Biomedical subjects

R K Prichard

Publications and source records attributed to R K Prichard.

At least 19 recordsLinked to original sources

PubMed1992

Mechanisms of inactivation of Schistosoma mansoni and mammalian glutathione S-transferase activity by the antischistosomal drug oltipraz.

Glutathione S-transferase (GST) purified from Schistosoma mansoni or human placenta was inhibited by the antischistosomal drug oltipraz (OPZ) in a time- and concentration-dependent manner. Inhibition of placenta GST was complete at a low concentration of drug, whereas that of parasite GST was incomplete and relatively high amounts of OPZ were needed to reach 50% inhibition. Complete reactivation of GST from placenta was achieved with dithiothreitol (DTT) and other sulfhydryl-containing compounds, while the inactivation of parasite GST was irreversible. The oxy-derivative of OPZ (RP 36,642), in which the thione sulfur is replaced with oxygen, did not inhibit GST activity. There were no differences between OPZ and RP 36,642 in their patterns of binding to the hydrophobic non-substrate site of GST. GST from the placenta incorporated much higher levels of [14C]N-ethylmaleimide compared to schistosome GST. The incorporation of [14C]N-ethylmaleimide by GST was inhibited by OPZ but not by RP 36,642. Yeast and S. mansoni hexokinases were similarly inhibited by OPZ but not by RP 36,642. Both hexokinase preparations recovered their activity following incubation with DTT. These data suggest that the inactivation of these enzymes by OPZ is a result of its interaction with their SH groups. Thus, the antischistosomal activity of OPZ may be accounted for by its interaction with the SH groups of macromolecules in general.

Animals↗

PubMed1992

Induction of glutathione S-transferase isoenzymes in mouse liver by 5-(2-pyrazynl)-4-methyl-1,2-dithiole-3-thione (oltipraz).

Treatment of mice with a single dose of oltipraz (OPZ) at 200 mg/kg led to a significant (P less than 0.05) increase in hepatic cytosolic glutathione S-transferase (GST) activity and content. GST activity monitored with 1,2-dichloro-4-nitrobenzene was increased 3.8-fold 3 days after treatment, suggesting the induction of mu class isoenzymes. Ethacrynic acid, a marker for pi class isoforms, showed only a slight increase in GST activity while no induction was observed with cumene hydroperoxide, an indicator for the alpha class. The increase in mu class isoenzymes was further confirmed by separation of the mouse liver affinity purified GST by chromatofocusing and also by resolving the GST subunits by reverse-phase high performance liquid chromatographic procedures. Therefore, OPZ induces mainly the mu class isoenzymes in mouse hepatic tissues.

Animals↗

PubMed1992

Methimazole increases the plasma concentrations of the albendazole metabolites of netobimin in sheep.

The influence of methimazole (MTZ) on the pharmacokinetics of netobimin (NTB) and its metabolites was investigated in adult sheep. NTB zwitterion suspension was administered at 20 mg kg-1 by intraruminal injection either alone or with simultaneous administration of MTZ intramuscularly at 1.5 mg kg-1. Blood samples were taken serially over a 120-h period and plasma was analysed by HPLC for NTB, albendazole (ABZ), albendazole sulphoxide (ABZSO), and albendazole sulphone (ABZSO2). NTB parent drug showed fast absorption, low area under the plasma concentration-time curve (AUC) and was rapidly removed from plasma after both treatments. The presence of MTZ did increase significantly the ABZ AUC (138 per cent) and mean residence time (MRT) (86 per cent). Concomitant treatment with MTZ resulted in a notably higher ABZSO plasma profile with significantly longer elimination half-life (t1/2 beta) (390 per cent) and MRT (252 per cent) and with significantly higher AUC (95 per cent). Also, MTZ induced significant increases in ABZSO2 t1/2 beta, AUC, and MRT. We have demonstrated a pharmacokinetic interaction between MTZ and NTB metabolites. MTZ may alter the liver biotransformation of ABZ metabolites which results in pronounced changes in the disposition kinetics of anthelmintically active metabolites.

Albendazole↗

PubMed1992

Identification of a novel Brugia pahangi beta-tubulin gene (beta 2) and a 22-nucleotide spliced leader sequence on beta 1-tubulin mRNA.

We have examined the expression of beta-tubulin genes in the parasitic nematode, Brugia pahangi. A genomic library was constructed and screened by hybridization with a Haemonchus contortus beta-tubulin cDNA fragment which recognizes several B. pahangi beta-tubulin sequences, including sequences which correspond to the previously characterized beta 1-tubulin gene. The B. pahangi beta 2-tubulin gene was isolated by selecting clones which hybridize to the H. contortus beta-tubulin gene but which do not hybridize to the beta 1-tubulin gene. A partial sequence of the beta 2-tubulin gene confirms that it codes for a distinct beta-tubulin. Southern hybridization analyses show that the beta 2-tubulin sequence exists as a single copy gene within the B. pahangi genome. Expression of the beta 2-tubulin gene is developmentally regulated and the message is found predominantly in adult male worms, whereas the beta 1-tubulin gene is expressed in microfilariae and approximately equal levels of the transcript are found in male and female adult worms. During mRNA maturation the beta 1-tubulin mRNA of microfilariae and adult worms acquires a trans-spliced leader identical to the SL1 of Caenorhabditis elegans.

Amino Acid Sequence↗

PubMed1992

Three beta-tubulin cDNAs from the parasitic nematode Haemonchus contortus.

Experimental evidence indicates that tubulin is the site of action of the anthelmintic benzimidazoles. Furthermore, certain residues of beta-tubulin seem to be critical for this mechanism. Although the benzimidazoles selectively affect nematode vs. mammalian beta-tubulin, the molecular basis for this differential action is not known. To enhance our understanding of this phenomenon, and to provide the basis for investigating benzimidazole resistance in parasitic nematodes, we undertook the cloning of beta-tubulin cDNAs from the ruminant parasite, Haemonchus contortus. We have cloned and sequenced three beta-tubulin cDNAs from this organism, beta 12-16, beta 12-164, and beta 8-9. The first 2 differ at only 23 nucleotides, which give rise to 4 amino acid changes. beta 8-9 represents a different isotype class from the other two, since it differs extensively in the carboxyterminus. By comparing the sequences of these and other nematode beta-tubulins with mammalian beta-tubulins, several regions of consistent difference can be recognized; the functional significance of these regional differences has not been defined. Sequences very similar or identical to beta 8-9 and beta 12-16 are present in both benzimidazole-sensitive and benzimidazole-resistant populations of H. contortus. However, it appears that drug-resistant organisms may differ in the presence of a gene product which is closely related to beta 8-9.

Amino Acid Sequence↗

PubMed1992

Epidemiological study of parasite infection in a cow-calf beef herd in Quebec.

The patterns of gastrointestinal and pulmonary nematode infections in a previously untreated Aberdeen Angus cow-calf herd were observed between May 1988 and December 1990. The cow-calf herd and replacement heifers were on separate pastures. The relatively high mean faecal egg counts of cows and heifers at the time of turnout were mainly owing to the maturation of hypobiotic worms. The strongyle egg counts of calves began to rise soon after turnout onto pasture and reached peak levels at the end of the grazing season. The number of infective larvae on pasture was highest during September/October. Ostertagia, Cooperia and Nematodirus were the most prevalent genera found at necropsy and on pasture. Larvae of these nematodes were able to overwinter on pasture and Ostertagia larvae, additionally, were able to overwinter in the host as arrested early fourth stage larvae. The high egg output of cows at the time of turnout may serve as a source of infection for their calves and be responsible for the late-season rise in pasture larval counts.

Animals↗

PubMed1992

Efficacy of moxidectin against naturally acquired nematode infections in cattle.

The efficacy of moxidectin, a new endectocide against natural nematode infections, was evaluated. Twenty-five calves were divided into two groups of eight calves each and a third group of nine calves. Moxidectin was administered s.c. to two groups (I, 0.2 mg kg-1 body weight (BW); II, 0.3 mg kg-1 BW) and the third group (III) received the vehicle (placebo) s.c. Two pre-treatment and one post-treatment faecal nematode egg count determinations were made from all calves, and they were necropsied 2 weeks after treatment for the identification and enumeration of nematode parasites. Group III calves, which received the drug-free vehicle, harboured eight species of nematodes (Ostertagia ostertagi, Trichostrongylus axei, Cooperia oncophora, Cooperia punctata, Nematodirus helvetianus, Trichuris discolor, Oesophagostomum radiatum and Dictyocaulus viviparus). The mean total worm burden for this group was 8935. There was a significant reduction in the numbers of many species of nematodes (Ostertagia, Trichostrongylus, Cooperia and Nematodirus) in both treated groups. Cooperia oncophora was reduced by 94% in Group I and by 96% in Group II, while all other nematode species were reduced by 99%. Immature stages of Ostertagia and Nematodirus were significantly reduced in the two treated groups. Two weeks after treatment, the mean faecal egg counts of both treated groups were reduced by more than 98%. There was no significant difference in mean total worm burdens or egg counts between the two treated groups.

Animals↗

PubMed1992

The effect of co-administration of parbendazole on the disposition of oxfendazole in sheep.

The effect of intraruminal administration of parbendazole (PBZ) on the flow rate of bile and the pharmacokinetic behaviour of oxfendazole (OFZ) was examined in sheep. PBZ given at 18, 9 and 4.5 mg/kg resulted in a dose-related reduction in bile flow rate which was also inversely related to changing concentration of PBZ and its metabolites in plasma. Co-administration of 4.5 mg PBZ/kg with 5.0 mg [14C]-OFZ/kg resulted in increased concentrations of fenbendazole (FBZ), OFZ and fenbendazole sulphone (FBZ-SO2) in plasma, although total 14C levels remained unchanged compared with that observed when OFZ alone was administered. The presence of PBZ also reduced biliary secretion of 14C by 22% and altered the relative proportions of OFZ metabolites in bile during the 72-h experimental period. The ratio of 4'-hydroxy-OFZ (OH-OFZ) to 4'-hydroxy-FBZ (OH-FBZ) changed from 7:1 in the absence of PBZ to approximately 1:1 in the presence of PBZ. There was no change in urinary or faecal 14C excretion. The PBZ-induced effects were temporary since the pharmacokinetic behaviour of OFZ given alone two weeks before was similar to that given two weeks after PBZ co-administration. It is suggested that the presence of PBZ temporarily slowed hepatic metabolism and biliary secretion of OFZ metabolites but concomitantly increased extra-biliary transfer of OFZ and/or its metabolites from plasma into the gastrointestinal tract. Elevated exposure of parasites in the gut wall to plasma-derived drug, coupled with higher concentrations of anthelmintically active OH-FBZ secreted in bile, could contribute to the previously reported increased efficacy of OFZ when co-administered with PBZ.

Analysis of Variance↗

PubMed1992

Morantel tartrate release from a long-acting intraruminal device in cattle: pharmacokinetics and gastrointestinal distribution.

The pharmacokinetics and gastrointestinal distribution of morantel tartrate release from a sustained release trilaminate bolus in cattle were investigated over a 98-day period post-treatment. Six Holstein calves (125-150 kg) had permanent indwelling fistulae surgically inserted into the rumen, abomasum and terminal ileum. Samples of jugular blood, feces and ruminal, abomasal and ileal fluids were taken on days -3, 1, 4, 7, 10, 14 and weekly up to 98 days post-bolus administration. Morantel tartrate concentrations were measured by HPLC after extraction and clean-up. Morantel was not detected in plasma at any time after bolus administration. High concentrations of morantel tartrate were found in ruminal, abomasal and ileal fluids and feces over 98 days post-treatment. The morantel peak concentration (Cmax) was achieved at Day 1 post-administration in each of these compartments. The steady-state morantel concentration (Css) was achieved at approximately 10 days post-treatment and maintained for 91-98 days post-treatment in these gastrointestinal compartments. The morantel Cmax, Css, area under the zero (AUC) and first moment (AUMC) of the concentration-time curve were significantly higher (P less than 0.01) in feces than in other compartments. The in vivo drug release profile of this device has been determined. Steady-state concentrations for from 91 to 98 days have been confirmed.

Abomasum↗

PubMed1992

Methimazole-mediated modulation of netobimin biotransformation in sheep: a pharmacokinetic assessment.

The effects of modulation of liver microsomal sulphoxidation on the disposition kinetics of netobimin (NTB) metabolites were investigated in sheep. A zwitterion suspension of NTB was given orally at 7.5 mg/kg to sheep either alone (control treatment) or co-administered with methimazole (MTZ) orally (NTB + MTZ oral treatment) or intra-muscularly (NTB + MTZ i.m.) at 3 mg/kg. Blood samples were taken serially over a 72 h period and plasma was analysed by HPLC for NTB and its major metabolites, i.e. albendazole (ABZ), albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2). Only trace amounts of NTB parent drug and ABZ were detected in the earliest samples after either treatment. There were significant modifications to the disposition kinetics of ABZSO in the presence of MTZ. ABZSO elimination half-life increased from 7.27 h (control treatment) to 14.57 h (NTB + MTZ oral) and to 11.39 h (NTB + MTZ i.m.). ABZSO AUCs were significantly higher (P less than 0.05) for the NTB + MTZ oral treatment (+55%) and for the NTB + MTZ i.m. treatment (+61%), compared with the NTB alone treatment. The mean residence times for ABZSO were 12.66 +/- 0.68 h (control treatment), 18.85 +/- 2.35 h (NTB + MTZ oral) and 17.02 +/- 0.90 h (NTB + MTZ i.m.). There were no major changes in the overall pharmacokinetics of ABZSO2 for the concomitant MTZ treatments. However, delayed appearance of this metabolite in the plasma resulted in longer ABZSO2 lag times and a delayed Tmax for treatments with MTZ.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

PubMed1992

Metabolism of albendazole and albendazole sulphoxide by ruminal and intestinal fluids of sheep and cattle.

1. The metabolism of albendazole (ABZ), albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2) by ruminal, abomasal and ileal fluids of sheep and cattle was investigated under anaerobic conditions in vitro. 2. None of the compounds was metabolically changed by incubation with abomasal fluids of sheep and cattle. 3. ABZ and ABZSO were extensively metabolized by sheep and cattle ruminal and ileal fluids. ABZSO2 was unaffected by incubation with these gastrointestinal fluids. 4. The rate of ABZ oxidation into ABZSO was greater for cattle ruminal and ileal fluids than for sheep fluids. 5. ABZSO was reduced back to ABZ by ruminal and ileal fluids of both species. This reducing activity was significantly higher for both ruminal and ileal fluids of sheep compared with those of cattle.

Abomasum↗

PubMed1992

Effects of methimazole on the kinetics of netobimin metabolites in cattle.

1. The effects of methimazole (MTZ) on the pharmacokinetic behaviour of netobimin (NTB) and its albendazole (ABZ) metabolites were studied in calves. NTB trisamine salt solution was given by subcutaneous (12.5 mg/kg) and oral (20 mg/kg) routes, either alone or co-administered with MTZ (1.5 mg/kg, intramuscularly). 2. NTB parent drug was detected only after s.c. treatments, showing rapid absorption, early Cmax and fast disposition. ABZ was not found in plasma at any time after either s.c. or oral treatments. 3. Concomitant treatment with MTZ significantly increased the albendazole sulphoxide (ABZSO) elimination half-life (t1/2 beta) (321%) and mean residence time (MRT) (170%) from the values obtained after s.c. treatment with NTB alone. 4. Oral treatment resulted in an ABZSO pharmacokinetic profile with an AUC 27% higher, a significantly longer t1/2 beta (151%) and MRT (124%) in the presence of MTZ. 5. We conclude that when co-administered with NTB in cattle, MTZ induces significant changes in the disposition kinetics of the anthelmintically active ABZSO metabolite.

Administration, Oral↗

PubMed1991

Differential effects of oltipraz and its oxy-analogue on the viability of Schistosoma mansoni and the activity of glutathione S-transferase.

Adult worms of Schistosoma mansoni recovered from mice treated with oltipraz (OPZ) showed a significant diminution in their ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan, a measure of parasite viability. Incubation of glutathione S-transferase (GST) from S. mansoni with OPZ resulted in a time- and concentration-dependent inhibition of enzyme activity. RP 36,642 (an inactive oxy-derivative of OPZ) had a minimal effect on the viability of the worms and no effect on GST activity. The structural integrity of OPZ, particularly the thione sulphur, appears to be necessary for expression of the antischistosomal effects of the drug. OPZ-induced inhibition of GST was non-competitive with either reduced glutathione (GSH) or 1-chloro-2,4-dinitrobenzene (CDNB), indicating that the drug is not a substrate for GST-catalysed conjugation reactions. In addition, the inhibition of GST could not be reversed by dialysis or repurification of the enzyme via a GSH-agarose affinity column. The effects of OPZ on GST activity could render the parasite vulnerable to damage by host-derived reactive oxygen species and aldehydic products of lipid peroxidation. The effects of OPZ on GST activity may play a role in the antischistosomal action of OPZ.

Animals↗

PubMed1991

Specific interaction of benzimidazole anthelmintics with tubulin from developing stages of thiabendazole-susceptible and -resistant Haemonchus contortus.

The low- and high-affinity binding of tritiated benzimidazole anthelmintics (mebendazole and oxibendazole) to tubulin-containing supernatants derived from unembryonated eggs, third stage larvae or adult worms of thiabendazole-susceptible and -resistant strains of Haemonchus contortus were examined and compared. The displacement of these radioligands by unlabelled benzimidazoles (mebendazole, fenbendazole, thiabendazole and oxibendazole) also was examined. The binding affinity, K alpha, and maximum binding, Bmax, for the high-affinity binding were calculated by non-linear least-square iterative curve fitting using a computer programme (LIGAND) based on the exact mathematical model of ligand-receptor interactions. The K alpha was of the same order of magnitude (x 10(7) M-1) for the susceptible and resistant eggs, larvae and worms. Resistance was associated with a loss of high-affinity binding. There was a 2- to 5-fold loss of Bmax by the resistant strain. The eggs showed greater high-affinity binding per milligram of protein than the larvae which, in turn, showed greater high-affinity binding than the adult worms. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis that the tubulin content per milligram of protein decreased from egg, through larvae to adult worm. Cross-displacement studies indicated that different benzimidazole drugs interacted with the same receptor (tubulin) and that a rank order of affinity of the benzimidazole drugs could be inferred.

Animals↗

PubMed1991

Interaction of benzimidazole anthelmintics with Haemonchus contortus tubulin: binding affinity and anthelmintic efficacy.

The ability of various benzimidazoles (BZs) to bind tubulin under different conditions was assessed by determining their IC50 values (the concentration of unlabeled drug required to inhibit 50% of the labeled drug binding), Ka (the apparent equilibrium association constant) and Bmax (the maximum binding at infinite [BZ] = [drug-receptor]). The ability of unlabeled benzimidazoles--fenbendazole, mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ), rycobendazole (albendazole sulfoxide, ABZSO), albendazole sulfone, oxfendazole (OFZ), and thiabendazole--to bind tubulin was determined from their ability to inhibit the binding of [3H]MBZ or [3H]OBZ to tubulin in supernatants derived from unembryonated eggs or adult worms of Haemonchus contortus. The binding constants (IC50, Ka, and Bmax) correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZ compounds except for OFZ and ABZSO whose Ka values were lower than could be expected from anthelmintic potency. The binding of [3H]ABZ or [3H]OFZ to tubulin in supernatants derived from BZ-susceptible and BZ-resistant H. contortus was compared. [3H]ABZ demonstrated saturable high-affinity binding but [3H]OFZ bound with low affinity. The high-affinity binding of [3H]ABZ was reduced for the R strain. Tubulin bound BZ drugs at 4 degrees C with lower apparent Ka than at 37 degrees C.

Albendazole↗

PubMed1991

Identification of tubulin isoforms in different tissues of Ascaris suum using anti-tubulin monoclonal antibodies.

Three monoclonal antibodies specific to alpha- and beta-tubulin were used to examine the expression of tubulin isoforms in the intestine, reproductive tract and body wall muscle of A. suum. The tubulins were found to be different in their isoelectric points, number of isoforms and peptide maps with Western blot analysis of one-dimensional polyacrylamide gel confirming the presence of alpha-, beta 1- and beta 2-tubulin. Commercial cross-reactive anti-alpha and anti-beta MAbs 356 and 357 recognized tubulin from A. suum tissues as well as from pig brain, whereas anti-A. suum beta-tubulin specific MAb P3D6 recognized tubulin from the A. suum tissues only. Two-dimensional gel analysis showed different isoform patterns in different A. suum tissues with anti-A. suum beta-tubulin MAb P3D6 and cross-reactive beta-tubulin MAb 357 recognizing 2-4 beta-tubulin isoforms and anti-alpha-tubulin MAb 356 recognizing 1-6 alpha-tubulin isoforms. Different peptide maps of tubulin were observed in the three tissues, when subjected to limited proteolysis followed by SDS-PAGE. The data indicate that different tubulins are found in different tissues of adult A. suum.

Animals↗

PubMed1991

Oltipraz-induced decrease in the activity of cytosolic glutathione S-transferase in Schistosoma mansoni.

The activity of glutathione S-transferase (GST) decreased progressively in Schistosoma mansoni from mice treated with oltipraz (OPZ). However, the peroxidase activity of GST (selenium-independent) and selenium-dependent glutathione peroxidase was not affected by OPZ treatment. Purification and quantification of GST from worms after OPZ treatment indicated that the decrease in enzyme activity was greater than could be accounted for by the decrease in GST protein content. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with GST isoenzyme specific antisera revealed a slight decrease in the quantity of both 26 and 28 kDa GSTs. Fractionation of cytosolic GSTs from male S. mansoni by chromatofocusing resolved three major isoenzymes (SmI, II and III) and a minor form which eluted first from the column. SmI, II and III all had a molecular weight of about 28 kDa on SDS-polyacrylamide gel electrophoresis. However, on electrophoresis in the absence of SDS, the three GST forms exhibited different mobilities. The pattern of SmI, II and III was similar in untreated and OPZ-treated worms, but the activities of the isoenzymes from treated worms were lower. The results suggest that OPZ interacts with the GST isoenzymes SmI, II and III in a similar manner; thus, the effects are not isoenzyme specific. Taken together, these results suggest that OPZ and/or its metabolites interact directly with GST resulting in inhibition of activity and reduction in total enzyme protein. This mechanism may be important in the antischistosomal action of OPZ.

Animals↗

PubMed1991

Enhancement of the plasma concentration of albendazole sulphoxide in sheep following coadministration of parenteral netobimin and liver oxidase inhibitors.

The effects of methimazole (MTZ), metyrapone (MTP) and quinine (QNE) on the pharmacokinetics and bioavailability of parenterally administered netobimin (NTB) and its major metabolites, albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2), were studied in sheep. NTB trisamine solution was first administered alone at 20 mg kg-1 by subcutaneous injection and then coadministered with either MTZ (1.5 mg kg-1 intramuscularly), MTP (20 mg kg-1 subcutaneously) or QNE (30 mg kg-1 intraruminally) in adult sheep. Blood samples were taken serially over a 120 hour period and plasma was analysed for NTB and its metabolites by high performance liquid chromatography. NTB parent drug showed a similar pharmacokinetic behaviour after all parenteral treatments. Both ABZSO AUCs (P less than 0.01) and Cmax (P less than 0.05) were significantly higher in the presence of MTZ and MTP than with the treatment with NTB alone. In the presence of each of the oxidation inhibitor compounds, the ratio of AUC ABZSO/ABZSO2 was significantly higher than with the NTB alone treatment. It has been demonstrated that the coadministration of substances which alter liver microsomal oxidation resulted in a modified pharmacokinetic pattern for the metabolites of NTB. Both NTB + MTZ and NTB + MTP treatments resulted in an improved pharmacokinetic profile for the anthelmintically active ABZSO metabolite.

Albendazole↗