PHA-408

PHA-408 is a novel, potent, highly selective, and ATP-competitive inhibitor of IKB kinase-2. The selective inhibitory activity of PHA-408 supports the use of PHA-408 in investigations of NF-κB signaling regulation, inflammatory signaling cascades, kinase biology, and cellular mechanisms involving IKB kinase-2.

Method: The inhibitory activity of PHA-408 against purified IκB kinase 2 was evaluated in a cell-free kinase assay.
Rresult: PHA-408 acted as a tight-binding, ATP-competitive inhibitor of IκB kinase 2 with an IC50 of 40 ± 2 nM and exhibited relatively slow dissociation kinetics.[1]
Method: The anti-inflammatory effects of PHA-408 were evaluated in cytokine-stimulated airway epithelial and inflammation-relevant cellular assays by measuring NF-κB pathway activation and inflammatory mediator production.
Rresult: PHA-408 inhibited IκBα phosphorylation and degradation, NF-κB activation, and inflammatory mediator production in a concentration-dependent manner.[2]
Method: Cultured mdx mouse myotubes were exposed to 20 μM PHA-408 for 52 h, and the subcellular distribution of p65 was evaluated by confocal immunofluorescence.
Rresult: PHA-408 substantially reduced nuclear p65 immunofluorescence in mdx myotubes.[5]
Method: Human umbilical vein endothelial cells were exposed to TNFα at 10 ng/mL together with 2 μM PHA-408 for 6 days, followed by a 3-day recovery period. NF-κB p65 translocation, reactive oxygen species, cell proliferation, senescence markers, and senescence-associated secretory phenotype factors were evaluated.
Rresult: PHA-408 largely prevented TNFα-induced nuclear translocation of p65, mitigated the later increase in reactive oxygen species, reduced growth impairment and cell-cycle arrest, attenuated the reduction in Ki-67-positive cells and increases in p16- and p21-positive cells, and reduced E-selectin, IGFBP-5, IL-6, and IL-8 responses associated with the senescence-associated secretory phenotype.[6]

Method: Rats with streptococcal cell wall-induced arthritis received PHA-408 orally at 30 mg/kg daily for 3 days, and IKKβ activity, paw swelling, inflammatory signaling, and joint pathology were evaluated.
Rresult: PHA-408 inhibited approximately 80% of IKKβ activity and markedly reduced paw swelling. It also suppressed IκBα phosphorylation and degradation, p65 phosphorylation and DNA-binding activity, inflammatory mediator expression, and joint pathology, without evident adverse effects at maximally efficacious doses.[1]
Method: PHA-408 was administered to rats exposed to inhaled lipopolysaccharide, and airway inflammatory cell infiltration and cytokine production were measured.
Rresult: PHA-408 dose-dependently attenuated lipopolysaccharide-induced inflammatory cell infiltration and cytokine production in the airways.[2]
Method: Adult Sprague-Dawley rats received PHA-408 orally at 15 or 45 mg/kg once daily for 3 days and were exposed to lipopolysaccharide aerosol or cigarette smoke. Animals were sacrificed 1, 4, or 24 h after the final exposure, and bronchoalveolar lavage and lung inflammatory responses were evaluated.
Rresult: PHA-408 significantly inhibited lipopolysaccharide- and cigarette smoke-induced neutrophil influx into bronchoalveolar lavage fluid; reduced CINC-1 in bronchoalveolar lavage fluid and IL-6, TNF-α, IL-1β, and GM-CSF in lung tissue; and decreased NF-κB nuclear translocation and DNA-binding activity.[3]
Method: Pharmacokinetic modeling was used to design an oral steady-state delivery regimen for PHA-408 in rat arthritis efficacy and safety studies. Plasma exposure, disease efficacy, and safety findings were evaluated under steady-state conditions.
Rresult: Steady-state delivery established a clear exposure-dependent relationship between plasma PHA-408 concentrations and efficacy in the rat arthritis model and enabled a more reliable assessment of target-related safety and the no-observed-adverse-effect level.[4]
Method: Young adult mdx mice received PHA-408 by oral gavage at 50 mg/kg daily for 30 days, a single oral dose of 100 mg/kg, or intraperitoneally at 0.8 mg/kg/day for 30 days in two daily injections. Nuclear p65 and cytosolic IκBα levels in costal diaphragm muscle were measured.
Rresult: Thirty-day oral treatment caused a small but significant 14% increase in cytosolic IκBα but did not reduce nuclear p65; a single 100 mg/kg oral dose also had no effect on nuclear p65. In contrast, prolonged intraperitoneal treatment reduced nuclear p65 by approximately 50%.[5]

DMSO: 40 mg/mL (71.43 mM), Sonication is recommended.