Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals ERICH W. ZINSER MARK L. WOLFE SUSAN J. ALEXANDER- BOWMAN EILEEN M. THOMAS JOHN P. DAVIS VINCENT E. GROPPI BYUNG H. LEE DAVID P. THOMPSON & TIMOTHY G. GEARY Pharmacia Animal Health, Kalamazoo, MI, USA Zinser, E. W., Wolfe, M. L., Alexander-Bowman, S. J., Thomas, E. M., Davis, J. P., Groppi, V. E., Lee, B. H., Thompson, D. P., Geary, T. G. Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals. J. vet. Pharmacol. Therap. 25, 241–250. Oxindole alkaloids in the paraherquamide ⁄ marcfortine family exhibit broad- spectrum anthelmintic activity that includes drug-resistant strains of nema- todes. Paraherquamide (PHQ), 2-deoxoparaherquamide (2DPHQ), and close structural analogs of these compounds rapidly induce flaccid paralysis in parasitic nematodes in vitro, without affecting adenosine triphosphate (ATP) levels. The mechanism of action of this anthelmintic class was investigated using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum. None of the compounds altered A. suum muscle tension or membrane potential. However, PHQ blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by acetylch- oline (ACh) and the nicotinic agonists levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic activity of PHQ and marcfortines is due to blockade of cholinergic neuromuscular transmission. The effects of these compounds were also examined on subtypes of human nicotinic ACh receptors expressed in mammalian cells with a Ca 2+ flux assay. 2DPHQ blocked nicotinic stimulation of cells expressing a3 ganglionic (IC50  9 lM) and muscle-type (IC50  3 lM) nicotinic cholinergic receptors, but was inactive at 100 lM vs. the a7 CNS subtype. PHQ anthelmintics are nicotinic cholinergic antagonists in both nematodes and mammals, and this mechanism appears to underlie both their efficacy and toxicity. (Paper received 1 February 2002; accepted for publication 1 July 2002) Timothy G. Geary, Pharmacia Animal Health, 7923-25-403, 7000 Portage Road, Kalamazoo, MI 49001-0199, USA. E-mail: timothy.g.geary@pharmacia.com INTRODUCTION Paraherquamide (PHQ) is an oxindole alkaloid originally isolated from cultures of Penicillium paraherquii (Yamazaki et al., 1981). The anthelmintic activity of PHQ was initially detected in a jird ⁄ Trichostrongylus colubriformis model (Ostlind et al., 1990). Subsequently, anthelmintic activity of the structurally related compound marcfortine A was identified in an in vitro test using Caenorhabditis elegans (unpublished observations). These com- pounds are less potent than the macrocyclic lactones, but are more potent than the benzimidazoles, imidazothiazoles and tetrahydropyrimidines (e.g. Shoop et al., 1990; Lee et al., 2002; our unpublished observations). Importantly, marcfortines and PHQ are effective against strains of parasites that are resistant to each of the known broad-spectrum anthelmintics, suggesting that they possess a novel mechanism(s) of action (Shoop et al., 1990). A medicinal chemistry program at Pharmacia Animal Health provided a number of other analogs, one of which, 2-deoxoparaherquamide (2DPHQ; Lee et al., 2002) was chosen for further study. Similarities in structure and anthelmintic spectra of the PHQ and marcfortines suggest that these compounds share an anthelmintic mechanism. This concept is supported by observa- tions that PHQ and 2DPHQ displace [ 3 H]marcfortine A in competition binding assays using membranes prepared from adult Haemonchus contortus; competition is also observed in binding assays using membranes prepared from the free-living nematode Panagrellus redivivus and [ 3 H]2DPHQ as ligand (unpublished observations). The clinical utility of PHQ is compromised by the fact that it is toxic to mice when administered in dimethyl sulfoxide (DMSO) ⁄ propylene gly- col ⁄ glycerol formal (1.0:1.2:1.8), with an LD50 value of 15 mg ⁄ kg. Lethality occurred in dogs dosed with PHQ in propylene glycol ⁄ glycerol formal (60:40) at £ 0.5 mg ⁄ kg J. vet. Pharmacol. Therap. 25, 241–250, 2002. ANTIINFECTANTS Ó 2002 Blackwell Science Ltd 241