H+/K+-ATPase or acid pump (pIC50 = ～6.0）

PF 03716556, with a pIC50 of 7.095 at pH 7.4, inhibits H+, K+-ATPase activity in porcine ion tight membrane vesicles in a concentration-dependent manner[1]. The affinities for a range of receptors, ion channels, and enzymes were determined to analyze selectivity profile. Acid secretion in Ghosh-Schild rats and Heidenhain pouch dogs were measured by titrating perfusate and gastric juice samples. PF-03716556 demonstrated 3-fold greater inhibitory activity than 5,6-dimethyl-2-(4-fluorophenylamino)-4-(1-methyl-1,2,3,4-tetrahydroisoquinoline-2-yl)pyrimidine (revaprazan), the only acid pump antagonist that has been available on the market, in ion-tight assay. The compound did not display any species differences, exhibiting highly selective profile including the canine kidney Na(+),K(+)-ATPase. Kinetics experiments revealed that PF-03716556 has a competitive and reversible mode of action.

Rats treated with PF 03716556 (1–10 mg/kg; intraduodenal; once; male Sprague-Dawley rats) exhibit dose-dependent inhibition of gastric acid output [1].More rapid onset of action than 5-methoxy-2-{[(4-methoxy-3,5-dimethyl-2-pyridyl)methyl]-sulfinyl}-benzimidazole (omeprazole) and 3-fold greater potency than revaprazan were observed in Ghosh-Schild rats and Heidenhain pouch dogs. PF-03716556, a novel acid pump antagonist, could improve upon or even replace current pharmacological treatment for gastroesophageal reflux disease.

Animal/Disease Models: Male SD (Sprague-Dawley) rats (250 -300 g) treated with Pentagastrin[1]
Doses: 1 mg/kg, 3 mg/kg, 10 mg/kg
Route of Administration: Intraduodenal administration; once
Experimental Results: Inhibited gastric acid secretion in a dose-dependent manner.

[1]. N-(2-hydroxyethyl)-N,2-dimethyl-8-{[(4R)-5-methyl-3,4-dihydro-2H-chromen-4-yl]amino}imidazo[1,2-a]pyridine-6-carboxamide (PF-03716556), a novel, potent, and selective acid pump antagonist for the treatment of gastroesophageal reflux disease. J Pharmacol Exp Ther. 2009 Feb;328(2):671-9.

In vitro inhibition of H+,K+-ATPase activity. PF-03716556 inhibited H+,K+-ATPase activity in porcine ionosphere vesicles in a concentration-dependent manner, with a pIC50 of 6.026 ± 0.112 at pH 6.4 (Figure 2A). In canine ionosphere vesicles and human recombinant cells, the pIC50 values at pH 6.4 were 6.038 ± 0.039 (Figure 2A) and 6.009 ± 0.209 (Figure 2B), respectively. In porcine ion-leaking membrane vesicles, rivarazine and omeprazole inhibited the activity of H+,K+-ATPase…[1]

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Gastric H+,K+-ATPase is a transmembrane enzyme present in parietal cells and is a target molecule of APA. In this study, we evaluated the inhibitory effect of PF-03716556 on gastric H+,K+-ATPase in either the enzyme fraction (ion-leaking assay) or within ion-closed vesicles (ion-closed assay). Isolated ion-closed vesicles have low ion permeability; since the binding sites of K+ and APA are both located inside the ion-closed vesicles, the enzymatic activity of gastric H+,K+-ATPase…[1]

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Inhibition of H+,K+-ATPase is considered the most effective way to control gastric acid secretion. However, current acid-suppressing therapies used to treat gastroesophageal reflux disease, including histamine H2 receptor antagonists and proton pump inhibitors, cannot fully meet the needs of all patients due to the limitations of their mechanisms of action. This study aimed to elucidate the in vitro and in vivo pharmacological properties of a novel acid pump antagonist, N-(2-hydroxyethyl)-N,2-dimethyl-8-{[(4R)-5-methyl-3,4-dihydro-2H-chromen-4-yl]amino}imidazo[1,2-a]pyridine-6-carboxamide (PF-03716556), and to compare it with other acid-suppressing agents. The activities of recombinant gastric H+,K+-ATPases in pigs, dogs, and humans were determined using ion leakage and ion-locking methods. To analyze the selectivity profile, the compound's affinity for a range of receptors, ion channels, and enzymes was determined. Gastric acid secretion was measured in Ghosh-Schild rats and Heidenhain kangaroo dogs by titrating perfusion and gastric fluid samples. In ion-tight assays, PF-03716556 exhibited 3-fold higher inhibitory activity than 5,6-dimethyl-2-(4-fluorophenylamino)-4-(1-methyl-1,2,3,4-tetrahydroisoquinoline-2-yl)pyrimidine (Revarrazan), the only currently available acid pump antagonist. This compound showed no species-specificity and high selectivity, including selectivity for canine renal Na(+),K(+)-ATPases. Kinetic experiments demonstrated that PF-03716556 possesses a competitive and reversible mechanism of action. In Ghosh-Schild rats and Heidenhain kangaroos, PF-03716556 was observed to have a faster onset of action than 5-methoxy-2-{[(4-methoxy-3,5-dimethyl-2-pyridyl)methyl]-sulfinyl}-benzimidazole (omeprazole) and was 3-fold more potent than Revarrazan. PF-03716556 is a novel acid pump antagonist that is expected to improve or even replace current drug treatments for gastroesophageal reflux disease. [1]

C22H26N4O3

394.47

394.2

C, 66.99; H, 6.64; N, 14.20; O, 12.17

928774-43-0

25134521

White to yellow solid powder

1.3±0.1 g/cm3

143-145°C

1.651

2.6

2

5

5

29

576

1

CC1=C2[C@@H](CCOC2=CC=C1)NC3=CC(=CN4C3=NC(=C4)C)C(=O)N(C)CCO

YBHKBMJREUZHOV-QGZVFWFLSA-N

InChI=1S/C22H26N4O3/c1-14-5-4-6-19-20(14)17(7-10-29-19)24-18-11-16(22(28)25(3)8-9-27)13-26-12-15(2)23-21(18)26/h4-6,11-13,17,24,27H,7-10H2,1-3H3/t17-/m1/s1

N-(2-hydroxyethyl)-N,2-dimethyl-8-{[(4R)-5-methyl-3,4-dihydro-2H-chromen-4-yl]amino}imidazo[1,2-a]pyridine-6-carboxamide

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)

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.)