The metabolism of the anthelmintics clioxanide and resorantel and related compounds in vitro by Moniezia expansa, Ascaris suum and mouse- and sheep-liver enzymes.

1. Clioxanide and related compounds were deacetylated by the cestode Moniezia expansa, the nematode Ascaris suum, by enzymes prepared from these species and by mouse- and sheep-liver homogenates. Deacetylase activity was found in the cytosol of cestode proglottids throughout the strobila, and in the cytosol of nematode intestinal cells and reproductive tract. 2. The O-deacetylases from both helminths showed similar pH optima of about 7.0. Activity was enhanced by Ca2+ and low molecular weight thiols. Cu2+, Cd2+, Hg2+, Zn2+, and La2+ inhibited the deacetylation of clioxanide. 3. Resorantel and clioxanide were not hydrolysed at the amide bond by helminth or mammalian enzymes. 4. Resorantel was hydroxylated by mammalian microsomal enzymes, but helminths did not modify the molecule.     

The metabolism of niclosamide and related compounds by Moniezia expansa, Ascaris lumbricoides var suum, and mouse- and sheep-liver enzymes.

1. Niclosamide and related nitro compounds were reduced to the corresponding amines by Moniezia expansa, Ascaris lumbricoides var suum and by enzymes prepared from these species and by mouse and sheep liver homogenates. The reduction of niclosamide by the helminths required as cofactors NADH2 and glutathione, but was inhibited 50% by 2 X 10(-7) M allopurinol. 2. Unlike benzanilide, niclosamide was not hydrolysed by either the helminths, or the mammalian liver preparations. Rates of hydrolysis of compounds related to niclosamide indicate that niclosamide was not hydrolysed because substituents in both benzene rings ortho to the amide bond sterically hinder the hydrolase. 3. Hydrolysis of benzanilide and related compounds was inhibited by anthelmintic organophosphates.     

The metabolism of foreign compounds in the cestode, Moniezia expansa, and the nematode, Ascaris lumbricoides var suum.

1. The ability of the cestode Moniezia expansa and the nematode Ascaris lumbricoides var suum to metabolize foreign compounds has been assessed. 2. Both species were unable to oxidase aldrin, aniline, biphenyl, butylbenzene and nitrobenzene or to demethylate aminopyrine, and 4-nitroanisole. 3. M. expansa and A. lumbricoides var suum readily induced 4-nitroanisole, nitrobenzene, 4-nitrobenzoic acid and 4-nitrophenol to the corresponding amines. Azobenzene, dimethylaminoazobenzene, and 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene were also reduced. 4. Hydrolysis of esters, acetanilide, acetylsalicylic acid, aryl sulphates and aryl phosphates took place readily. However, beta-glucuronides were not hydrolysed. 5. The following reactions were not detected in either species: phosphate, sulphate, beta-glucuronide or beta-glycoside conjugation of phenolic compounds; acetylation of amino compounds, or the formation of glycine conjugates with 4-aminobenzoic acid or benzoic acid. 6. Male nematodes showed a higher rate of drug metabolism than female nematodes.     

The Metabolism of Niclosamide and Related Compounds by Moniezia expansa,Ascaris lumbricoides var suum,and Mouse- and Sheep-liver Enzymes

1. Niclosamide and related nitro compounds were reduced to the corresponding amines by Moniezia expansa, Ascaris lumbricoides var suum and by enzymes prepared from these species and by mouse and sheep liver homogenates. The reduction of niclosamide by the helminths required as cofactors NADH₂ and glutathione, but was inhibited 50% by 2 × 10^-7 M allopurinol.

2. Unlike benzanilide, niclosamide was not hydrolysed by either the helminths, or the mammalian liver preparations. Rates of hydrolysis of compounds related to niclosamide indicate that niclosamide was not hydrolysed because substituents in both benzene rings ortho to the amide bond sterically hinder the hydrolase.

3. Hydrolysis of benzanilide and related compounds was inhibited by anthelmintic organophosphates.     

The Metabolism of Foreign Compounds in the Cestode,Moniezia expansa,and the Nematode,Ascaris lumbricoides var suum

1. The ability of the cestode Moniezia expansa and the nematode Ascaris lumbricoides var suum to metabolize foreign compounds has been assessed.

2. Both species were unable to oxidase aldrin, aniline, biphenyl, butyl-benzene and nitrobenzene or to demethylate aminopyrine, and 4-nitroanisole.

3. M. expansa and A. lumbricoides var suum readily reduced 4-nitroanisole, nitrobenzene, 4-nitrobenzoic acid and 4-nitrophenol to the corresponding amines. Azobenzene, dimethylaminoazobenzene, and 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene were also reduced.

4. Hydrolysis of esters, acetanilide, acetylsalicylic acid, aryl sulphates and aryl phosphates took place readily. However, β-glucuronides were not hydrolysed.

5. The following reactions were not detected in either species: phosphate, sulphate, β-glucuronide or β-glycoside conjugation of phenolic compounds; acetylation of amino compounds, or the formation of glycine conjugates with 4-aminobenzoic acid or benzoic acid.

6. Male nematodes showed a higher rate of drug metabolism than female nematodes.     

The localization and some properties of the acetylsalicylic acid O-deacetylases of Ascaris lumbricoides var suum and Moniezia expansa

1. Enzymes hydrolysing acetylsalicylic acid were found in the cytosol of the cestode, Moniezia expansa, and in the cytosol of the intestinal epithelial cells and cytosol of the reproductive tract of the nematode, Ascaris lumbricoides var suum. 2. Enzymes hydrolysing 2-naphthyl acetate and 4-methylumbelliferyl acetate were found throughout the proglottid of the cestode and in the reproductive tract, intestine, mesenchyme fluid and cuticle of the nematode. These enzymes had mol. wt. of 30 000-300 000 whereas those hydrolysing acetylsalicylic acid in both species had mol. wt. of about 87 000. 3. The acetylsalicylic acid hydrolases from both helminths showed pH optima of about 7.0, and activity was enhanced by Ca2+ and low-mol. wt. thiols. Cu2+, Cd2+, Hg2+, Zn2+, La3+, F- and EDTA at 1 mM inhibited activity. N-Ethylmaleimide, p-chloromercuribenzoate, haloxon and paraoxon also inhibited hydrolase activity.     

The role of cyclic AMP-mediated regulation of glycogen metabolism in levamisole-perfused Ascaris suum muscle

The effects of levamisole on muscle contraction and glycogen metabolism have been examined in isolated muscle-cuticle sections of the roundworm Ascaris suum. Muscle contraction occurred when various levels of levamisole were perfused through the preparation. At a levamisole concentration of 0.42 mM, the period of contraction lasted only about 6 min and was followed by a period of relaxation. During this relaxation period, there was an activation of glycogen synthase (EC 2.4.1.11), as evidenced by a decrease in the Ka values of glucose 6-phosphate for glycogen synthase to 0.26 mM from control values of 0.50 mM. The glycogen phosphorylase (EC 2.4.1.1) activity ratio decreased from 0.85 to 0.65, which indicated an inactivation of this enzyme. Concomitant with this activation of glycogen synthase and inactivation of phosphorylase there was an increased synthesis of glycogen. In addition, the presence of levamisole prevented both the serotonin-induced cyclic AMP accumulation and the activation of the cyclic AMP-dependent protein kinase (EC 2.7.1.37). However, levamisole did not significantly affect the changes in glycogen synthase and phosphorylase brought about by perfusion with the neurostimulator acetylcholine. Collectively, the data indicated that levamisole caused a transient muscle contraction followed by muscle relaxation, and the muscle relaxation effect appeared to be the result of a levamisole-inhibited cyclic AMP-mediated pathway of glycogen utilization.     

Experimental studies on anthelmintics (XXVI). Biochemical and pharmacological studies of 4-iodothymol on Ascaris lumbricoides suum]

We have shown previously that 4-iodothymol (IT) produces a contraction in Ascaris muscle, probably due to the myogenic action. In this paper, the effects of IT on the carbohydrate metabolism in Ascaris muscle have been investigated in comparison with those of hexylresorcinol (Hex), santonin (S) and piperazine (Pip). (1) Hex (200 approximately 400 mug/ml) showed a strong nonspecific inhibition on the formation of succinate from fumarate in muscle homogenate, the phosphofructokinase (PFK) activity in cytoplasm, and the electron transfer activity in mitochondria. (2) S(100 approximately 400 mug/ml) and Pip (100 approximately 400 mug/ml) were inneffective on these activities. (3) IT inhibited the formation of succinate from glucose and fumarate in muscle homogenate (100 approximately 400 mug/ml), the PFK activity in cytoplasm (400 mug/ml), and the mitochondrial succinate oxidase system (25 approximately 400 mug/ml). These results suggest that IT elicits the wormcidal action by inhibiting the energy metabolism of Ascaris muscle mitochondria.     

The interaction between anthelmintic drugs and histamine in Ascaris suum.

1 Piperazine reduced the histamine content of Ascaris suum, yet it greatly increased the uptake of histamine from the surrounding medium, the neuromuscular structures of the nematode preferentially increasing in amount. 2 Bephenium reduced the histamine content of Ascaris and the uptake of histamine from the surrounding medium. However, the relative amount in the neuromuscular structures increased. 3 The flaccid paralysing action of piperazine may thus involve increased histamine absorption whereas the spastic paralysing action of bephenium may be independent of histamine.     

Inhibitory reflex during bronchoconstrictions induced by histamine and Ascaris suum antigen.

We investigated the involvements of sympathetic and nonadrenergic nervous systems in the inhibitory reflex following bronchoconstriction in dogs. Inhalations of a 0.00125% solution of histamine and Ascaris suum antigen (3 mg protein) to the bronchial side induced reflex tracheal constriction following bronchoconstriction. An intra-arterial infusion of 5 micrograms/min of atropine to the tracheal site changed the reflex tracheal constrictions by histamine and antigen inhalations into tracheal dilatations. The reflex tracheal dilatations were abolished by the combination of intra-arterial propranolol (100 micrograms) and transections of both the bilateral superior laryngeal nerves and the spinal cord at the C1 level. The reflex tracheal constrictions induced by histamine and antigen inhalations were increased with 100 micrograms propranolol. Furthermore, the reflex tracheal constrictions were enhanced by the combination of 100 micrograms propranolol and transection of the spinal cord. These findings indicate that during the constriction of the bronchial smooth muscle, not only a reflex tracheal constriction mechanism but also one of reflex dilatation operates and that the latter reflex response may be mainly mediated by the sympathetic nerves, with partial involvement of the nonadrenergic nerves. This inhibitory reflex may attenuate asthmatic bronchoconstriction.     

Decursin and decursinol angelate selectively inhibit NADH-fumarate reductase of Ascaris suum

NADH-fumarate reductase (NFRD) is a key enzyme in many anaerobic helminths. Decursin and decursinol angelate have been isolated from the roots of ANGELICA GIGAS Nakai (Apiaceae) as NFRD inhibitors. They inhibited ASCARIS SUUM NFRD with IC (50) values of 1.1 and 2.7 microM, respectively. Their target is the electron transport enzyme complex I. Since the inhibitory activities of decursin against bovine heart complexes are weak, it is a selective inhibitor of the nematode complex I. In contrast, decursinol angelate moderately inhibits bovine heart complexes II and III. Decursinol inhibits A. SUUM NFRD to a similar extent, but its target is complex II. It also inhibits bovine heart complexes II and III.     

Azo- and nitro-reductases of the cestode Moniezia expansa. Substrate specificity, reaction products and the effects of flavins and other compounds.

1. The effects of various inhibitors and activators on the azo- and nitro-reductases of Moniezia expansa have been studied. Both reductions were partially inhibited by FAD, FMN, riboflavin, allopurinol, dicoumarol, 5-nitro-2-furaldehyde, azide and cyanide at 1 mM. Both reactions were stimulated by hypoxanthine. Menadione, nitrofurantoin, SKF 525-A (2-diethylaminoethyl 2,2-diphenylvalerate) and fluoride were without effect. 2. Xanthine- and aldehyde-oxidase activities were not detected in the enzyme preparation. 3. The substrate specificity of the azo- and nitro-reductases were determined. Azobenzene, 4-dimethylamino-azobenzene and 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene, nitrobenzene, 4-nitrohippuric acid and the isomers of nitrophenol, nitroanisole, nitrobenzoic acid, nitrobenzaldehyde and nitrobenzyl alcohol were reduced. Nitrobenzaldehyde isomers were not reduced to the alcohols and the coumaric acids were not reduced to the phenylpropionic acids. 4. The products of azo- and nitro-reduction were the corresponding amines; hydroxylamino- and hydrazo-compounds were not detected. 5. The pH optima and cofactor requirements were the same for both azo- and nitro-reduction. Neither reaction was inhibited by oxygen.     

The pharmacology of the cholinoceptor in muscle preparations of Ascaris lumbricoides var. suum

1. The preparation of a muscle strip of Ascaris lumbricoides for the study of the effect of drugs in vitro is described.2. Stimulant drugs which are classified as nicotine-like in mammalian pharmacology increased the isometric tension of this preparation. These drugs were, in descending order of potency: dimethylphenylpiperazinium, nicotine, acetylcholine, carbachol, decamethonium and pyridine-2-aldoxime methiodide.3. Muscarine-like drugs (oxotremorine, methacholine, pilocarpine) had no activity.4. Potassium and barium ions stimulated the tissue, while the anti-cholinesterases, dichlorvos and eserine, increased the resting tension of the preparation and potentiated the responses to acetylcholine.5. Adrenaline neither stimulated the tissue nor affected the responses to nicotine-like drugs.6. The relative potency of several blocking agents which antagonize the responses to nicotine-like drugs was assayed. These blocking agents were, in descending order of potency: mecamylamine, (+)-tubocurarine, hexamethonium, atropine and piperazine. Acetylcholine, dimethylphenylpiperazinium and pyridine-2-aldoxime methiodide apparently act on a common receptor, for each blocking agent had a similar molar inhibitory concentration against these stimulants.7. It is concluded that the cholinoceptor in muscle preparations of Ascaris lumbricoides is pharmacologically similar to that of the mammalian autonomic ganglion.     

Electrophysiological effects of piperazine and diethylcarbamazine on Ascaris suum somatic muscle.

1 Electrophysiological recordings were made from the bag region of Ascaris suum muscle. Membrane potential and input conductance or membrane current under voltage clamp were measured. 2 In high-Cl- Ringer, bath-applied piperazine, at concentrations greater than 10(-4)M, produced a dose-dependent and reversible increase in input conductance associated with a hyperpolarizing potential. The increase in input conductance was reduced when the preparations were bathed in low-Cl- Ringer. Gamma-Aminobutyric acid (GABA) and piperazine reversal potentials were measured with a voltage clamp on the same cells using iontophoretic application of the agonists. The reversal potentials were the same and close to the predicted Nernst Cl- potential (-65 mV). When GABA and piperazine were applied simultaneously piperazine reversibly reduced the amplitude of the control outward GABA current response. It was concluded that piperazine acts as a GABA agonist of low potency on the extra-synaptic GABA receptors of the bag, mediating an increase in Cl- conductance. 3 Acetylcholine was applied iontophoretically within 100 micron of the bag region while the preparation was bathed in a low-Ca2+, low-Cl- Ringer. The response under voltage clamp was a dose-dependent inward current associated with an increase in input conductance. This response was reversibly antagonized by 3 X 10(-5)M tubocurarine, high concentrations of diethylcarbamazine (10(-3) to 10(-2)M) but not high concentrations of piperazine (10(-3) to 10(-2)M). It was concluded that there are extra-synaptic acetylcholine receptors on the bag region of Ascaris muscle and that diethylcarbamazine but not piperazine acts as an antagonist. 4 Bath-applied diethylcarbamazine (10(-4) to 2 X 10(-3)M) produced a reversible dose-dependent depolarization of the membrane potential which was associated with an increase in the amplitude and frequency of spontaneous depolarizing potentials in active preparations at 32 degrees C to 35 degrees C in high-Cl- Ringer. The excitatory action of diethylcarbamazine was not blocked by 3 X 10(-5)M tubocurarine. Diethylcarbamazine (10(-4) to 10(-3)M) had no effect on the outward current response to GABA iontophoresis. Diethylcarbamazine (10(-4) to 10(-2)M) reversibly antagonized in a dose-dependent manner the delayed rectification of the bag membrane. In a low-Ca2+, low-Cl- Ringer, diethylcarbamazine (10(-4) to 2 X 10(-3)M) reversibly antagonized the voltage-sensitive outward current of the bag. This effect was mimicked by high-K+ Ringer or perfusion with 4-aminopyridine (10(-3) to 2 X 10(-3)M). It was concluded that diethylcarbamazine did not react with the GABA receptor but antagonized a voltage-sensitive K+ conductance.     

The binding and subsequent inhibition of tubulin polymerization in Ascaris suum (in vitro) by benzimidazole anthelmintics

Benzimidazole anthelmintics may act by interfering with the microtubule system in Ascaris suum. Binding of benzimidazole anthelmintics, and their inactive metabolites, to A. suum tubulin was demonstrated by the inhibition of intestinal extracts of the nematode to bind [³H]colchicine. In addition, these compounds inhibited the polymerization of tubulin into microtubules in vitro.     

The effects of chlorpheniramine and antiallergic drugs on Ascaris suum antigen-induced active cutaneous anaphylaxis in dogs

Effects of chlorpheniramine and two antiallergic drugs on the active cutaneous anaphylaxis (ACA) reaction induced by intradermal injection of Ascaris suum antigen in naturally sensitized dogs were investigated. Chlorpheniramine (10 mg/kg, intraduodenally (i.d.)) almost abolished the ACA reaction. NCO-650 (100 mg/kg, i.d.) had no inhibitory effect, while tranilast (300 mg/kg, i.d.) showed a weak inhibitory effect. These findings show that the ACA reaction is almost totally mediated by histamine and ACA reaction is considerably resistant to antiallergic drugs such as tranilast and NCO-650.     

The nematode neuropeptide, AF2 (KHEYLRF-NH2), increases voltage-activated calcium currents in Ascaris suum muscle

BACKGROUND AND PURPOSE: Resistance to all the classes of anti-nematodal drugs like the benzimidazoles, cholinergic agonists and avermectins, has now been recorded in parasites of animals and/or humans. The development of novel anthelmintics is an urgent and imperative need. Receptors of nematode neuropeptides have been suggested to be suitable target sites for novel anthelmintic drugs. EXPERIMENTAL APPROACH: To investigate the effect of AF2 on calcium-currents in Ascaris suum somatic muscle cells we employed the two-micropipette current-clamp and voltage-clamp techniques and a brief application of AF2. KEY RESULTS: Here we report the isolation of voltage-activated, transient, inward calcium currents. These currents are similar in characteristics to Caenorhabditis elegans UNC-2 type currents, non-L-type calcium currents. Following a 2-minute application of 1 microM AF2 , there was a significant long-lasting increase in the transient inward calcium current; AF2 increased the maximum current (from -84 nA to -158 nA) by shifting the threshold in the hyperpolarising direction (V (50) changed from -7.2 to -12.8 mV) and increasing the maximum conductance change from 1.91 to 2.94 microS. CONCLUSION AND IMPLICATIONS: These studies demonstrate a mechanism by which AF2 increased the excitability of the neuromuscular system by modulating calcium currents in nematodes. A selective small molecule agonist of the AF2 receptor is predicted to increase the contraction and act synergistically with cholinergic anthelmintics and could counter resistance to these compounds.     

Shortening of the induction period of allergic asthma in cynomolgus monkeys by Ascaris suum and house dust mite

The development of non-human primate models of asthma requires a period of time (e.g., 0.5-1 year). To develop the models in a short period, male cynomolgus monkeys were sensitized with dinitrophenyl-Ascaris suum (DNP-As) allergen by intraperitoneal and intramuscular injection and by intratracheal inhalation. All sensitized animals developed positive intradermal skin reaction to DNP-As. Sensitization elevated allergen-specific IgE levels in serum, the number of CCR4-positive T helper lymphocytes in peripheral blood, and IL-4 and IL-5 releases from phorbol 12-myristate 13-acetate- and ionomycin-stimulated peripheral blood. In addition, allergen challenge induced increases in lung resistance, airway inflammation, and hyperresponsiveness to inhaled methacholine. Next, animals were sensitized with house dust mite extracts (HDM) under the similar procedure. In these animals sensitized with DNP-As or HDM, inhaled fluticasone propionate and oral prednisolone inhibited the allergen-induced airway hyperresponsiveness. Taken together, monkey asthma models were successfully developed by sensitization with DNP-As or HDM under a short-term protocol (within 7 weeks). These models should be useful for the evaluation of anti-inflammatory drugs for asthma treatment.