Identification of phase-I metabolites and chronic toxicity study of the Kv1.3 blocker PAP-1 (5-(4-phenoxybutoxy)psoralen) in the rat

Abstract

PAP-1 (5-(4-phenoxybutoxy)psoralen), a potent small-molecule blocker of the voltage-gated potassium Kv1.3 channel, is currently in pre-clinical development for psoriasis. The present study was undertaken to identify the major phase-I metabolites of PAP-1 in rats. Following oral administration at 50 mg/kg, bile, plasma, urine and feces were collected, and separated by reversed-phase HPLC after sample preparation by solid-phase extraction. Five phase-I metabolites, i.e., 5-(oxybutyric-acid)psoralen (M1), 5-[4-(4-hydroxybutoxy)]psoralen (M2), 5-[4-(4-hydroxyphenoxy)]psoralen (M3), 5-[4-(3-hydroxyphenoxy)]psoralen (M4), 8-hydroxyl-5-(4-phenoxybutoxy)psoralen (M5), were isolated and identified by mass spectrometry and NMR spectroscopy. M3, M4 and M5 were hydroxylated products, while M1 and M2 were O-dealkylation products. Incubation of PAP-1 with rat liver microsomes rendered the same five major metabolites in a NADPH-dependent manner suggesting that cytochrome P450 (CYP) enzymes are involved in PAP-1 metabolism. Inhibitors of rat CYP1A1/2 (alpha-naphthoflavone) and CYP3A (ketoconazole) but not CYP2D6, CYP2E or CYP2C9 blocked the metabolism of PAP-1 in rat microsomes. Of the five metabolites M3, M4 and M5 were found to inhibit Kv1.3 currents with nanomolar IC50s, while M1 and M2 were inactive. Our results identified the Kv1.3-inactive M1 as the major phase-I metabolite, and suggest that hydroxylation and O-dealkylation are the major pathways of PAP-1 metabolism principally via CYP1A1/2 and CYP3A. We further conducted a 6-months repeat-dose toxicity study with PAP-1 at 50 mg/kg in both male and female rats and did not observe any changes in hematology, blood chemistry, body weight, histology of any major organ, or speeding up of metabolism suggesting induction of its metabolizing enzymes (presumably CYPs).

Targeting Potassium Channels Kv1.3 and KCa3.1: Routes to Selective Immunomodulators in Autoimmune Disorder Treatment?

“…Some Kv channel blockers are too unstable for oral delivery without appropriate protection (e.g., Stichodactyla helianthus toxin [ShK]‐ L5) . Table summarizes the Kv1.3 and K Ca 3.1 blockers with improved ion channel selectivity and stability and potential clinical use as immunomodulators . It is encouraging that some pharmaceutical companies are actively involved in drug development of Kv1.3 and K Ca 3.1 blockers.…”

“…15 Table 1 summarizes the Kv1.3 and K Ca 3.1 blockers with improved ion channel selectivity and stability and potential clinical use as immunomodulators. 9,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] It is encouraging that some pharmaceutical companies are actively involved in drug development of Kv1.3 and K Ca 3.1 blockers. Preclinical studies have been conducted with the injectable Kv1.3 blocker ShK-186, 11 which is currently being evaluated in firstin-man phase 1 trials.…”

Insight into the mechanism of cytotoxicity of membrane-permeant psoralenic Kv1.3 channel inhibitors by chemical dissection of a novel member of the family

“…The synthesis of PAP-1-MHEG (IUPAC name: 4-(4-(4-(2,5,8,11,14,17-hexaoxanonadecan-19-yloxy)phenoxy)butoxy)-7H-furo[3,2-g]chromen-7-one) and of the other compounds used is described in the Materials and Methods section. As control for our experiments, we used either PAP-1, or PAP-OH (IUPAC name: 4-(4-(4-hydroxyphenoxy)butoxy)-7H-furo[3,2-g]benzopiren-7-one), the major active metabolite of PAP-1 forming in vivo [ 22 ]. PAP-1 is insoluble in water, while it can reach a concentration of 140 mM in pure DMSO.…”

“…Regarding the mechanism of action of PAP-1, Zimin and colleagues [ 20 ] used experimental and computational approaches – the latter based on the known structure of the Kv1.2 channel [ 21 ] – to identify a cooperative action by the two major parts of the molecule. The phenoxybutoxy side-chain of PAP-1 inserts into the lipophilic interphase between transmembrane segments S5 and S6 of the channel: lack of the phenyl ring seems to disrupt these hydrophobic interactions and prevents inhibition [ 22 ]. The coumarin moiety interacts instead with a second site, touching the P-loop and obstructing the channel conduit.…”

Effects of psoralen on the pharmacokinetics of anastrozole in rats

“…Psoralen is the main active ingredient extracted from the natural products of Psoralea corylifolia L. (Leguminosae) and it has been commonly used in traditional Chinese medicine for the treatment of osteoporosis, osteosarcoma, bone fracture and osteomalacia (Hao et al. 2011 ; Chen et al. 2017 ; Du et al.…”