Iptakalim hydrochloride is an ATP-sensitive potassium channel (K_ATP) opener and an antagonist of α4β2-containing nicotinic acetylcholine receptors (nAChRs). It inhibits nicotine-induced dopamine and glutamate release in the nucleus accumbens. No specific IC50, Ki, or EC50 values for these targets are reported in this study. [3]

It has been demonstrated that itakalim hydrochloride (Ipt) reverses the remodeling of the pulmonary resistance vascular system, inhibits the growth of smooth muscle cells in the pulmonary artery (PASMC) and airway (ASMC), and shields PAEC from pathogenic stimuli [1]. Through the activation of KATP channels, iptakalim inhibits the rise in [Ca2+]cyt and pulmonary artery SMC proliferation that is produced by endothelin 1. Iptakalim (10 μM, 10 min) pretreatment of pulmonary artery SMC effectively inhibited the transient rise in [Ca2+] cells produced by endothelin-1 [2]. At doses of 0.1, 1 and 10 AM, iptakalim decreased [3H]thymidine incorporation by 19.75±4.60% (n = 10), 41.20±9.49% (n = 10), and 54.74±10.11% (n = 10). ), in contrast to cells that were only treated with endothelin-1 [2].

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Iptakalim hydrochloride (at concentrations ranging from 1 nM to 0.1 mM) did not inhibit dopamine transporter (DAT) or serotonin transporter (SERT) function in rat striatal and hippocampal synaptosomal preparations, as measured by [³H]dopamine and [³H]serotonin uptake assays. [3]

Pretreatment with Iptakalim (60 mg/kg) considerably reduced activity (LSD comparison). Iptakalim (10-60 mg/kg) attenuates chamber activity during these nicotine tests. Iptakalim also attenuates nicotine-induced conditioned responses [3].

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In rats, pretreatment with iptakalim hydrochloride (10, 30, 60 mg/kg, IP) dose-dependently attenuated nicotine-evoked conditioned responding in a discriminated goal-tracking task, with significant reductions at 30 and 60 mg/kg. [3]
In a separate study, pretreatment with iptakalim hydrochloride (1, 3, 6 mg/kg, IV) decreased nicotine self-administration in rats, reducing active lever pressing without affecting inactive lever responding. [3]

Inhibition of [³H]dopamine and [³H]serotonin uptake by iptakalim hydrochloride was determined using rat striatal (for dopamine) and hippocampal (for serotonin) synaptosomal preparations. Freshly dissected tissue was homogenized in ice-cold 0.32 M sucrose buffer at pH 7.4 and centrifuged at 800×g for 12 minutes at 4°C. The resulting supernatant was further centrifuged at 22,000×g for 15 minutes at 4°C. The pellet was resuspended in an ice-cold assay buffer containing NaCl, KCl, CaCl₂, sodium phosphate, MgSO₄, glucose, ascorbic acid (pH 7.4), and 1 µM pargyline. Iptakalim hydrochloride (1 nM to 1 mM) was added to the assay tubes. Samples were incubated at 37°C for 10 minutes, and the reaction was initiated by adding [³H]dopamine (final concentration 0.5 nM) or [³H]serotonin (final concentration 5 nM). After a 3-minute incubation, the reaction was stopped on ice. The mixture was filtered through GF/B filter paper pre-soaked in 0.05% polyethyleneimine. The filter was rapidly washed three times with 3 ml of ice-cold 0.32 M sucrose buffer. For [³H]dopamine uptake, non-specific binding was determined in the presence of 50 µM cocaine. For [³H]serotonin uptake, non-specific binding was determined in the presence of 10 µM fluoxetine. Radioactivity retained on the filter was measured using a liquid scintillation counter. [3]

For the CCK-8 assay, human ECFCs were plated in 96-well plates at a density of 1×10⁴ cells/well in 200 µL of complete endothelial basal medium (EBM-2). After 24h, cells were pre-treated with iptakalim (10⁻⁷ M to 10⁻⁴ M) for 1h before being exposed to hypoxic conditions (2% O₂) for 12h. In some groups, cells were pre-incubated with glibenclamide (10⁻⁵ M) for 30 min before iptakalim treatment. CCK-8 reagent was added according to the manufacturer's protocol, and absorbance was measured at 450 nm using a microplate reader. [1]
For the EdU proliferation assay, ECFCs were pre-treated with iptakalim (10⁻⁵ M) for 2h or glibenclamide (10⁻⁵ M) for 30 min before being exposed to hypoxia for 12h. Cells were then incubated with 50 µM EdU for 4h. EdU incorporation was detected according to the kit protocol, and the percentage of EdU-positive nuclei was calculated from five random high-power fields per well using fluorescence microscopy. [1]
For the migration assay, ECFCs were detached and resuspended in serum-free EBM-2. A total of 1×10⁵ cells, with or without iptakalim (10⁻⁵ M), were added to the upper chamber of a 24-well Transwell insert. The lower chamber contained 600 µL of EBM-2 medium with 10% FBS. After 24h incubation under normoxic or hypoxic conditions, cells on the top membrane were removed. Migrated cells on the lower side were fixed with 4% formaldehyde, stained with 5% crystal violet, and counted in five random fields using a phase-contrast microscope. Glibenclamide (10⁻⁵ M) was also used to assess its effect. [1]
For the tube formation assay, 10 µL of Matrigel was added to an angiogenesis slide and allowed to solidify at 37°C for 1h. Then, 50 µL of ECFC suspension (1.5×10⁵ cells/mL) in complete EBM-2 medium, with or without iptakalim (10⁻⁵ M) or glibenclamide (10⁻⁵ M), was seeded onto the solidified Matrigel. Cells were incubated under normoxic (21% O₂) or hypoxic (2% O₂) conditions for 12h. Tube formation was imaged, and the total tube length per image was quantified using image analysis software. [1]
For the apoptosis assay, starved ECFCs were treated with iptakalim (10⁻⁵ M) or glibenclamide (10⁻⁵ M) under hypoxic conditions (2% O₂) for 12h. Cells were collected, washed with PBS, and stained with Annexin V FITC and PI according to the protocol. Apoptotic cells were detected using a flow cytometer. [1]
For NO measurement, ECFCs were seeded in 96-well plates and pre-treated with iptakalim (10⁻⁵ M) for 1h before hypoxic stimulation. The culture supernatants were collected, and the levels of nitrite/nitrate (NO₂⁻/NO₃⁻), as stable metabolites of NO, were determined using a commercial Nitric Oxide Assay Kit according to the manufacturer's instructions. Absorbance was measured at 540 nm. [1]
For Western blot analysis, ECFCs were lysed in RIPA buffer with protease inhibitors. Protein concentrations were measured using a BCA assay. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% BSA. Membranes were incubated with primary antibodies against Akt, phospho-Akt (Ser473), eNOS, and β-actin, followed by HRP-conjugated secondary antibodies. Signals were detected using enhanced chemiluminescence. [1]

Animal/Disease Models: 30 male SD (SD (Sprague-Dawley)) rats, weighing 275-299 grams [3].
Doses: 0, 10, 30 or 60 mg/kg.
Route of Administration: IP, 4 minutes 10 minutes before test session starts
Experimental Results: diminished chamber activity. Dipper entries after pretreatment with 30 and 60 mg/kg Iptakalim were Dramatically lower than those pretreated with 0 (saline) or 10 mg/kg Iptakalim (Tukey HSD test).

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For the discriminated goal-tracking (DGT) task, male Sprague-Dawley rats were trained in intermixed sessions receiving either nicotine (0.4 mg/kg, SC) or saline. Sucrose reward was delivered only during nicotine sessions. On test days, rats were pretreated with iptakalim hydrochloride (0, 10, 30, or 60 mg/kg, IP) 10 minutes before a 4-minute test session. Nicotine (0.4 mg/kg, SC) was administered 5 minutes before the test. Head entries into the sucrose dipper receptacle and general chamber activity were recorded. [3]
For the nicotine self-administration task, rats were surgically implanted with a jugular vein catheter. After recovery, they were trained to press a lever for nicotine infusions (0.03 mg/kg/infusion) on a variable ratio 3 (VR3) schedule. For testing, rats were pretreated with iptakalim hydrochloride (0, 1, 3, or 6 mg/kg, IV) 5 minutes before the start of the 60-minute self-administration session. Active and inactive lever presses were recorded. Iptakalim hydrochloride and nicotine were dissolved in 0.9% saline. The nicotine solution pH was adjusted to 7.0 ± 0.2 with dilute NaOH. [3]

In the target tracking task, intraperitoneal injection of 60 mg/kg of ipulcaline hydrochloride reduced the overall activity level in mice, but this movement effect could not explain the reduction in nicotine-induced behavior. [3]

[1]. Iptakalim ameliorates hypoxia-impaired human endothelial colony-forming cells proliferation, migration, and angiogenesis via Akt/eNOS pathways. Pulm Circ. 2019 Oct 18;9(3):2045894019875417.

[2]. Anti-proliferating effect of iptakalim, a novel KATP channel opener, in cultured rabbit pulmonary arterial smooth muscle cells. Eur J Pharmacol. 2005 Mar 28;511(2-3):81-7.

[3]. Iptakalim attenuates self-administration and acquired goal-tracking behavior controlled by nicotine. Neuropharmacology. 2013 Dec;75:138-44.

Iptakalim hydrochloride has been approved in China for the treatment of hypertension. Its potential as a nicotine-dependent drug is supported by preclinical model studies that have shown that Iptakalim can block the interoceptive and reinforcing effects of nicotine, possibly by antagonizing α4β2 nAChRs and opening K_ATP channels. [3] Studies have found that the behavioral effects of Iptakalim hydrochloride are not due to nonspecific motor dysfunction or DAT/SERT function inhibition. [3]

C9H21N.HCL

179.731

642407-63-4

642407-44-1(free base);642407-63-4 (HCl);

11446679

Yellow to orange solid powder

2

1

3

11

92.9

0

CC(C)C(C)(C)NC(C)C.[H]Cl

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)

H2O : ~25 mg/mL (~139.10 mM)

Solubility in Formulation 1: 100 mg/mL (556.39 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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