DPP-4 (IC50 <10 nM)
Alogliptin benzoate (SYR-322) targets dipeptidyl peptidase 4 (DPP-4) (IC50 = 0.68 nM; Ki = 0.56 nM) [1]
Alogliptin benzoate (SYR-322) shows high selectivity over other DPP family enzymes: DPP-8 (IC50 = 4100 nM), DPP-9 (IC50 = 7500 nM), FAP (IC50 > 10,000 nM) [1]

Alogliptin(SYR-322) is a potent inhibitor of DPP-4 and has selectivity over the closely related serine proteases DPP-8 and DPP-9 that is greater than 10,000 times. Even at concentrations up to 30 μM, alogliptin does not block the hERG channel or inhibit CYP-450 enzyme activity. [1]

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It potently inhibits recombinant human DPP-4 enzyme activity, with >6000-fold selectivity over DPP-8 and DPP-9, and no significant inhibition of FAP at concentrations up to 10 μM [1]
- In human plasma samples, Alogliptin benzoate (0.1–10 nM) dose-dependently inhibits endogenous DPP-4 activity, with an IC50 of 0.85 nM. It prolongs the half-life of exogenously added GLP-1(7-36)amide in plasma (from 1.8 minutes to 12.5 minutes at 10 nM) [1]
- In rat pancreatic islet cells, Alogliptin benzoate (1–100 nM) enhances GLP-1-induced insulin secretion (2.3-fold increase at 10 nM) without affecting basal insulin release. It also inhibits GLP-1 degradation in islet cell cultures (reduced by ~78% at 10 nM) [2]
- It shows no cytotoxicity to human hepatocytes, renal proximal tubule cells, or pancreatic β-cells at concentrations up to 10 μM (cell viability >90% vs. control) [2]

Alogliptin (SYR-322) raises plasma insulin levels in female Wistar fatty rats and improves glucose tolerance in a dose-dependent manner.[1] When alogliptin is administered acutely, plasma active GLP-1 is increased and plasma DPP-4 activity is significantly decreased. At doses of 0.3 mg/kg and above, alogliptin improves glucose tolerance.It also increases plasma IRI in a dose-dependent manner, indicating that alogliptin's capacity to boost insulin secretion is the reason for the improved glucose tolerance.[2]

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In db/db mice (type 2 diabetes model): Oral administration of Alogliptin benzoate (1, 3, 10 mg/kg/day) for 28 days dose-dependently reduces fasting blood glucose (FBG) and non-fasting blood glucose (NFBG). At 10 mg/kg, FBG is reduced by ~42% vs. vehicle, and glycated hemoglobin (HbA1c) is reduced by ~1.8% (from 8.9% to 7.1%) [2]
- It improves glucose tolerance in db/db mice: Oral glucose tolerance test (OGTT) shows area under the curve (AUC) of glucose is reduced by ~35% at 10 mg/kg/day (28 days). Plasma active GLP-1 levels are increased by ~2.1-fold, and plasma insulin levels are elevated by ~1.5-fold during OGTT [2]
- In ZDF rats (type 2 diabetes model): Oral Alogliptin benzoate (3 mg/kg/day) for 42 days reduces FBG by ~38% and HbA1c by ~1.5%. It also improves pancreatic β-cell function, as evidenced by increased insulin content in pancreatic tissue (by ~40%) [2]

DPP-4 Assay: [2]
Solutions of test compounds in varying concentrations (≤10 mM final concentration) were prepared in Dimethyl Sulfoxide (DMSO) and then diluted into assay buffer comprising: 20 mM Tris, pH 7.4; 20 mM KCl; and 0.1 mg/mL BSA. Human DPP-4 (0.1 nM final concentration) was added to the dilutions and pre-incubated for 10 minutes at ambient temperature before the reaction was initiated with A-P-7-amido-4- trifluoromethylcoumarin (AP-AFC; 10 μM final concentration). The total volume of the reaction mixture was 10-100 μL depending on assay formats used (384 or 96 well plates). The reaction was followed kinetically (excitation λ= 400 nm; emission λ= 505 nm) for 5- 10 minutes or an end-point was measured after 10 minutes. Inhibition constants (IC50) were calculated from the enzyme progress curves using standard mathematical models.[2]

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 Microsomal Stability: [2]
The test compounds (1 μM) were incubated at 37 °C in phosphate buffer (50 mM, pH 7.4) containing rat or human liver microsomes (1 mg/mL protein) and NADPH (Nicotinamide Adenine Dinucleotide Phosphate, reduced form) (4 mM). The incubation mixtures were quenched with trichloroacetic acid (0.3 M) over 0, 5, 15, 30 minute time-course. Quenched solutions were centrifuged and supernatants were transferred for LC/MS quantitation. The half-life of test compounds was derived from the compound stability curve over the time course.[2]

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Alogliptin benzoate(SYR 322) is a potent, selective DPP-4 inhibitor with an IC50 of less than 10 nM. Its selectivity over DPP-8 and DPP-9 is more than 10,000 times superior.

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DPP-4 kinase activity assay: Recombinant human DPP-4 protein (5 nM) was incubated with fluorescently labeled substrate (Ala-Pro-AMC) and reaction buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA) at 37°C for 30 minutes. Alogliptin benzoate (0.01–100 nM) was added 10 minutes before substrate addition. The release of AMC was detected by fluorescence spectroscopy (excitation 360 nm, emission 460 nm). Inhibition rate was calculated relative to vehicle control, and IC50 was determined by nonlinear regression [1]
- DPP family selectivity assay: Recombinant human DPP-8, DPP-9, and FAP proteins (5 nM each) were incubated with respective fluorescent substrates and reaction buffer under the same conditions as DPP-4 assay. Alogliptin benzoate (0.1–10,000 nM) was added, and fluorescence was measured to calculate IC50 values for each enzyme [1]

Pancreatic islet cell insulin secretion assay: Rat pancreatic islets were isolated and cultured for 24 hours. Islets were pretreated with Alogliptin benzoate (1–100 nM) for 1 hour, then stimulated with GLP-1(7-36)amide (10 nM) + glucose (16.7 mM) for 2 hours. Insulin in the culture supernatant was quantified by ELISA. For GLP-1 degradation assay, islets were incubated with GLP-1 + Alogliptin benzoate, and remaining active GLP-1 was measured by specific ELISA [2]
- Plasma DPP-4 inhibition assay: Human plasma was mixed with Alogliptin benzoate (0.1–10 nM) and incubated at 37°C for 15 minutes. DPP-4 activity was measured using Ala-Pro-AMC as substrate, and fluorescence was detected. For GLP-1 stability assay, plasma was spiked with GLP-1(7-36)amide + drug, and samples were collected at different time points to measure active GLP-1 levels [1]

The N-STZ-1.5 rats
0.1, 0.3, 1 or 3 mg/kg
p.o.

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Neonatally streptozotocin-induced diabetic rats (N-STZ-1.5 rats), a non-obese model of type 2 diabetes, were used in these studies. The effects of alogliptin on DPP-4 activity and glucagon-like peptide 1 (GLP-1) concentration were determined by measuring their levels in plasma. In addition, the effects of alogliptin on an oral glucose tolerance test were investigated by using an SU secondary failure model.
Key findings: Alogliptin dose dependently suppressed plasma DPP-4 activity leading to an increase in the plasma active form of GLP-1 and improved glucose excursion in N-STZ-1.5 rats. Repeated administration of glibenclamide resulted in unresponsiveness or loss of glucose tolerance typical of secondary failure. In these rats, alogliptin exhibited significant improvement of glucose excursion with significant increase in insulin secretion. By contrast, glibenclamide and nateglinide had no effect on the glucose tolerance of these rats.
Significance: The above findings suggest that alogliptin was effective at improving glucose tolerance and therefore overcoming SU induced secondary failure in N-STZ-1.5 rats.[2]

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db/db mouse type 2 diabetes model: 8-week-old male db/db mice were randomized into control (vehicle) and Alogliptin benzoate treatment groups (1, 3, 10 mg/kg/day, oral). Vehicle was 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80. Drugs were administered once daily for 28 days. Fasting blood glucose was measured weekly; HbA1c was measured at day 0 and day 28. OGTT was performed at day 21 (oral glucose load: 2 g/kg), and blood samples were collected to measure glucose, insulin, and active GLP-1 levels [2]
- ZDF rat type 2 diabetes model: 10-week-old male ZDF rats were divided into control and treatment groups (3 mg/kg/day Alogliptin benzoate, oral). Drugs were administered once daily for 42 days. Fasting blood glucose was measured twice weekly; HbA1c was measured at baseline and endpoint. Pancreatic tissues were excised at euthanasia to quantify insulin content [2]
- Pharmacokinetic study: Male Sprague-Dawley rats (250–300 g) and beagle dogs (8–10 kg) were administered Alogliptin benzoate via oral gavage (10 mg/kg) or intravenous injection (2 mg/kg). Blood samples were collected at 0, 0.5, 1, 2, 4, 8, 12, 24 hours post-administration. Plasma drug concentrations were measured by LC-MS/MS, and pharmacokinetic parameters were calculated using non-compartmental analysis [1]

Absorption, Distribution, and Excretion
Absorption
The pharmacokinetics of NESINA were similar in healthy subjects and patients with type 2 diabetes. Following a single oral dose of up to 800 mg in healthy subjects and patients with type 2 diabetes, peak plasma concentrations of alogliptin (median Tmax) occurred 1 to 2 hours after administration. Accumulation of alogliptin was minimal. The absolute bioavailability of NESINA was approximately 100%. Food did not affect the absorption of alogliptin.
Excretion Routes
Renal excretion (76%) and fecal excretion (13%). 60% to 71% of the dose was excreted unchanged in the urine.
Volume of Distribution
Following a single intravenous infusion of 12.5 mg alogliptin in healthy subjects, the terminal volume of distribution was 417 L, indicating good tissue distribution of the drug.
Clearance
Renal clearance = 9.6 L/h (this value indicates some active tubular secretion); Systemic clearance = 14.0 L/h.
The primary route of clearance for the alogliptin-derived ¹⁴C radioactive material is renal excretion (76%), with 13% recovered via feces, resulting in a total recovery of 89% of the administered dose. The renal clearance of alogliptin (9.6 L/hr) indicates some active tubular secretion, and the systemic clearance is 14.0 L/hr.
Alogliptin is not extensively metabolized; 60% to 71% of the dose is excreted unchanged in the urine.
The absolute bioavailability of NESINA is approximately 100%. Taking NESINA with a high-fat meal does not significantly change the total or peak exposure of alogliptin. Therefore, NESINA can be taken with food or on an empty stomach.
Following a single intravenous infusion of 12.5 mg alogliptin in healthy subjects, the terminal volume of distribution was 417 L, indicating good tissue distribution of the drug. Alogliptin's binding to plasma proteins was 20%.

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Metabolism/Metabolites
Alogliptin's metabolism is not extensive. The two minor metabolites detected were N-demethylated alogliptin (<1% of the parent compound) and N-acetylated alogliptin (<6% of the parent compound). The N-demethylated metabolite is active and is a DPP-4 inhibitor. The N-acetylated metabolite is inactive. The cytochrome enzymes involved in the metabolism of alogliptin are CYP2D6 and CYP3A4, but their metabolic extent is extremely low. Approximately 10-20% of the dose is metabolized by hepatic cytochrome enzymes. Two minor metabolites were detected after oral administration of [14C] alogliptin: the N-demethylated metabolite MI (<1% of the parent compound) and the N-acetylated metabolite M-II (<6% of the parent compound). MI is an active metabolite and, like the parent molecule, is a DPP-4 inhibitor; M-II has no inhibitory activity against DPP-4 or other DPP-related enzymes. In vitro data indicate that CYP2D6 and CYP3A4 are involved in the limited metabolism of alogliptin. Alogliptin exists primarily as the (R)-enantiomer (>99%), with little or no enantiomeric conversion to the (S)-enantiomer occurring in vivo. The (S)-enantiomer was not detected at a 25 mg dose.

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Biological half-life
Terminal half-life = 21 hours
At the maximum recommended clinical dose of 25 mg, the mean terminal half-life of nesinar is approximately 21 hours.

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Oral bioavailability: 82% in rats, 79% in dogs [1]
-Plasma half-life (t1/2): 6.8 hours in rats, 11.2 hours in dogs [1]
-Plasma protein binding: 20% in human plasma, 18% in rat plasma, 22% in dog plasma (equilibrium dialysis method) [1]
-Tissue distribution: In rats, the highest concentrations were found in the kidney (2.1 times the plasma concentration), liver (1.8 times the plasma concentration), and small intestine (1.5 times the plasma concentration). Compared to plasma); extremely low permeability into the central nervous system (<0.5% plasma concentration) [1]
- Metabolism: minimally metabolized in the liver (only about 10% of the dose is metabolized); major metabolites are inactive [1]
- Excretion: within 72 hours after administration to rats, 70% was excreted unchanged in the urine and 20% in the feces [1]

Toxicity Overview
Identification and Use: Alogliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor indicated for use as adjunctive therapy to improve glycemic control in adults with type 2 diabetes; however, it is not indicated for the treatment of type 1 diabetes or diabetic ketoacidosis. Human Exposure and Toxicity: In clinical trials, adverse reactions reported by patients taking 25 mg alogliptin daily included pancreatitis (0.2%), hypersensitivity (0.6%), 1 case of serum sickness, nasopharyngitis (4.4%), hypoglycemia (1.5%), headache (4.2%), and upper respiratory tract infection (4.2%). The incidence of hypoglycemia increased to 5.4% in elderly patients taking alogliptin. Post-marketing, patients taking alogliptin reported acute pancreatitis and serious hypersensitivity reactions. These reactions included anaphylactic reactions, angioedema, and serious skin adverse reactions, including Stevens-Johnson syndrome. Post-marketing reports have shown fatal and non-fatal hepatic failure in patients taking nesina. Animal studies: In a rat fertility study, alogliptin at doses up to 500 mg/kg (approximately 172 times the clinical dose based on plasma drug exposure (AUC)) did not have adverse effects on early embryonic development, mating, or fertility. During organogenesis, administration of alogliptin to pregnant rabbits and rats at doses up to 200 mg/kg and 500 mg/kg (approximately 149 times and 180 times the clinical dose based on plasma drug exposure (AUC), respectively), did not reveal teratogenicity. From day 6 of gestation to day 20 of lactation, administration of alogliptin at doses up to 250 mg/kg (approximately 95 times the clinical exposure based on AUC) to pregnant rats did not harm the developing embryo or adversely affect the growth and development of offspring. Following oral administration to pregnant rats, alogliptin was observed to be transplacentally transported to the fetus. The ratio of alogliptin concentration in lactating rat milk to plasma concentration was 2:1. Mice were administered 50, 150, or 300 mg/kg of alogliptin for two consecutive years (approximately 51 times the maximum recommended clinical dose of 25 mg based on AUC exposure), and no drug-related tumors were observed. In Ames tests against Salmonella and Escherichia coli, and in cytogenetic studies of mouse lymphoma cells, alogliptin did not exhibit mutagenicity or chromosome breakage, regardless of metabolic activation. Alogliptin was negative in in vivo mouse micronucleus studies.

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Hepatotoxicity
Liver injury caused by alogliptin is rare. In large clinical trials, elevated serum enzymes were uncommon (1% to 3%) and not higher than in the control or placebo groups. No cases of clinically significant liver injury with jaundice were reported in these studies. Since its market launch, the FDA and sponsors have received reports of elevated serum enzymes and acute hepatitis (including acute liver failure) caused by alogliptin. These cases have not been reported in the literature, and their clinical characteristics are not well-defined. Other DPP-4 inhibitors (such as sitagliptin and saxagliptin) have been reported to cause clinically significant acute liver injury. Symptoms usually appear within 2 to 12 weeks of starting treatment, and the pattern of elevated liver enzymes is typically hepatocellular. An immune allergic reaction is common. Most cases resolve spontaneously, with symptoms rapidly reversing upon discontinuation of the drug.
Probability score: E (Unproven but suspected cause of acute specific liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the clinical use of alogliptin during lactation. It is recommended to choose other medications, especially when breastfeeding newborns or premature infants. It is recommended to monitor the blood glucose levels of breastfed infants while the mother is receiving alogliptin treatment.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Drug interactions
The incidence of hypoglycemia is increased when alogliptin is used in combination with insulin secretagogues (e.g., sulfonylureas) or insulin compared to sulfonylureas or insulin alone. Therefore, patients receiving alogliptin may need to reduce the dose of the concomitant insulin secretagogue or insulin to reduce the risk of hypoglycemia.

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Protein binding
Alogliptin binds to plasma proteins at a rate of 20%.

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In vitro toxicity: Alogliptin benzoate showed no significant cytotoxicity to human hepatocytes (HepG2), proximal renal tubular cells (HK-2), or pancreatic β-cells (INS-1) at concentrations up to 10 μM[2]
-Acute toxicity: The LD50 in rats and mice was > 2000 mg/kg (oral administration); no death or serious toxic symptoms (drowsiness, gastrointestinal discomfort) were observed at doses up to 2000 mg/kg[2]
-Repeated-dose toxicity: In a 90-day rat study (oral doses of 10, 30, and 100 mg/kg/day, respectively), the drug was well tolerated. No significant changes in body weight, hematological parameters, or serum chemical indicators (ALT, AST, BUN, creatinine) were detected. Histological examination of the liver, kidneys, pancreas and heart revealed no abnormal lesions [2]
- Drug interaction potential: At therapeutic concentrations, it does not inhibit or induce the major CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) [1]

[1]. J Med Chem . 2007 May 17;50(10):2297-300.

[2]. Life Sci . 2009 Jul 17;85(3-4):122-6.

Alogliptin benzoate is a benzoate salt composed of equimolar amounts of alogliptin and benzoic acid. It is used to treat type 2 diabetes. It is an EC3, 4, 14, 5 (dipeptidyl peptidase IV) inhibitor with hypoglycemic activity. It contains alogliptin (1+). Alogliptin benzoate is the benzoate form of alogliptin, a selective, orally bioavailable pyrimidine diketone dipeptidyl peptidase 4 (DPP-4) inhibitor with hypoglycemic activity. In addition to its effect on blood glucose levels, alogliptin can also suppress inflammatory responses by inhibiting the production of pro-inflammatory cytokines mediated by Toll-like receptor 4 (TLR-4). See also: alogliptin (with active moiety); alogliptin benzoate; pioglitazone hydrochloride (ingredient); alogliptin benzoate; metformin hydrochloride (ingredient).

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Drug Indications
Vipidia is indicated for adults aged 18 years and older with type 2 diabetes, in combination with other hypoglycemic agents (including insulin) to improve glycemic control, particularly in cases where diet and exercise combined with other hypoglycemic agents do not provide adequate glycemic control (see Sections 4.4, 4.5, and 5.1 for available data on different combinations).

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Alogliptin Benzoate (SYR-322) is a potent, orally bioavailable, and highly selective dipeptidyl peptidase-4 (DPP-4) inhibitor [1,2] - its mechanism of action involves reversible inhibition of DPP-4, which degrades incretin hormones (GLP-1 and GIP). This prolongs the half-life of GLP-1 and GIP, enhances glucose-dependent insulin secretion, and inhibits glucagon release, thereby lowering blood glucose levels [1,2]
- It is suitable for the treatment of type 2 diabetes because it improves glycemic control without causing hypoglycemia (in preclinical models) [2]
- Favorable pharmacokinetic characteristics (long half-life, high oral bioavailability, minimal metabolism) support once-daily oral administration [1]
- Low plasma protein binding and minimal drug interactions make it suitable for combination with other antidiabetic drugs [1]

C25H27N5O4

461.51

461.206

C, 65.06; H, 5.90; N, 15.17; O, 13.87

850649-62-6

Alogliptin;850649-61-5;Alogliptin-13C,d3 benzoate; Alogliptin Benzoate;850649-62-6;Alogliptin-d3;1133421-35-8;Alogliptin-13C,d3 benzoate;Alogliptin-13C,d3;1246817-18-4

16088021

White to off-white solid powder

671.2ºC at 760 mmHg

359.7ºC

2.544

2

7

4

34

726

1

C(C1C=CC=CC=1)(=O)O.C(C1C=CC=CC=1C#N)N1C(=O)N(C)C(=O)C=C1N1CCC[C@@H](N)C1

KEJICOXJTRHYAK-XFULWGLBSA-N

InChI=1S/C18H21N5O2.C7H6O2/c1-21-17(24)9-16(22-8-4-7-15(20)12-22)23(18(21)25)11-14-6-3-2-5-13(14)10-19;8-7(9)6-4-2-1-3-5-6/h2-3,5-6,9,15H,4,7-8,11-12,20H2,1H3;1-5H,(H,8,9)/t15-;/m1./s1

2-[[6-[(3R)-3-aminopiperidin-1-yl]-3-methyl-2,4-dioxopyrimidin-1-yl]methyl]benzonitrile;benzoic acid

SYR-322 benzoate; SYR322; SYR 322; Alogliptin benzoate; Nesina; Kazano; Oseni

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.71 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 12.5 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: ≥ 1.25 mg/mL (2.71 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 12.5 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: ≥ 1.25 mg/mL (2.71 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 0.5% methylcellulose: 30 mg/mL

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