To assess the efficacy of vadadustat in the presence or absence of ERFE and CKD, we evaluated the effects of 3 weeks of daily vadadustat treatment in WT and EKO mice with and without adenine diet-induced CKD. In the non-CKD model, in which the mice were not anemic, vadadustat induced similar increases in hemoglobin concentrations from pretreatment values in the WT and EKO groups (Supplementary Figure S1). In the CKD model, as expected, both WT and EKO groups developed moderate anemia...[3]

Absorption, Distribution and Excretion
Vadadustat is rapidly absorbed after both single and repeated administrations. The time to peak concentration (Tmax) of vadadustat is 2 to 3 hours. No significant drug accumulation was detected after repeated administrations of vadadustat in healthy subjects. Compared to the fasting state, Cmax and AUC decreased by 27% and 6%, respectively, after administration of 450 mg vadadustat tablets with a standard high-fat meal. Vadadustat can be taken on an empty stomach or with food. The mean plasma concentration/dose ratio of vadadustat increased from 0.50 to 0.55, indicating extremely low retention of vadadustat in erythrocytes. Following a single administration of 650 mg of radiolabeled vadadustat in healthy subjects, 85.9% of the dose was recovered, with 58.9% occurring in the urine and 26.9% in the feces. A small amount of unmetabolized vadadustat is excreted in urine and feces (<1% and 9%, respectively). The apparent volume of distribution of vadadustat in patients with chronic kidney disease is 11.6 L. In healthy subjects, the apparent total clearance after a single 300 mg dose of vadadustat ranges from 1.7 L/h to 1.9 L/h. The clearance of vadadustat in patients with chronic kidney disease is 0.8 L/h. Metabolism/Metabolites Vadadustat is primarily metabolized by UDP-glucuronyltransferase (UGT) via direct glucuronidation to form O-glucuronide conjugates. Metabolism by cytochrome P450 (CYP) is minimal. The major metabolite of vadadustat is vadadustat-O-glucuronide, which accounts for 15% of the total plasma radioactivity AUC and is catalyzed by various UGT enzymes, including UGT1A1, UGT1A7, UGT1A8, and UGT1A9. Watastat acyl glucuronide is a minor metabolite, accounting for 0.047% of total plasma radioactivity. All metabolites of wadastat are inactive.
Biological half-life
In patients with dialysis-dependent chronic kidney disease, the half-life of wadastat is 9.2 hours.
In a phase 1a single-dose study in healthy men, the half-life of wadastat was approximately 4.5 hours. [2]

Protein Binding
In human plasma, vadadustat has a protein binding rate ≥99.5%. In the phase IIb study conducted by Pergola et al., the most common drug-related adverse events in the vadadustat group were diarrhea (4.3%) and nausea (4.3%). The incidence of hypertension was higher in the vadadustat group compared to the placebo group, but all patients reporting hypertension had a history of hypertension. No effect on serum cholesterol levels was observed. [2] In a trial involving 94 hemodialysis patients, 78 adverse events (83.0%) and 13 serious adverse events (13.8%) were reported, all unrelated to the drug. [2]

[1]. Vadadustat, a novel oral HIF stabilizer, provides effective anemia treatment in nondialysis-dependent chronic kidney disease. Kidney Int. 2016 Nov;90(5):1115-1122.

[2]. Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors: A Potential New Treatment for Anemia in Patients With CKD. Am J Kidney Dis. 2017 Feb 24. pii: S0272-6386(17)30110-5.

[3]. Amelioration of chronic kidney disease-associated anemia by vadadustat in mice is not dependent on erythroferrone. Kidney Int. 2021 Jul;100(1):79-89.

[4]. Vadadustat, a HIF Prolyl Hydroxylase Inhibitor, Improves Immunomodulatory Properties of Human Mesenchymal Stromal Cells. Cells. 2020 Nov 1;9(11):2396..

One of the most common symptoms of advanced kidney disease is anemia, primarily caused by the kidneys' inability to respond to the anemic condition and thus fail to increase erythropoietin (EPO) production accordingly. Traditionally, treatment for anemia associated with chronic kidney disease (CKD) involves the use of exogenous erythropoietin (ESAs), such as dabepone alfa, to counteract the reduction in endogenous EPO production. While ESAs are effective, overuse has been associated with increased cardiovascular complications, CKD progression, and overall mortality. A relatively new alternative treatment for CKD-related anemia is the use of hypoxia-inducible factor prolyl hydroxylase (HIF-PH) small molecule inhibitors. These drugs inhibit the oxygen sensor in the prolyl hydroxylase domain, mimicking a hypoxic environment and activating hypoxia-inducible factors. These transcription factors play multiple roles, including stimulating erythropoiesis. Vadadustat, an oral HIF-PH inhibitor with a safety and efficacy non-inferior to dabepone alfa, is used to treat anemia in dialysis patients with chronic kidney disease (CKD). This drug was initially approved in Japan and received European Medicines Agency (EMA) approval in April 2023 for the treatment of symptomatic anemia in adult CKD patients undergoing long-term maintenance dialysis. Vadadustat is currently awaiting approval from the U.S. Food and Drug Administration (FDA). Vadadustat did not meet the pre-specified non-inferiority criteria for cardiovascular safety in non-dialysis-dependent CKD patients. Vadadustat is an orally bioavailable hypoxia-inducible factor prolyl hydroxylase (HIF-PHI) inhibitor with potential anti-anemic and anti-inflammatory activities. After administration, vadadustat binds to HIF-PH and inhibits its activity. HIF-PH is an enzyme responsible for degrading HIF family transcription factors under normoxic conditions. This prevents HIF degradation and promotes HIF activity. Increased HIF activity leads to increased endogenous erythropoietin production, thereby enhancing erythropoiesis. In addition, it can reduce the expression of the peptide hormone hepcidin, improve iron utilization, and increase hemoglobin (Hb) levels. HIFs can regulate gene expression in response to hypoxia levels, including genes required for erythropoiesis and iron metabolism. Furthermore, HIF 1-α (HIF1A) may reduce inflammation during acute lung injury (ALI) by HIF-dependently regulating glucose metabolism in alveolar epithelial cells.
Drug Indications
Vadadustat is indicated for the treatment of symptomatic anemia associated with chronic kidney disease (CKD) in adults undergoing long-term maintenance dialysis.
Vafseo is indicated for the treatment of symptomatic anemia associated with chronic kidney disease (CKD) in adults undergoing long-term maintenance dialysis.
Treatment of Anemia Due to Chronic Disease

Mechanism of Action
Hypoxia-inducible factor (HIF) is a transcription factor responsible for cellular survival under hypoxic conditions. They regulate a variety of processes, including angiogenesis, cell growth and differentiation, various metabolic processes, and erythropoiesis. Under normoxic conditions, HIF is degraded by hydroxylation by prolyl hydroxylase dioxygenase. Vadadustat is a HIF-prolyl hydroxylase (HIF-PHI) inhibitor that promotes increased HIF activity under non-hypoxic conditions. The Vadadustat-induced increase in HIF levels stimulates the production of endogenous erythropoietin, increasing iron mobilization, thereby promoting a gradual increase in hemoglobin levels and correcting iron metabolism. In patients with chronic kidney disease and anemia (whose normal erythropoiesis is impaired), this corrects the anemia.

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Pharmacodynamics
This study compared the efficacy of vadadustat with dabepoetin alpha in treating anemia in adult patients with dialysis-dependent chronic kidney disease. Vadadustat was non-inferior to dabepoetin alpha and met the primary hemoglobin level endpoint. In healthy subjects, administration of vadadustat from 600 mg to 1200 mg was not associated with clinically significant QTc interval prolongation. Compared with dabepoetin α, patients with dialysis-dependent chronic kidney disease treated with vadadustat had similar risks of death, myocardial infarction, and stroke. Vadadustat use may also lead to thromboembolic events, liver damage, hepatotoxicity, seizures, and elevated blood pressure. Vadadustat is a selective HIF prolyl hydroxylase inhibitor that can simulate hypoxia under normoxic conditions. It is currently in a phase III clinical trial for the treatment of secondary anemia in chronic kidney disease. Pretreatment of MSCs with Vadadustat may enhance their immunomodulatory properties, thereby potentially improving their therapeutic efficacy in autoimmune diseases. [4]

C14H11CLN2O4

306.7011

306.041

C, 54.83; H, 3.62; Cl, 11.56; N, 9.13; O, 20.87

1000025-07-9

23634441

White to off-white solid powder

2.312

3

5

4

21

393

0

C1=CC(=CC(=C1)Cl)C2=CC(=C(N=C2)C(=O)NCC(=O)O)O

JGRXMPYUTJLTKT-UHFFFAOYSA-N

InChI=1S/C14H11ClN2O4/c15-10-3-1-2-8(4-10)9-5-11(18)13(16-6-9)14(21)17-7-12(19)20/h1-6,18H,7H2,(H,17,21)(H,19,20)

(5-(3-chlorophenyl)-3-hydroxypicolinoyl)glycine

AKB-6548; PG1016548; B-506; AKB 6548; AKB-6548; Vafseo; PG-1016548; Vadadustat [USAN]; B506; PG1016548; PG 1016548; B506; AKB6548; PG-1016548;Vadadustat

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 : ~100 mg/mL (~326.05 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)