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

Abstract

1. PAP-1 (5-(4-phenoxybutoxy)psoralen), a potent small-molecule blocker of the voltage-gated potassium Kv1.3 channel, is currently in preclinical development for psoriasis. This study was undertaken to identify the major phase I metabolites of PAP-1 in Sprague-Dawley (SD) rats.

2. Five phase I metabolites, that is 5-(oxybutyric-acid)psoralen (M1), 5-[4-(4-hydroxybutoxy)]psoralen (M2), 5-[4-(4-hydroxyphenoxy)butoxy]psoralen (M3), 5-[4-(3-hydroxyphenoxy)butoxy]psoralen (M4), and 8-hydroxyl-5-(4-phenoxybutoxy)psoralen (M5), were isolated from the bile of rats and identified by mass spectrometry and NMR spectroscopy. The last four metabolites are new compounds.

3. Incubation of PAP-1 with SD rat liver microsomes rendered the same five major metabolites in a nicotinamide adenine dinucleotide phosphate (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 (quinidine), CYP2E (diethyldithiocarbamate), or CYP2C9 (sulphaphenazole) blocked the metabolism of PAP-1 in rat microsomes.

4. 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.

5. We further conducted a 6-month repeat-dose toxicity study with PAP-1 at 50 mg/kg in both male and female Lewis rats and did not observe any toxic effects.

Keywords::

• 5-(4-phenoxybutoxy)psoralen (PAP-1)
• CYP isoenzyme
• Kv1.3 activity
• Kv1.3 blocker
• metabolite
• O-dealkylation.

Acknowledgement

We thank Jieli Wu from the Analysis Center at Shanghai Jiao Tong University for excellent technical assistance with the NMR assay.This work was supported by the National Natural Science Foundation of China (No: 30973581), the National Basic Research ‘‘973” Program, Ministry of Science and Technology, China (No: 2009CB521900, PI: Dr. Ding-Feng Su), a Howard Hughes Medical Institute HMI Predoctoral Fellowship to Pavel I. Zimin and a National Institute of Health RO1 grant (GM076063, PI: Heike Wulff).

Declaration of interest

B.H., Z.C., X.Z., Y.W. and P.I.Z. report no conflict of interest. H.W. is an inventor on the University of California patent claiming PAP-1 and related KV1.3 blockers for immunosuppression. She is a scientific founder of Airmid, a start-up company that is aiming to develop KV1.3 blockers as immunosuppressants. G.M. is the CEO and a full-time employee of Airmid. The authors alone are responsible for the content and writing of the paper.