Transtegumental diffusion of benzimidazole anthelmintics into Moniezia benedeni: correlation with their octanol–water partition coefficients

Abstract

The experiments described here report on the correlation between the ex vivo diffusion of different benzimidazole (BZD) anthelmintics into the cestode parasite Moniezia benedeni, and their octanol–water partition coefficients (P.C.). The characterisation of the drug diffusion process into target parasites is relevant to understand the mechanism of drug penetration and the pharmacological activity of anthelmintic drugs. Specimens of the tapeworm M. benedeni, used as a helminth parasite model, were obtained from untreated cattle killed at the local abattoir. The collected parasites were incubated (5–210 min) with either fenbendazole (FBZ), albendazole (ABZ), ricobendazole (RBZ), oxfendazole (OFZ), mebendazole (MBZ), oxibendazole (OBZ), or thiabendazole (TBZ), in a Kreb’s Ringer Tris buffer medium at a final concentration of 5 nmol/ml. After the incubation time elapsed, samples of parasite material were chemically extracted and prepared for high performance liquid chromatography (HPLC) analysis to measure drug/metabolite concentrations. Additionally, the octanol–water P.C. for each molecule was estimated as an indicator of drug lipophilicity, using reversed phase HPLC analysis. All the incubated drugs were recovered from the tapeworms as early as 5 min post incubation. There was a high correlation (r=0.87) between drug lipophilicity, expressed as octanol–water P.C. (Log P), and drug availability within the parasite. The most lipophilic BZD compounds (FBZ, ABZ, and MBZ), with P.C. values higher than 3.7, were measured at significative higher concentrations within the tapeworm compared to those drugs with the lowest P.C. values. Considering the results from the current and previous studies, it is clear that passive diffusion is a major mechanism of BZD penetration into cestode parasites, where lipid solubility is a determinant factor influencing the diffusion of these anthelmintic molecules through the parasite tegument.
Index Descriptors and Abbreviations: Transtegumental diffusion; Benzimidazole anthelmintics; Cestodes; Moniezia benedeni; Lipophilicity; Octanol–water partition coefficient

Introduction

Helminth parasites infect a quarter of the world’s total population and are a major cause of morbidity (Colley et al., 2001). Moreover, helminth infections are the most important cause of productivity losses in livestock worldwide (Parkins and Holmes, 1989). Although alternative control methods have been developed, chemically-based anthelmintic treatments are the most important tool to control parasitism in livestock (Martin, 1985). Benzimidazole (BZD) anthelmintics are effective against nematodes, cestodes and trematodes (Campbell, 1990; McKellar and Scott, 1990). Their pharmacological activity is based on the binding to β-tubulin, which produces subsequent disruption of the tubulin–microtubule dynamic equilibrium (Lacey, 1990). Thus, all the functions ascribed to microtubules at the cellular level are altered (cell division, maintenance of cell shape, cell motility, cellular secretion, nutrient absorption and intracellular transport) (Lacey, 1988). The introduction of thiabendazole (TBZ) in the 1960s was followed by the development of a series of BZD and BZD prodrugs (pro-BZD) as anthelmintics. Each drug represented an improvement in efficacy and spectrum of activity which firmly established the predominance of BZD anthelmintics within the chemotherapy arsenal (Lacey, 1988). BZD anthelmintics are chemically classified as BZD thiazols (TBZ and cambendazole); halogenated BZD thiols (triclabendazole); pro-BZD (febantel, netobimin, and thiophanate); and BZD methylcarbamates (albendazole [ABZ], ricobendazole [RBZ], fenbendazole [FBZ], oxfendazole [OFZ], mebendazole [MBZ], flubendazole [FLBZ], oxibendazole [OBZ], etc.). RBZ and OFZ are the active sulphoxide metabolites of ABZ and FBZ, respectively. Although they are chemically similar, their physico-chemical properties (Lacey, 1988), tubulin binding affinity (Lubega and Prichard, 1991) and pharmacological potency (Petersen et al., 1997) are different.

The anthelmintic activity of BZD compounds is dependent on two main factors: their affinity for a specific receptor (β-tubulin); and the transport properties that allow the delivery of effective concentrations of the compound at the receptor within the parasite cells, in sufficient time, to cause the therapeutic effect (Thompson et al., 1993). Anthelmintic drugs can reach target helminth parasites by either oral ingestion (from host’s blood and/or intestinal contents) or by diffusion through the external surface (named cuticle in nematodes or tegument in cestodes and trematodes), or some combination of both routes (Thompson and Geary, 1995; Thompson et al., 1993). Cestode parasites do not have a gut such as the gastrodermis in trematodes or the intestine in nematodes, and each proglottis functions as an individual unit. The absence of a digestive tract facilitates the interpretation of the data obtained from drug transport studies (Thompson and Geary, 1995). Therefore, it is of interest that the only way that a given drug molecule can reach its receptor is by passing through the cestode’s tegument. If passive diffusion is the main mechanism of entry of BZD anthelmintics (over active transport), restrictions imposed by tegumental lipid barriers will probably be similar to those of standard cellular membranes. Consequently, the rate of anthelmintic drug penetration will depend mainly on their lipophilicity (Thompson et al., 1993).

Moniezia benedeni, a sheep and cattle intestinal cestode parasite, was used as a parasite model to compare the ex vivo patterns of diffusion of different BZD anthelmintics in the current experiments. The results obtained from the transtegumental diffusion kinetic studies were correlated with the lipid-to-water partition coefficient (P.C.) of the anthelmintic drugs assayed. The logarithm of the octanol–water P.C. (Log P) was chosen as an indicator of drug lipophilicity, since is the most frequently used parameter for defining the lipophilic character of a given drug molecule (Péhourcq et al., 2000).