GPR109A (HCA2, HM74A) – human: pEC50 = 7.5 (EC50 ≈ 31.6 nM) for GTPγS binding; rat orthologue: pEC50 = 6.9 (EC50 ≈ 126 nM) [2]
HCA3 (human): pEC50 = 5.6 (EC50 ≈ 2.5 μM) [2]

GSK256073 acted as a full and potent agonist at recombinant human HCA2 in GTPγS binding assays using CHO cell membranes, with pEC50 = 7.5 ± 0.04 (mean ± S.E.M., n=18-20), compared to niacin pEC50 = 6.7. It also showed good activity at rat orthologue of HCA2 (pEC50 = 6.9) vs niacin (pEC50 = 6.4). At human HCA3, GSK256073 had a mean pEC50 of 5.6 (acifran standard pEC50 = 4.8). [2]
In human adipocyte membranes, GSK256073 was a full agonist in [35S]GTPγS binding assay, with pEC50 = 6.48 ± 0.09 and maximum response 142 ± 10% of niacin maximum (n=10). [2]
In rat primary adipocytes, pretreatment with GSK256073 (100 μM) suppressed isoprenaline (200 nM)-induced cAMP elevation. [2]
Selectivity screening against a panel of 7-transmembrane receptors, ion channels, transporters, and phosphodiesterases showed no significant activity; GSK256073 was selective versus adenosine receptors and PDEs despite its xanthine structure. [2]

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In membranes made from Chinese hamster experimental cells expressing recombinant human HCA2, GSK256073 is about ten times more potent than nicotinic acid against human HCA2 (pEC50 value of 7.5 compared to 6.7 for nicotinic acid), with a good activity value of 6.9 compared to the HCA2 proportional ortholog (pEC50) acid 6.4 [2]. Using the primary adipocyte kit, propylene glycol isoform (100 nM) is converted to GSK256073 (100 μM) [2].

In fasted rats, oral GSK256073 (3, 10, 30 mg/kg) produced rapid, dose-related decreases in plasma NEFA (max inhibition 74%, 81%, 88% respectively). Parallel decreases in triglycerides were observed, with 91% reduction still present at 6 h post-dose at 10 mg/kg. [2]
In tyloxapol-treated rats (to block triglyceride clearance), GSK256073 (0.03, 0.3, 3, 30 mg/kg p.o. given 30 min before tyloxapol) dose-dependently inhibited triglyceride accumulation by 7%, 30%, 51%, and 78% respectively at 4 h after tyloxapol. [2]
In anesthetized guinea pigs, i.v. GSK256073 (1-10 mg/kg) dose-dependently reduced NEFA; at 10 mg/kg, NEFA decrease was similar to niacin 10 mg/kg i.v., but ear temperature increase (flush surrogate) was only 40% of that induced by niacin. [2]
In conscious guinea pigs, oral GSK256073 (30 mg/kg) produced a very small (<1°C) ear temperature increase at 1 min post-dose, returning to baseline within 5 min; niacin 10 mg/kg caused >5°C increase. Antilipolytic effect of GSK256073 30 mg/kg was superior to niacin 10 mg/kg. [2]
In type 2 diabetes subjects (39 enrolled, 35 completed), GSK256073 administered for 2 days (5 mg BID, 10 mg QD, 25 mg BID, 50 mg QD) significantly reduced weighted mean glucose concentration from baseline (24-48 h interval) vs placebo, with max reduction -0.87 mmol/l (-1.20, -0.52) for 25 mg BID. Sustained suppression of NEFA and glycerol occurred across all doses. Serum insulin and C-peptide decreased, and HOMA-IR scores decreased 27-47%. No marked differences between QD vs BID or 10 vs 50 mg total daily dose. [1]

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NEFA recovery began quickly in topology when GSK256073 (sidewall; 1, 30, and 100 mg/kg) was administered; peak dose-related inhibition rates were 74%, 81%, and 88%, respectively. Even though the pattern of triglyceride reduction was longer, 91% of the reduction was still evident 6 hours after 10 mg/kg [2]. GSK256073 (IV; 1–10 mg/kg) decreases NEFA in a dose-related manner. ear temperature increased by 40% when 10 mg/kg iv niacin and 10 mg/kg iv GSK256073 were administered intravenously [2].

GTPγS binding assay using membranes from CHO cells stably expressing human HCA2, human HCA3, or rat HCA2: Membranes were incubated with increasing ligand concentrations. GTPγ[35S] binding was measured by scintillation proximity assay with PV-WCA beads. Assay buffer contained 20 mM HEPES, 100 mM NaCl, 10 mM MgCl2 pH7.4, plus saponin (60 μg/ml), WGA beads (250 μg/well), and 10 μM GDP. After adding radioligand, plates were incubated 4 h at room temperature then read on a microplate imager. [2]
GTPγS binding assay using human primary adipocyte membranes: Human adipocytes were digested with collagenase, filtered, washed, and homogenized. Membranes were prepared by differential centrifugation (100,000g, 20 min, 4°C). The assay was performed similarly as above. [2]
cAMP suppression assay in rat primary adipocytes: Rat fat pads were digested with collagenase, filtered, and adipocytes were floated by centrifugation. Adipocytes were incubated with test compound (100 μM) and 200 nM isoprenaline for 10 min at 37°C. Cells were lysed with dilute HCl, and cAMP was measured by ELISA. [2]

Rat antilipolytic activity: Male Sprague-Dawley rats (200-250 g) fasted ≥12 h. Compounds were given orally at 3, 10, 30 mg/kg (volume 10 ml/kg). Vehicle: 10% DMSO + 90% sodium bicarbonate aqueous solution (1.5 mg/ml) in sterile water. Blood samples (0.3 ml) from tail vein collected pre-dose and up to 8 h post-dose. Plasma NEFA measured. [2]
Rat triglyceride synthesis (tyloxapol model): Fasted male rats (n=4 per group) received oral GSK256073 (0.03-30 mg/kg) or vehicle. After 30 min, all animals received i.v. tyloxapol (300 mg/kg). Blood collected 4 h after tyloxapol, triglycerides quantified. [2]
Anesthetized guinea pig flushing model: Male Dunkin Hartley guinea pigs fasted 12 h, anesthetized with ketamine/xylazine and pentobarbitone. Tracheostomy, mechanical ventilation, jugular/carotid cannulation. Test compounds (niacin or GSK256073 1-10 mg/kg i.v.) given. Ear temperature measured by infrared probe every 1 min from 5 min pre-dose to 40 min post-dose. Blood taken for NEFA. [2]
Conscious guinea pig oral flushing model: Fasted guinea pigs dosed orally (5 ml/kg) with vehicle, niacin (1, 10 mg/kg), or GSK256073 (1, 30, 100 mg/kg). Ear temperature measured pre-dose and at intervals up to 2 h. Blood collected by cardiac puncture under terminal anesthesia at 0.5, 1, 2, 3, 4 h. [2]
Clinical trial in type 2 diabetes (NCT01147861): 39 subjects, randomized single-blind placebo-controlled three-period crossover. Each subject received placebo and two of four regimens: GSK256073 5 mg BID, 10 mg QD, 25 mg BID, 50 mg QD for 2 days. Subjects received 2200 kcal/day (25% breakfast, 35% lunch, 40% supper; 45% carb/35% fat/20% protein). Washout 5-12 days between periods. [1]
First-in-human study (healthy males): Part A – randomized single-blind double-dummy single-dose dose-rising three-period crossover, 5 cohorts of 8 subjects. Doses: GSK256073 5,25,50,100,150 mg; immediate release niacin 100,200,300,400 mg; placebo. Washout ≥7 days. Part B – open-label single-dose two-period crossover, GSK256073 50 mg fasted vs fed. [2]

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Animal/Disease Models: SD rat [2]
Doses: 1, 30 and 100 mg/kg
Route of Administration: Oral administration
Experimental Results: Inhibited NEFA expression in a dose-dependent manner.

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Animal/Disease Models: guinea pig [2]
Doses: 10 mg/kg
Route of Administration: intravenous (iv) (iv)injection
Experimental Results: It has the anti-lipolysis and flushing effects of HCA2 agonist.

In healthy male volunteers (Part A), single oral doses of GSK256073 (5, 25, 50, 100, 150 mg) were absorbed with median Tmax 2.01-5.00 h. Geometric mean AUC(0-∞) ranged from 7296 to 130864 ng·h/ml; Cmax from 394 to 6256 ng/ml. Mean half-life ranged 12.9-17.0 h. 70-95% of dose excreted unchanged in urine. Dose-proportional increase in AUC and Cmax. [2]
In fed state (50 mg with high-fat meal), AUC and Cmax were ~68% and ~20% higher than fasted, Tmax delayed from median 3 h (fasted) to 8 h (fed). [2]
In type 2 diabetes subjects (clinical trial), GSK256073 PK parameters: For 5 mg BID, day 2 AUC(0-24) geometric mean 14720 ng·h/ml, Cmax 988 ng/ml, median tmax 2.03 h. For 10 mg QD, day 2 AUC(0-24) 12758 ng·h/ml, Cmax 1152 ng/ml, tmax 2.00 h. For 25 mg BID, day 2 AUC(0-24) 68337 ng·h/ml, Cmax 4960 ng/ml, tmax 2.00 h. For 50 mg QD, day 2 AUC(0-24) 86895 ng·h/ml, Cmax 6815 ng/ml, tmax 2.00 h. Moderate to high inter-subject variability (39-68% CV). Day 2 exposures greater than day 1. [1]

In the type 2 diabetes clinical trial, no dose-related trends or clinically relevant changes in ECG, heart rate, blood pressure. Most common drug-related adverse events across GSK256073 groups: headache, dizziness, salt-craving, nausea (majority mild/moderate). No adverse events of flushing, erythema, tingling, pruritus. No serious adverse events. One subject withdrawn for decreased hemoglobin (protocol-required blood withdrawal ~500 ml over 3 weeks). Hypoglycemia (<3.9 mmol/l) did not occur. [1]
In first-in-human study, single doses of GSK256073 (5-150 mg) were well tolerated. No serious adverse events or withdrawals due to AEs. Flushing reported by 65% (22/34) of subjects following immediate release niacin, 2% (1/47) following GSK256073, and 5% (2/41) following placebo. [2]

[1]. GSK256073, a selective agonist of G-protein coupled receptor 109A (GPR109A) reduces serum glucose in subjects with type 2 diabetes mellitus.Diabetes Obes Metab. 2013 Nov;15(11):1013-21.

[2]. Discovery and characterization of GSK256073, a non-flushing hydroxy-carboxylic acid receptor 2 (HCA2) agonist.Eur J Pharmacol. 2015 Jun 5;756:1-7.

Mechanism of action: GSK256073 activates GPR109A on adipocytes, leading to Gi-mediated inhibition of adenylyl cyclase, reduced cAMP, decreased PKA activity, and reduced phosphorylation of perilipin and hormone-sensitive lipase (HSL). This inhibits lipolysis, lowering circulating NEFA. Reduced NEFA availability in liver and muscle decreases intracellular DAG and LCFA-CoA, reducing PKC activity and increasing insulin receptor tyrosine phosphorylation, thereby improving insulin sensitivity. Reduced glycerol release limits hepatic gluconeogenic substrate. [1]
GSK256073 did not lower mean serum triglyceride concentrations over 24 h in diabetic subjects (unlike niacin). [1]
In longer-term studies in dyslipidemic patients, GSK256073 did not increase HDLc, indicating dissociation of HDL-raising effect from HCA2 stimulation and NEFA lowering. [2]
The compound was evaluated as a potential treatment for type 2 diabetes, showing acute glucose-lowering effects. However, longer-term studies are needed to understand full clinical benefits and risks. [1]

C10H13CLN4O2

256.68882060051

256.073

C, 46.79; H, 5.10; Cl, 13.81; N, 21.83; O, 12.47

862892-90-8

1228650-42-7 (tris);862892-90-8 (free);

46215799

White to off-white solid powder

1.668

2

3

4

17

325

0

O=C1C2=C(N=C(N2)Cl)N(CCCCC)C(=O)N1

CGAMDQCXAAOFSR-UHFFFAOYSA-N

InChI=1S/C10H13ClN4O2/c1-2-3-4-5-15-7-6(12-9(11)13-7)8(16)14-10(15)17/h2-5H2,1H3,(H,12,13)(H,14,16,17)

8-chloro-3-pentyl-7H-purine-2,6-dione

GSK-256073; GSK 256073; GSK256,073; 862892-90-8; 8-chloro-3-pentyl-7H-purine-2,6-dione; GSK-256073; 1VI94C980K; GSK256073

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 : ~16.7 mg/mL (~64.9 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (6.51 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 16.7 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.)