Gastric H/K-ATPase (Ki = 120 nM)

SCH28080 [1] has a Ki of 0.12 μM and competitively inhibits K+-stimulated ATP hydrolysis. SCH28080 has an IC50 of 0.029 μM, which blocks the absorption of [14C]aminopyrine into isolated rabbit parietal cells caused by histamine [1]. Cell viability is reduced by SCH28080 in a dose-dependent manner. The IC50 values were 22.9 µM and 15.3 µM, respectively, after 2 and 24 hours of treatment. The cell viability at 100 µM was less than 10% after two hours of treatment [2]. Higher doses of SCH28080 cause apoptosis and make it cytotoxic [2]. By activating IK ATP and blocking L-type voltage-gated Ca2+ channels, SCH28080 suppresses insulin production, lowers cell viability, and causes apoptosis/necrosis in a dose-dependent manner [2].

SCH28080 (20 mg/kg; intraperitoneal injection) can suppress gastric ulcer produced by pyloric ligation in rats [3].

The novel antisecretory agents SCH 32651 and SCH 28080 were evaluated for their antisecretory activities in vitro as well as for their abilities to inhibit the (H+ + K+)-ATPase enzyme activity in preparations of microsomal membranes from rabbit fundic mucosa. SCH 32651 and SCH 28080 inhibited both the histamine- and dibutyryl cAMP-stimulated uptake of [14C]-aminopyrine into isolated parietal cells with IC50 values of about 1.5 and 0.02 microM respectively. SCH 32651 and SCH 28080 competitively inhibited the K+-stimulated hydrolysis of ATP catalyzed by the (H+ + K+)-ATPase with Ki values of 16.3 and 0.12 microM respectively. The inhibition of the enzyme by both compounds was not affected by the addition of the sulfhydryl reducing agents dithiothreitol or beta-mercaptoethanol, was readily reversible by dilution or washing, and was dependent upon the concentration of KCl used to stimulate the enzyme. These data suggest that SCH 32651 and SCH 28080 are reversible, competitive inhibitors of the K+-stimulated hydrolysis of ATP [1].

Cell viability assay [2]
Cell Types: INS-1E Cell
Tested Concentrations: 3.1 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM
Incubation Duration: 2 hrs (hours), 24 hrs (hours)
Experimental Results: Caused dose-dependent reduction of cell viability .

Animal/Disease Models: Male Wistar rat (280-350g), Shay rat model [3]
Doses: 20mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: 91% inhibitory effect on gastric ulcer in pyloric ligated rats.

[1]. Studies on the mechanism of action of the gastric microsomal (H+ + K+)-ATPase inhibitors SCH 32651 and SCH 28080. Biochem Pharmacol. 1987 Jan 1;36(1):97-104.

[2]. The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells. Cell Physiol Biochem. 2017;43(3):1037-1051.

[3]. Inhibitory Effects of Copiamycin A, a Macrocyclic Lactone Antibiotic, on Gastric H+,K(+)-ATPase, Acid Secretion and Ulcer Formation. Jpn J Pharmacol. 1991 Feb;55(2):283-6.

2-(2-methyl-8-phenylmethoxy-3-imidazo[1,2-a]pyridinyl)acetonitrile is an imidazopyridine.

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Background/aims: Glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem), activation of voltage-activated Ca2+ currents (ICav) and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi) or cell volume, which also affect β-cell viability, can elicit or modify insulin release. In β-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. [2]
Methods: In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi) recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. [2]
Results: We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICav blocker nifedipine caused a full inhibition of GSIS at 10 and 20 µM. At 20 µM, SCH-28080 inhibited ICav comparable to 20 µM nifedipine and in addition augmented IKATP recorded at -60 mV and hyperpolarized Vmem by ∼15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC50 values of 22.9 µM and 15.3 µM, respectively. At 20 µM the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40-50 µM, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of ΔΨm within 4 h under 50 and 100 µM while 10 and 20 µM had no effect on ΔΨm within 24 h. [2]
Conclusion: We demonstrate expression of HKα2 in rat INS-1E cells. However, the pump is apparently non-functional under the given conditions. Nonetheless the H+/K+ ATPase blocker SCH-28080 inhibits insulin secretion and induces cell death. Importantly, we show that SCH-28080 inhibits ICav - and activates KATP channels identifying them as novel "off-targets" of the inhibitor, causing hyperpolarization of Vmem and inhibition of insulin secretion.

C17H15N3O

277.3205

277.122

C, 73.63; H, 5.45; N, 15.15; O, 5.77

76081-98-6

108137

Light yellow to brown solid powder

1.16g/cm3

1.615

3.287

0

3

4

21

385

0

PYKJFEPAUKAXNN-UHFFFAOYSA-N

InChI=1S/C17H15N3O/c1-13-15(9-10-18)20-11-5-8-16(17(20)19-13)21-12-14-6-3-2-4-7-14/h2-8,11H,9,12H2,1H3

Imidazo(1,2-a)pyridine-3-acetonitrile, 2-methyl-8-(phenylmethoxy)-

SCH28080; SCH-28080; 76081-98-6; Sch 28080; Sch-28080; 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile; SCH28080; Schering compound 28080; S28080; 2-(8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl)acetonitrile; SCH 28080.

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 (~360.59 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.01 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 (9.01 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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 (Please use freshly prepared in vivo formulations for optimal results.)