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R K Prichard

Publications and source records attributed to R K Prichard.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cloning, sequencing, and developmental expression levels of a novel glutamate-gated chloride channel homologue in the parasitic nematode Haemonchus contortus.

Glutamate-gated chloride channels (GluCls) are the proposed site of action for macrocyclic lactone anthelminthics such as ivermectin (IVM) and the milbemycins such as moxidectin (MOX). The importance of this interaction between macrocyclic lactones and GluCls is strengthened by the recent genetic evidence that GluCls are involved in IVM and MOX resistance in the parasitic nematode Haemonchus contortus (1). We have cloned two full length GluCl putative alpha-subunit cDNAs from H. contortus (HcGluCla and b) that exhibit different sized ligand binding domains. Phylogenetic analysis of the conserved regions of the amino acid sequence of HcGluCla suggests that it is a member of the GluCl family but forms a distinct subbranch within this family. The expression level of HcGluCla was examined in different developmental stages of H. contortus (eggs, L3, and adults) and found to be significantly downregulated in eggs compared to adults.

Alternative Splicing↗
PubMed1999

Haemonchus contortus: characterization of a glutamate binding site in unselected and ivermectin-selected larvae and adults.

A specific ivermectin-sensitive, glutamate binding site has been identified in the parasitic nematode Haemonchus contortus. Glutamate binding in H. contortus was saturable and occurred in a single class of high-affinity binding sites which appeared to have pharmacological properties different from those of mammalian glutamate receptors. Adult and larval forms of H. contortus had dramatically different glutamate binding kinetics, the larvae showing nearly up to 200-fold higher Bmax values and up to 9-fold increases in Kd values compared to adults. Treatment of adult H. contortus with the anthelmintic, ivermectin, decreased the Bmax value for glutamate binding in the susceptible strain but not in the resistant parasites. Furthermore, selection for ivermectin resistance was associated with a significant increase in Bmax for glutamate binding in adults and a similarly significant increase in glutamate binding affinity in larvae. These results suggest that the H. contortus glutamate binding site identified in this study may be involved in the phenomenon of ivermectin resistance.

Animals↗
PubMed1999

Haemonchus contortus: effects of glutamate, ivermectin, and moxidectin on inulin uptake activity in unselected and ivermectin-selected adults.

Using [(3)H]inulin uptake as a measure of pharyngeal pumping activity, we have investigated and compared the effects of glutamate, ivermectin, and moxidectin on inulin uptake in susceptible and ivermectin-selected Haemonchus contortus. Inulin uptake is inhibited by glutamate, ivermectin, and moxidectin, at biologically relevant concentrations. Glutamate influences the responses to both ivermectin and moxidectin, suggesting that these three substances share a common mechanism of action. The effects of ivermectin on inulin uptake, but not moxidectin, are significantly altered as a result of selection with ivermectin. These results suggest that ivermectin and moxidectin may differ, to some extent, in their mode of action responses or mechanisms of resistance.

Animals↗
PubMed1999

Effects of the multidrug-resistance-reversing agents verapamil and CL 347,099 on the efficacy of ivermectin or moxidectin against unselected and drug-selected strains of Haemonchus contortus in jirds (Meriones unguiculatus).

The development of anthelmintic resistance is making parasite control in small ruminants problematic. Following the discovery that the drug transporter P-glycoprotein may be involved in macrocyclic lactone resistance in Haemonchus contortus, we determined the effect of two multidrug-resistance modulators, verapamil and CL347,099, on the efficacy of ivermectin and moxidectin against unselected and drug-selected strains of H. contortus. CL347,099 is an analog of verapamil that has multidrug-resistance properties but weaker calcium-channel-blocking activity than the parent drug. The combinations of verapamil with either ivermectin or moxidectin significantly reduced worm counts of the selected strains as compared with the untreated controls, whereas ivermectin or moxidectin alone did not significantly reduce worm counts as compared with the untreated controls. The CL347,099 plus moxidectin combination was significantly more efficacious than moxidectin alone against the ivermectin-selected strain. The drug-combination regimes were without adverse effect on the jirds. However, higher levels of verapamil (> or =40 mg/kg) produced some toxicity.

Animals↗
PubMed1999

Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro.

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.

Animals↗
PubMed1999

Persistent efficacy of doramectin pour-on against artificially induced infections of nematodes in cattle.

Two studies were conducted to determine the persistent efficacy of doramectin pour-on against an artificial, trickle challenge of mixed nematodes in calves. In each study, 42, 4-8 months old calves were randomly assigned into four groups of 10 animals each (T1-T4), plus two larval-viability monitor animals. All animals were treated with fenbendazole (10 mg kg(-1)) 14 days prior to the start of the study to clear any existing infection. Doramectin pour-on at 500 microg kg(-1) was used on each animal in Groups T2, T3, and T4 with intervals of 1 week (Day 0, 7, and 14, respectively). Calves in Group T1 were treated with saline solution on Day 0 and at the same volumetric rate (1 ml 10 kg(-1)) as the doramectin treated animals. All treatments were applied in a single passage along the midline of the back, from the withers to the tailhead. Subsequently, trickle inoculations with infective larvae were administered to all calves for 22 consecutive days (Days 14-35). Doramectin pour-on provided > or = 91.9% efficacy against challenge with Dictyocaulus viviparus, Haemonchus spp., and Ostertagia ostertagi for up to 35 days post-treatment and against challenge with Cooperia oncophora, Cooperia punctata, and Oesophagostomum radiatum for up to 28 days post-treatment.

Abomasum↗
PubMed1998

Selection at a P-glycoprotein gene in ivermectin- and moxidectin-selected strains of Haemonchus contortus.

Resistance to anthelmintics that are used to control parasite populations in domestic animals has become a serious problem worldwide. The development of resistance is an evolutionary process that leads to genetic changes in parasite populations in response to drug exposure. The anthelmintic ivermectin is known to bind to the human membrane transport protein, P-glycoprotein, and P-glycoprotein-deficient mice treated with ivermectin have shown signs of neurotoxicity. P-glycoprotein is believed to be involved in the multidrug resistance phenotype seen in some human cancers and for drug resistance in some protists. We have examined the genetic variation of a P-glycoprotein homologue from the nematode Haemonchus contortus to see if an association exists between specific alleles of this gene and survival to exposure to ivermectin or moxidectin. Two parasite strains passaged without drug treatment and three strains, subjected to anthelmintic selection and derived from the unselected strains, were examined. Allelic variation in the unselected strains showed this locus to be highly polymorphic. chi 2 analyses of allele frequencies showed significant differences between the unselected and the drug-selected derived strains. In all three drug-selected strains, an apparent selection for the same allele was observed. These findings suggest that P-glycoprotein may be involved in resistance to both ivermectin and moxidectin in H. contortus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Haemonchus contortus: selection at a glutamate-gated chloride channel gene in ivermectin- and moxidectin-selected strains.

Anthelmintic resistance in nematode parasites of livestock is a serious problem worldwide. Ivermectin, an avermectin, and moxidectin, a milbemycin, are potent endectocides commonly used to control these parasites. The proposed mode of action of avermectins and possibly the milbemycins involves the binding of the drug to the alpha-subunit of a glutamate-gated chloride channel, which opens or potentiates gating of the channel and leads to the hyperpolarization of the target neuromuscular cell. Glutamate gates the channel by binding to the beta-subunit. We have cloned a fragment of a putative alpha-subunit gene from Haemonchus contortus. The sequence of the beta subunit is available from GenBank. Genetic variability of this fragment was analysed by single-strand conformation polymorphism in five strains of H. contortus: two strains passaged without drug selection, two strains selected with ivermectin, and one strain selected with moxidectin. One allele of the putative alpha-subunit gene appeared to be associated with resistance to the drugs, increasing in frequency in the three drug-selected strains relative to the unselected strains. Another allele appeared to be associated with susceptibility, decreasing in frequency in the three drug-selected strains relative to the unselected strains. A similar analysis of the beta-subunit gene showed no significant differences in allele frequencies between the unselected and drug-selected strains. Our findings suggest a correlation between changes in allele frequencies of the putative alpha-subunit gene and resistance to ivermectin and moxidectin.

Amino Acid Sequence↗
PubMed1998

Hypobiosis of Haemonchus contortus in natural infections of sheep and goats in a semi-arid area of Kenya.

A total of 42 lambs, 42 kids, 21 ewes and 21 does were necropsied during an investigation of the epidemiology of Haemonchus contortus infection of sheep (Red Maasai) and goats (Small East African Goat) in a semi-arid area of Kenya. Availability and establishment of the infective stages were monitored by the necropsy of 21 tracer lambs and 21 tracer kids. Prevalence of H. contortus was over 90% in both sheep and goats and this species contributed to about 80% of the total worm burden. Only about 10% of the hypobiotic larvae were recovered from the mucosal digest whereas about 90% were recovered from the abomasal contents and washings, thereby suggesting that hypobiotic larvae may be loosely attached to the abomasal mucosa from which they may be dislodged during the processing of the abomasa for examination. Throughout the study, both adult worms and hypobiotic larvae were found in proportions that varied with seasons. Statistically, a higher proportion of hypobiotic larvae was found during the dry months than during the wet months, an indication that hypobiosis was an important feature in the survival of H. contortus during the dry months. Negligible worm burdens were acquired by the tracers during the short rains, suggesting that few H. contortus larvae survived on pasture in this season. The effectiveness of strategic control using ivermectin varied according to the timing in relation to the wet season. Treatment did not influence the seasonal pattern of hypobiosis but the treatment administered before the onset of the rains significantly reduced the numbers of both hypobiotic larvae and the adult worms. Treatment during the rains conferred a temporary relief of adult worm burden but had no impact on hypobiotic larvae.

Animals↗
PubMed1998

Cloning and characterization of cDNAs encoding beta-tubulin from Dirofilaria immitis and Onchocerca volvulus.

Beta-Tubulin is the target for the benzimidazole anthelmintics. Unfortunately, none of these drugs is clinically useful against adult filariae. However, beta-tubulin has been shown to be a target for antibody-based toxicity to Brugia pahangi. We cloned and characterized cDNAs encoding beta-tubulin from 2 filariae, Dirofilaria immitis and Onchocerca volvulus, to explore possible explanations for benzimidazole insensitivity among adult filariae and the likelihood that epitopes of beta-tubulin could be used as antigens for a broad-spectrum filarial vaccine. The proteins predicted by these cDNAs were almost identical to the beta-tubulin previously reported from B. pahangi but were less similar to a beta-tubulin cDNA from Onchocerca gibsoni. We cloned the genomic locus for the O. volvulus beta-tubulin cDNA and compared its organization to the reported genomic loci for beta-tubulin in B. pahangi and O. gibsoni. The comparison reinforces the conclusion that the published O. gibsoni gene is in a different family, possibly the beta2 family previously described in B. pahangi. The substitution of tyr for phe at position 200 of beta-tubulin is associated with benzimidazole resistance. All 4 filarial beta-tubulins are predicted to encode phe at this position, suggesting that filarial beta-tubulin is not inherently insensitive to the benzimidazoles. A monoclonal antibody that recognizes the COOH terminus of B. pahangi beta-tubulin is lethal to this parasite in culture. The COOH terminal region is the most variable among the different isotypes of beta-tubulin and distinguishes mammalian from nematode tubulins. This region is highly conserved in 3 of the filarial beta-tubulins.

Amino Acid Sequence↗
PubMed1997

Reversal of P-glycoprotein-associated multidrug resistance by ivermectin.

P-Glycoprotein (P-gp) causes a multidrug resistance (MDR) phenotype in tumour cells. In some cancers, the expression of P-gp has been correlated with low clinical response to chemotherapy and survival of patients. Previous studies have shown that certain lipophilic drugs bind to P-gp and reverse the MDR phenotype of tumour cells. In this study, we extend that list of compounds and present evidence for the capacity of a potent and clinically safe anthelmintic, ivermectin (IVM), as an MDR-reversing drug. Using a highly drug-resistant human cell line, we compared IVM with other MDR-reversing agents and showed that IVM is 4- and 9-fold more potent than cyclosporin A and verapamil, respectively. The capacity of IVM to inhibit iodoaryl-azidoprazosin photolabeling of P-gp is consistent with direct binding to P-gp. Studies showed that [3H]IVM binding to membranes from resistant cells is specific and saturable with KD and Bmax values of 10.6 nM and 19.8 pmol/mg, respectively. However, while cyclosporin A or vinblastine inhibited [3H]IVM binding to membranes from drug-resistant but not drug-sensitive cells, neither verapamil nor colchicine had any effect. Furthermore, both IVM and cyclosporin A and, to a lesser extent, verapamil also inhibited [3H]vinblastine binding to membranes from drug-resistant cells. Drug transport studies showed that [3H]IVM is a substrate for the P-gp drug efflux pump. However, it was transported less efficiently by P-gp than [3H]vinblastine. Moreover, only cyclosporin A was effective in potentiating the accumulation of [3H]IVM in drug-resistant cells. Taken together, the high efficiency of MDR reversal by IVM combined with its low toxicity are consistent with the properties of an ideal MDR-reversing agent.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Effects of three nematode anthelmintic treatment regimes on flock performance of sheep and goats under extensive management in semi-arid Kenya.

A study was undertaken in a semi-arid area of Kenya between August 1991 and June 1993 to evaluate the effects of anthelmintic treatment using ivermectin before or during the rains, on performance of mixed sheep and goat flocks, in comparison with an untreated flock. Performance parameters measured included age and weight of dams at first parturition, parturition intervals, body weights of dams and offspring, and birth weights, growth rates, and mortality rates of offspring. Among these parameters, birth weights and growth rates of offspring were found to be significantly improved by the treatment administered before the rains compared with the other two treatments. Mortality was lower in lambs and kids with high birth weights. Treatment, either before or during the rains, significantly reduced the faecal egg output and improved body weight, packed cell volume and flock fertility. Liveweight was confirmed to be a better measure of sexual maturity than age. It was further shown that lambs and kids, born of dams at their first lambing or kidding, experienced higher mortality rates than lambs and kids born of dams in their second and subsequent parturitions. Overall, treatment with ivermectin before the onset of rains was equal to or better, in terms of the performance parameters measured, than treatment during the rains, whilst treatment compared with no treatment increased performance in almost all of the parameters measured.

Albendazole↗
PubMed1997

Nematode reinfection following treatment of cattle with doramectin and ivermectin.

A study was conducted to investigate reinfection with nematodes in calves following treatment with doramectin or ivermectin administered subcutaneously at a dose rate of 200 micrograms kg-1 of body weight under conditions of natural challenge. Thirty calves were allocated to three groups of 10 calves each based on body weight, sex, breed and pre-treatment faecal egg counts (FEC) after grazing together on a common pasture for three months. Treatments were doramectin, ivermectin and no treatment. Calves were returned to the same pasture for 56 days, placed on dry lot from days 56 to 63 and then necropsied over days 64-66. Faecal egg counts were done at days -1 and 0, then bi-weekly from day 14 to 56 and day 63. Mean FEC in control calves continued to rise throughout the grazing period. Trichostrongyle FEC were significantly (P < 0.05) greater in the control group compared to either treated group at each sample time following treatment. At necropsy, a total geometric mean of 19,847 nematodes per calf was recovered from untreated controls, of which eight genera were identified. The predominant nematode genera were Ostertagia (8749), Nematodirus (3702) and Cooperia (1927). In the ivermectin-treated calves, geometric mean worm burden was similar to that of the untreated controls: A total of 20,349 nematodes per calf was present including the genera Nematodirus (8633), Ostertagia (4700) and Cooperia (1740). In contrast, the geometric mean worm burden in doramectin-treated calves was 12,173, including Ostertagia (4310), Cooperia (1141) and Nematodirus (1667). Doramectin was more effective than ivermectin in protecting calves from reinfection over a 56-day post-treatment grazing period under conditions of natural challenge as measured by accumulated mean worm burdens.

Animals↗
PubMed1996

p-Azidosalicyl-5-amino-6-phenoxybenzimidazole photolabels the N-terminal 63-103 amino acids of Haemonchus contortus beta-tubulin 1.

Benzimidazoles (BZ) are broad spectrum anthelmintics thought to exert their effects by interacting with and disrupting the functions of microtubules. However, direct biochemical evidence for binding between BZ and tubulin has not been shown nor is it known what sequences in tubulin interact with BZ. In this study, a photoactive analogue of 2-acetamido-5-(3-aminophenoxy)benzimidaz ole that has biological activity similar to other benzimidazoles was synthesized and used to photoaffinity label cell lysates from the parasitic nematode of sheep Haemonchus contortus. The photoactive analogue, 2-acetamido-5-[3-(4-azido-3-125I-salicyl amido)phenoxy]benzimida zol e or 125I-ASA-BZ, was shown to photolabel a 54-kDa protein that was specifically immunoprecipitated with anti-tubulin monoclonal antibodies. Tubulin photoaffinity labeling by 125I-ASA-BZ was also inhibited with molar excess of various BZ analogues and colchicine. Interestingly, 125I-ASA-BZ photoaffinity-labeled the beta- and not the alpha-subunits of tubulin. Proteolytic digestion of 125I-ASA-BZ-labeled tubulin with Staphylococcus aureus V8 proteinase revealed one major peptide with an apparent molecular mass of 3.5 kDa. Exhaustive digestion of 125I-ASA-BZ-labeled beta-tubulin with trypsin resulted in two fractions containing radioactive peptides. Protein sequencing of the high performance liquid chromatography-purified tryptic ASA-BZ-photolabeled peptides identified the N-terminal 63-77 and 78-103 sequences as the BZ binding domain.

Affinity Labels↗
PubMed1995

Influence on the antithyroid compound methimazole on the plasma disposition of fenbendazole and oxfendazole in sheep.

The influence of methimazole on the plasma disposition kinetics of fenbendazole, oxfendazole and their metabolites, was investigated in adult sheep. The two anthelmintics were administered by oral drench at 5 mg kg-1 either alone (control treatments) or together with methimazole given orally at 3 mg kg-1. Blood samples were taken serially for 144 hours. Fenbendazole parent drug and its sulphoxide and sulphone metabolites were the three analytes observed by high performance liquid chromatography (HPLC) after the administration of both anthelmintics. The disposition of each analyte followed a similar pattern after the administration of the two anthelmintics alone. Oxfendazole was the main component recovered in plasma between four and 120 to 144 hours after the administration of both anthelmintics either with or without methimazole. A modified pattern of disposition, with significantly higher Cmax and AUC values for fenbendazole parent drug, and a delayed appearance in plasma with retarded Tmax values for the sulphoxide and sulphone metabolites, were the main pharmacokinetic changes observed when the drugs were administered with methimazole.

Animals↗
PubMed1995

Comparative plasma disposition kinetics of albendazole, fenbendazole, oxfendazole and their metabolites in adult sheep.

The comparative plasma disposition kinetics of albendazole (ABZ), fenbendazole (FBZ) and oxfendazole (OFZ) following their oral administration (5 mg/kg) to adult sheep was characterized. Jugular blood samples were taken serially over a 144 h period and plasma was analysed by high performance liquid chromatography (HPLC) for ABZ, ABZ sulphoxide (ABZSO) and ABZ sulphone (ABZSO2) (ABZ treatment), and for FBZ, OFZ and FBZ sulphone (FBZSO2) (FBZ and OFZ treatments). While the ABZ parent drug was not detected at any time post-treatment, ABZSO and ABZSO2 were the analytes recovered in plasma after oral administration of ABZ to sheep. The active ABZSO metabolite was the main analyte recovered in plasma (between 0.25 and 60 h post-treatment), accounting for 71% of the total AUC. FBZ, OFZ and FBZSO2 were the analytes detected in plasma following the oral administration of both FBZ and OFZ to sheep. Low concentrations of FBZ were found in plasma between 4 (FBZ treatment) or 8 h (OFZ treatment) and 72 h post-treatment. The plasma profile of each analyte followed a similar pattern after both treatments; OFZ being the main component detected in plasma. The plasma disposition of ABZ metabolites was markedly different to that of FBZ derivatives. ABZSO exhibited faster absorption and a higher Cmax than OFZ (both treatments). Furthermore, while ABZSO declined relatively rapidly in plasma reaching non-detectable concentrations at 60 h post-ABZ administration, OFZ was found in plasma for up to 120 (FBZ treatment) and 144 h (OFZ treatment).(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1994

Benzimidazoles, potent anti-mitotic drugs: substrates for the P-glycoprotein transporter in multidrug-resistant cells.

P-glycoprotein is though to mediate the energy-dependent efflux of many structurally and functionally unrelated lipophilic compounds. Presently, the molecular mechanism underlying the binding and efflux of drugs by P-glycoprotein is not well understood. However, it has been suggested that two planar benzene ring structures and a cationic charge are commonly found in many drugs that interact with P-glycoprotein. The benzimidazoles (BZs) are potent anti-tumour, anti-fungal and anti-parasitic agents, whose mode of action is thought to result from their inhibition of microtubule functions. Although other classes of microtubule inhibitors, such as colchicine and vinblastine, have been studied extensively with respect to their interaction and efflux by P-glycoprotein, the BZ group of drugs has not been characterized. In this study, we have characterized the interaction of BZ with multidrug-resistant cells and found that resistant cells accumulated substantially less BZ compared with drug-sensitive cells. Furthermore, BZ was more toxic to sensitive than to drug-resistant cells, suggesting that BZ is likely to be a substrate for the P-glycoprotein drug efflux pump. In addition, we used a photoactive analogue of BZ ([125I]ASA-BZ) to demonstrate a direct binding between BZ and P-glycoprotein. Results showing that a molar excess of vinblastine, unmodified BZ, verapamil and rhodamine 123, but not colchicine, inhibited the photoaffinity labelling of P-glycoprotein by [125I]ASA-BZ confirmed the binding specificity of BZ to P-glycoprotein. Protease digestion of [125I]ASA-BZ photoaffinity labelled P-glycoprotein yielded two peptides that were similar to those obtained with other P-glycoprotein-associated drugs, e.g. azidopine and iodoaryl azidoprazosin. Taken together, these results demonstrate a direct and specific interaction between P-glycoprotein and BZ in a manner that is probably similar to other previously characterized P-glycoprotein-associated drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗