Titration of subcutaneously administered eprinomectin against mature and immature nematodes in cattle.

Eprinomectin has been approved for use as a topically applied endectocide for beef and dairy cattle. To determine if eprinomectin has utility as an injectable anthelmintic, it was titrated at 0.05, 0.1, and 0.2 mg/kg s.c. against adult (Trial 1) and at 0.05, 0.1, 0.14, and 0.2 mg/kg s.c. against immature (Trial 2) stages of lung and gastrointestinal nematodes in cattle. In Trial 1, every dose of subcutaneously delivered eprinomectin showed maximal or near-maximal (> or = 99%) efficacy against Haemonchus placei, Ostertagia ostertagi, Trichostrongylus axei, T colubriformis, Cooperia punctata, Nematodirus helvetianus, Oesophagostomum radiatum, and Dictyocaulus viviparus. Adult C. oncophora was the only exception. However, even against this species, the lowest dose of 0.05 mg/kg showed 93% efficacy, and the efficacious dose necessary to kill 95% (ED95) of adults was 0.056 mg/kg. In Trial 2, every dose of subcutaneously delivered eprinomectin showed maximal or near-maximal (> or = 99%) efficacy against the immature stages of all of the above species of endoparasites. As a result, ED95 values could not be calculated. Consequently, the exquisite potency against endoparasites through parenteral administration suggests that eprinomectin may also have potential utility as an injectable product for cattle.

Synthesis of gem-difluoro-avermectin derivatives: potent anthelmintic and anticonvulsant agents.

A series of gem-difluoro-avermectin derivatives was synthesized from the corresponding ketones at positions 4", 4', 13, and 23 using diethylaminosulfur trifluoride (DAST). These fluorinated avermectins exhibit potent antiparasitic activity in a new Haemonchus contortus larval assay and are equipotent to ivermectin. In addition, 23-gem-difluoro-ivermectin displays useful anticonvulsant activity in mouse models.

Efficacy in sheep and pharmacokinetics in cattle that led to the selection of eprinomectin as a topical endectocide for cattle.

Eprinomectin (MK-397 or 4"-epi-acetylamino-4"-deoxy-avermectin B1) is a novel avermectin selected for development as a topical endectocide for all cattle, including lactating cows. The initial efficacy assessments were made in sheep to identify subclasses of the avermectin/milbemycins that possessed inherent activity against a spectrum of nematode parasites. This included examination of several hundred analogs each given orally to a single sheep experimentally infected with a range of parasitic nematodes. Representatives of several subclasses, most notably the 4"-epi-amino avermectin B1 subclass, were identified as possessing potent, broad-spectrum activity against the endoparasites, whereas subclasses such as those with a variety of synthetic substitutions at C-4a or oximes at C-5 were among the least potent. Eprinomectin, a member of the 4"-epi-amino subclass, possessed potent activity against the range of nematodes when tested at 0.025 mg kg-1 per os. Milk and plasma concentration profiles were also made for these and other selected avermectin/milbemycins following topical administration to lactating dairy cattle. The molecular structure of each compound had a significant effect on the milk to plasma ratio, but the ratio of each was constant over time, implying an equilibrium between the 2 compartments. Compounds that were saturated at the C-22,23 bond had milk to plasma ratios > or = 1.0, whereas those unsaturated at this bond were generally < or = 1.0. The milk to plasma ratio of eprinomectin was < or = 0.2. Therefore, not only is eprinomectin the most potent broad-spectrum avermectin/milbemycin identified to date, but it also possesses one of the lowest milk partitioning coefficients in this class of antiparasitics.

Eprinomectin: a novel avermectin for use as a topical endectocide for cattle.

Eprinomectin (MK-397 or 4"-epi-acetylamino-4"-deoxy-avermectin B1) is a novel avermectin selected for development as a topical endectocide for all cattle, including lactating dairy cows. Herein, we show its anthelmintic, insecticidal and miticidal activity. To determine its anthelmintic capabilities, eprinomectin was tested topically on Jersey calves at 0.08, 0.2, or 0.5 mg kg-1 in a probe formulation against experimental infections of adult Haemonchus placei, ostertagia ostertagi, Trichostrongylus axei, T. colubriformis, Cooperia oncophora, C. punctata, Nematodirus helvetianus, Oesophagostomum radiatum and Dictyocaulus viviparus. Eprinomectin removed > or = 99% and > or = 98% of the adult stage of every species at the 0.5 and 0.2 mg kg-1 dosage levels, respectively. The lowest dosage (0.08 mg kg-1) produced maximal or near maximal efficacy against most of the adult endoparasites with the exception of T. colubriformis (87%) and C. oncophora (88%). In a separate test, eprinomectin was evaluated topically against the immature stages of species at the same dosages. Results showed > or = 99% and > or = 98% removal of the immature stages of each species at the 0.5 and 0.2 mg kg-1 dosage levels, respectively. The 0.08 mg kg-1 dosage maintained > or = 97% efficacy against 6 species with reduced activity against H. placei (42%) and N. helvetianus (66%). For ectoparasites, eprinomectin was tested topically at 0.16, 0.24, 0.32 or 0.5 mg kg-1 on mixed breed cattle naturally infested with the sucking louse, Linognathus vituli. Complete elimination of lice at all dosages was observed by day 14. Topical delivery of eprinomectin at 0.16, 0.24, 0.32 or 0.5 mg kg-1 to Holstein calves experimentally challenged with horn fly, Haematobia irritans, produced 100% efficacy to challenge by week 2 post-treatment in all dosages groups and 94% and 99% efficacy to challenge at the 0.32 and 0.5 mg kg-1 dosage groups, respectively, at week 4. Topical delivery of eprinomectin at 0.16, 0.24 or 0.5 mg kg-1 to Deutsches Fleckvieh cattle infested with mange mites, Chorioptes bovis, produced > or = 95% control at all dosages levels by day 14 post-treatment and was maintained at or near this efficacious level for the 6-week duration of the trial. No adverse reaction was observed in any animal in any of these tests. In summary, these experimental data indicate that eprinomectin is an excellent broad-spectrum endectocide for cattle and is suitable for topical delivery.

Structure and activity of avermectins and milbemycins in animal health.

The avermectins and, to a lesser extent, the milbemycins, have revolutionized antiparasitic and antipest control over the last decade. Both avermectins and milbemycins have macrocyclic lactone structures that are superimposable, they are produced by the same genus of soil dwelling organisms, they have the same mode of action, they exert this action against the same nematode/acarine/insect spectrum of targets, and they show the same mechanism-based toxicity in mammals. Reports suggesting that milbemycins have a different mode of action from avermectins with implications that there will be no mutual resistance to the groups have been shown to be false. Contributing to the belief that there were differences in mode of action between the two groups are the vague definitions of resistance presently in use which rely on the ability of the parasite to survive treatment at the manufacturer's recommended use level. More appropriately, drug resistance should be defined as 'a change in gene frequency of a population, produced by drug selection, which renders the minimal, effective dosage previously used to kill a defined portion (e.g. 95%) of the population no longer equally effective'. This type of definition would allow us to detect changes in susceptibility of a population earlier and is essential when comparing different chemicals to determine if there is mutual resistance to them. It is concluded that much effort has been expended by pharmaceutical, government, and academic scientists searching for broad-spectrum second generation avermectin and milbemycin products, but none has exceeded the original avermectin in any fundamental way. The newer avermectin and milbemycin compounds that have appeared claim niches in the market place based on emphasis of certain narrow parts of the overall spectrum. Consequently, there are no second generation avermectins and milbemycins at present and all newer compounds from this mode of action class are viewed as siblings of the first generation.

Avermectins and milbemycins against Fasciola hepatica: in vivo drug efficacy and in vitro receptor binding.

Few studies have examined activity against trematodes for the avermectin/milbemycin class of anthelmintics. To gain insight into this, 12 different members of the avermectin/milbemycin mode of action class were tested against juvenile Fasciola hepatica in a mouse model. The compounds chosen were Avermectin A1, Avermectin A2, Avermectin B1, Avermectin B2, Ivermectin, Ivermectin monosaccharide, Ivermectin aglycone, 13-deoxy ivermectin aglycone, Moxidectin, 13-O-methoxyethoxymethyl ivermectin aglycone, 4"-deoxy-4"-epi-methylamino avermectin B1, and 4"-deoxy-4"-epi-acetylamino avermectin B1 5-oxime. Each of these compounds was administered orally to 4 mice at 2.0 mg kg-1. These mice had been administered 3 metacercariae of F. hepatica 14 days prior to treatment and all mice were necropsied 4 days after treatment. At necropsy, none of the individual avermectin or milbemycin-treated groups showed any significant activity (P > 0.05) against juvenile F. hepatica relative to a vehicle-treated control. In a receptor binding study, adult F. hepatica that had been obtained from sheep were homogenized, their membranes incubated in the presence of 3H-ivermectin, and then measured for high affinity binding sites. The same was done with the free-living nematode, Caenorhabditis elegans. While the C. elegans membranes displayed high affinity 3H-ivermectin binding sites over the range of ivermectin concentrations tested (5-100 nM), no significant 3H-ivermectin binding sites were detected in the F. hepatica membranes. Based on these data, it seems unlikely that any avermectin or milbemycin will show activity against F. hepatica, and certainly makes one pessimistic about possible activity of this mode of action class against trematodes in general.

The mechanism of action of avermectins in Caenorhabditis elegans: correlation between activation of glutamate-sensitive chloride current, membrane binding, and biological activity.

Xenopus laevis oocytes were injected with mRNA isolated from the free-living nematode Caenorhabditis elegans and the activation and potentiation of a glutamate-sensitive chloride current by a series of avermectin analogs and milbemycin D were determined. There was a strong correlation between the EC50 value determined for current activation in oocytes, the LD95 value for nematocidal activity, and also for the Ki value determined in a [3H]ivermectin competition binding assay. Four of the analogs were tested for potentiation of glutamate-sensitive current and the rank order for potentiation correlated with the EC50 for direct activation of current. We conclude that avermectins and milbemycins mediate their nematocidal effects on C. elegans via an interaction with a common receptor molecule, glutamate-gated chloride channels.

Radical-induced oxidation of oxahydrindene (hexahydrobenzofuran) portion of 22,23-dihydroavermectin B1a: identification of autooxidation products.

Three products resulting from free-radical-induced oxidation of the oxahydrindene portion of 22,23-dihydroavermectin B1a (H2B1a) are 5-oxo-H2B1a, 8a-oxo-H2B1a, and 5,8a-bisoxo-H2B1a. The last of these compounds has not been reported previously.

Synthesis and biological activity of 13-epi-avermectins: potent anthelmintic agents with an increased margin of safety.

Chemical conversion of the potent anthelmintic natural products avermectin B1 (1) and avermectin B2 (3) to the corresponding 13-epi analogs (15 and 9) is described. The novel analogs retain the full potency of the natural products but are substantially safer.

Affinity probes for the avermectin binding proteins.

The design and synthesis of a series of avermectin affinity probes used in the identification and purification of the avermectin binding proteins is described. These modified avermectins fall into two design classes: ligands to covalently modify specific avermectin binding proteins [an 125I-labeled aryl azide photoprobe (15) and a tritiated aziridine analog (6)] and ligands for affinity chromatography applications [three biotinylated compounds (10, 12, and 13) and one resin-bound derivative (9)]. The binding affinities of these compounds for the Caenorhabditis elegans avermectin binding protein is presented as well as their biological activities against C. elegans and Artemia salina.

Photoaffinity labeling of avermectin binding sites from Caenorhabditis elegans and Drosophila melanogaster.

An azido-avermectin analog [4'' alpha-(4-azidosalicylamido-epsilon-caproylamido-beta-alan ylamido)-4''-deoxyavermectin B1a; azido-AVM] was synthesized and used to photoaffinity label avermectin binding sites present in the membranes of Caenorhabditis elegans and Drosophila melanogaster. Azido-AVM was biologically active and behaved like a competitive inhibitor of [3H]ivermectin binding to C. elegans membranes (Ki = 0.2 nM). Radiolabeled azido-AVM bound specifically and with high affinity to C. elegans membranes (Kd = 0.14 nM) and, upon photoactivation, became covalently linked to three C. elegans polypeptides of 53, 47, and 8 kDa. Photoaffinity labeling of a membrane preparation from D. melanogaster heads resulted in labeling of a single major polypeptide of approximately 47 kDa. The proteins that were covalently tagged in these experiments are believed to be associated with avermectin-sensitive chloride channels present in the neuromuscular systems of C. elegans and D. melanogaster. Azido-AVM did not bind to rat brain membranes and therefore was selective for the nematode and insect receptors.

[3H]paraherquamide binding to Caenorhabditis elegans. Studies on a potent new anthelmintic agent.

Paraherquamide was identified recently as a potent anthelmintic agent. In this paper we describe the identification and characterization of a specific, high-affinity paraherquamide binding site in a membrane preparation isolated from the free-living nematode, Caenorhabditis elegans. [3H] Paraherquamide bound specifically to C. elegans membranes with an apparent dissociation constant, Kd, of 263 nM. A series of paraherquamide analogs were examined, and their relative affinity for the paraherquamide binding site correlated with their nematocidal activity. Phenothiazines were the only other class of anthelmintics tested which inhibited specific [3H]paraherquamide binding. These results suggest that the anthelmintic activity of paraherquamide and phenothiazine is mediated via an interaction with a common binding site.

A chemiluminescent assay for measuring avermectin binding sites.

Avermectins are potent, broad spectrum anthelmintics. We describe the synthesis of a biologically active chemiluminescent compound, 22,23-dihydroavermectin B1a, 4"-[4-[[4-[ethyl(1,2,3,4-tetrahydro-1,4-dioxo-6- phthalazinyl)amino]butyl]amino]-4-oxobutanoate] (ivermectin-luminol). This compound retains its anthelmintic properties and provides an extremely sensitive nonradioactive probe to study avermectin binding sites.

Syntheses and biological activities of 13-substituted avermectin aglycons.

The reactions of sulfonate esters of the allylic/homoallylic 13-alcohol of 5-O-(tert-butyldimethylsilyl)-22,23-dihydroavermectin B1a aglycon (1a) were investigated. Nucleophilic substitution gave 13 beta-chloro and 13 beta-iodo derivatives, while solvolytic reaction conditions yielded 13 alpha-methoxy, 13 alpha-fluoro, and 13 alpha-chloro products. A mixture of 13 alpha- and 13 beta-fluorides was obtained upon reaction with DAST. The 13 beta-iodide gave, upon elimination with lutidine, the 8(9),10(11),12(13),14(15)-tetraene. The 13 beta-alcohol and the rearranged 15-ol 13(14)-ene and 15-amino 13(14)-ene derivatives were obtained by substitution via the allylic carbonium ion. MEM ethers 11 and 12 of the two epimeric 13-ols were prepared by alkylation with MEM chloride. In contrast, methylation of 1a with MeI and Ag2O in CH2Cl2 occurred exclusively at the tertiary 7-hydroxy group and not at the secondary 13 alpha-ol. Oxidation of the allylic alcohol 1a proceeded under Swern conditions but not with MnO2 to the 13-oxo aglycon, which was reduced by NaBH4 exclusively to the natural 13 alpha-ol, while reductive amination with NaCNBH3-NH4OAc gave the 13 alpha-amine. The methoxime derivative was obtained in the form of the two geometric isomers. Anthelmintic activities against the sheep nematode Trichostrongylus colubriformis, miticidal activities against the two-spotted spider mite (Tetranychus urticae), and insecticidal activities against the southern armyworm (Spodoptera eridania) as well as the binding constants to a free living nematode (Caenorhabditis elegans) derived receptor assay were obtained and compared to avermectin B1a, 22,23-dihydroavermectin B1a, and the 13-deoxy-22,23-dihydroavermectin B1 aglycon related to the milbemycins. None of the newly prepared derivatives exceeded the potency of the three reference compounds. Lipophilic 13-substituents such as halogen, alkoxy, and methoxime retained high biological activities in all assays, while the more polar substituents hydroxy and amino had weaker activities. Rearranged 15-substituted 13(14)-ene derivatives were completely inactive. The 13-oxo and the 12,13-dehydro analogues were only weakly active in vivo despite having good binding affinity to the receptor, possibly due to instability or poor absorption.

Effect of liver enzymes on the mutagenicity of nitroheterocyclic compounds: activation of 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)- 1,2-benzisoxazole and deactivation of nitrofurans and nitroimidazoles in the Ames test.

The effect of liver enzymes (S9) on the mutagenic response of nitroimidazoles and nitrofurans in the Ames test was evaluated with strain TA100. A diminished response was observed with a 5-nitroimidazole and 5-nitrofurans when the S9 preparation was incorporated in the agar layer. Preincubation with S9 under anaerobic conditions prior to adding the bacteria resulted in a greater and sometimes complete loss of the mutagenic effect. The loss of mutagenic potency was dependent on both incubation time and quantity of the S9 preparation. These results suggest that metabolites formed after reductive metabolism are neither mutagenic (presumably due to the loss of the nitro group) nor capable of activation to mutagenic metabolites. One 5-nitroimidazole, 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro -1H-imidazol-2-yl)-1,2-benzisoxazole (MK-0436), gave an increased response in the presence of S9 in both the plate test and when preincubated under aerobic conditions. 7 metabolites were produced by the incubation. 4 monooxygenated metabolites were isolated and found to possess significant mutagenic activity. 2 synthetic dihydroxy analogs were more mutagenic than MK-0436. Similar results were obtained with S9 preparations from human liver and the livers of control, phenobarbital and Aroclor-1254 pretreated rats.

Avermectin acyl derivatives with anthelmintic activity.

Avermectins A2a, B1a, and B2a (1, 2, and 3) were acetylated to give 4"- and 23-acetates 4 and 5 and 4",23-diacetate 6 from 1, the 4"-and 5-acetates 7 and 8 and 4",5-diacetate 9 from 2, and triacetate 10 from 3. Structure proof by 300-MHz 1H NMR and mass spectral fragmentation is discussed for 10. Forcing acetylation conditions generated from both 1 and 3 the identical aromatic diacetate 11. Good anthelmintic activities in gerbils and sheep for 4"-acetylated derivatives 4 and especially 7 prompted the preparation of additional 4"-acylated derivatives of 2 with pivaloyl, n-octanoyl, succinoyl, carbamoyl, dimethylcarbamoyl and N-acetylglycyl substituents, prepared from the 5-O-tert-butyldimethylsilyl-protected intermediate 12. Other key intermediates were the trichloroethyoxysuccinoyl derivative 18 and 4-nitrophenyl carbonate 21. Anthelmintic activities against Trichostrongylus colubriformis in gerbils comparable in potency to the natural product 2 are shown by the more polar substituted derivatives 20, 23, and 27. Substitution of the 5-hydroxy group or its loss due to aromatization results in drastically reduced anthelmintic potency.

Substituted imidazo[2,3-alpha]pyridine-2-carbamate anthelmintics.

Anthelmintic efficacies of a series of 6-substituted methyl imidazo[1,2-alpha]pyridine-2-carbamates were compared to similarly substituted benzimidazole-2-carbamates. With only one exception, methyl 6-benzoylimidazo[1,2-alpha]pyridine-2-carbamate, both classes of compounds exhibited similar activity vs. Nematospiroides dubius in mice. Preliminary screening indicated methyl 6-(1,2,2-trichloroethenyl)imidazo[1,2-alpha]pyridine-2-carbamate to be the most potent derivative in the series. However, evaluation in sheep indicated that its anthelmintic spectrum was inferior to methyl 6-(phenylsulfinyl)imidazo[1,2-alpha]pyridine-2-carbamate.

Ivermectin, a new broad-spectrum antiparasitic agent.

22,23-Dihydroavermectin B1, ivermectin, derived from avermectin B1 by selective hydrogenation using Wilkinson's homogenous catalyst [Ph3P)3RhCl], was shown to be a highly effective drug for the treatment of a wide variety of metazoan parasitic diseases in animals.