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Nodulisporic acid A (1) is a structurally complex fungal metabolite that exhibits systemic efficacy against fleas via modulation of an invertebrate specific glutamate-gated ion channel. In order to identify a nodulisporamide suitable for monthly oral dosing in dogs, a library of 335 nodulisporamides was examined in an artificial flea feeding system for intrinsic systemic potency as well as in a mouse/bedbug assay for systemic efficacy and safety. A cohort of 66 nodulisporamides were selected for evaluation in a dog/flea model; pharmacokinetic analysis correlated plasma levels with flea efficacy. These efforts resulted in the identification of the development candidate N-tert-butyl nodulisporamide (3) as a potent and efficacious once monthly oral agent for the control of fleas and ticks on dogs and cats which was directly compared to the topical agents fipronil and imidacloprid, with favorable results obtained. Multidose studies over 3 months confirmed the in vivo ectoparasiticidal efficacy and established that 3 lacked overt mammalian toxicity. Tissue distribution studies in mice using [(14)C]-labeled 3 indicate that adipose beds serve as ligand depots, contributing to the long terminal half-lives of these compounds.

The worldwide spread of antibiotic resistance in pathogenic bacteria raises serious concerns that today's front-line antibiotics may become ineffective. So the discovery of a new class of compound with enormous potential as antibiotics is of great interest. Platensimycin is the first member of a novel class of natural products that selectively kills bacteria by inhibiting FabF/FabB proteins, a mechanism of action that is not used by any agents in clinical use. It is a potent antibiotic with broad-spectrum activity against Gram-positive bacteria, including clinically relevant drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. The future of platensimycins is considered in the News pages, and see the antimicrobials Web Focus on http://tinyurl.com/g8omx. A new class of antibiotics that targets bacterial lipid biosynthesis is isolated from Streptomyces platensis, and demonstrates potent in vitro and in vivo activity against Gram-positive bacteria — including methicillin-resistant Staphylococcus species. Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s1,2. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of β-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.

The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by gamma-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free alpha-carboxyl groups, despite reports that this chemistry may lead to chain scission. We have purified the high molecular mass capsule to >95% homogeneity and have demonstrated that the polymer contains >99% poly-gamma-D-glutamic acid. The predominant structure of the polymer as assessed by circular dichroism and multiangle laser light scattering was unordered at near-neutral pH. We investigated the effects of various activation chemistries, and we demonstrated that carbodiimide treatment under aqueous conditions results in significant cleavage of the gamma-peptidyl bond, whereas scission is significantly reduced in nonaqueous polar solvents, although undesired side chain modification was still observed. An activation chemistry was developed using the triazine-based reagent 4-(4,6-dimethoxy (1,3,5)triazin-2-yl)-4-methylmorpholinium chloride, which allowed for controlled and reproducible derivatization of alpha-carbonyls. In a two-pot reaction scheme, activated capsule was derivatized with a sulfhydryl-reactive heterobifunctional moiety and was subsequently coupled to thiolated carrier protein. This conjugate elicited very high capsule-specific immune titers in mice. More importantly, mice immunized with conjugated capsule exhibited good protection against lethal challenge from a virulent B. anthracis strain in two models of infection. We also showed, for the first time, that treatment of capsule with carbodiimide significantly reduced recognition by capsule-specific antisera concurrent with the reagent-induced reduction of polymer mass. The data suggested that for vaccine development, maintenance of the high mass of the polymer may be important.

Quinolinones and naphthyridinones with C7 N-t-butyl piperidine substituents were found to be potent p38 MAP kinase inhibitors. These compounds significantly suppress TNF-alpha release in both cellular and LPS-stimulated whole blood assays. They also displayed excellent PK profiles across three animal species. Quinolinone at 10 mpk showed comparable oral efficacy to that of dexamethasone at 1 mpk in a murine collagen-induced arthritis model.

Background: The mode of action of triptolide, the active ingredient of an anti-inflammatory Chinese herbal remedy, has been investigated in vitro and in vivo. Prior reports suggested that triptolide specifically inhibits the nuclear transcription factor (NF)-κB. . Methods: In vitro effects of triptoilde on cytokine release and cellular transcription were measured in A549 and THP-1 cells by enzyme-linked immunosorbent assay and incorporation of radiolabeled uridine, respectively. Levels and translocation of the transcription factor NF-kB in vitro were monitored by western blot. Arthritic mice were treated with triptolide, up to 0.5 mg/kg/day for 21 days, and inflammation was evaluated. Taqman analysis was performed on RNA isolated from the arthritic paws to determine relative levels of various cytokines in response to in vivo triptolide treatment. Results: In vitro, triptolide inhibited cellular transcription in A549 and THP-1 cells with IC50 values of 139 nM and 105 nM, respectively, similar to that for i...

The development of anthelmintic resistance by nematode parasites is a growing problem for veterinarians, pet owners, and producers. The intensive use of the macrocyclic lactones for the treatment of a variety of parasitic diseases has hastened the development of resistance to this family of parasiticides. As a result, resistance to ivermectin, moxidectin, nemadectin, and doramectin by Haemonchus contortus has been documented throughout the world. Sensory neurons located in the cephalic end of nematodes are in close contact with the external environment. Through these neurons, important chemical and thermal cues are gathered by the parasite. Examination of serial electron micrographs of ivermectin-susceptible and ivermectin-resistant H. contortus allows for comparison of neuronal structure, arrangement of neurons within the amphidial channel, and distance of the tip of the dendritic processes to the amphidial pore. The latter of these characteristics provides a useful means by which to compare the association between the neurons and the external environment of the worm. Comparison of parental laboratory strains of ivermectin-susceptible strains of H. contortus with related selected, ivermectin-resistant strains and with a wild-type ivermectin-susceptible field strain of H. contortus from Louisiana reveal that the ivermectin-resistant worms examined have markedly shorter sensory cilia than their ivermectin-susceptible parental counterparts. Additionally, the amphidial neurons of ivermectin-resistant worms are characterized by generalized degeneration and loss of detail, whereas other neurons outside of the channels, such as the labial and cephalic neurons, are normal in structure. These findings raise a number of questions regarding the relationship between amphidial structure and ivermectin resistance as well as the role of amphids as a means of entry for ivermectin. While shortened amphidial sensilla are associated with ivermectin resistance, it remains unclear if such a structural modification facilitates survival of nematodes exposed to macrocyclic lactones.

During the re-isolation of the lead compound nodulisporic acid A ( 1a ) and targeted chemical screening for related compounds, we discovered a series of 1′-deoxy congeners named herein nodulisporic acids B ( 1b ), B 1 ( 2b ), and B 2 ( 3b ). In comparison with nodulisporic acid A, these compounds were less active and were chemically unstable resulting into formation of Δ 23 dehydro derivatives. Therefore, these compounds were stabilized and isolated as sodium salts and methyl ester. Nodulisporic acid B is 100-fold less active than nodulisporic acid A against fleas. The isolation, structure elucidation, and biological activities of these compounds are described.

A series of new, diene-modified nodulisporic acid analogues ( 2 ) bearing diverse functionality at the 3″- and 4″-sites was efficiently prepared from the 3″-aldehyde 3 . Biological evaluation of these synthetic nodulisporic acid analogues for systemic flea efficacy identified potent compounds and further clarified the structural requirements for ectoparasite activity.

The discovery of the natural product nodulisporic acid A (NsA A) and its potent, systemic insecticidal activity at Merck Research Laboratories in 1992 stimulated intense scientific scrutiny. Interest in this new class of indole diterpenes led to extensive delineation of its properties, both chemical and biological. Synthetic modification of NsA A served to identify its pharmacologically permissive and non-permissive regions, produced semisynthetic derivatives with enhanced adulticidal flea efficacy (both in vitro and in vivo), and provided useful tools to support biological studies. Early observations in rodent models showed a wide therapeutic index for NsA A and detailed mechanism of action investigations demonstrated that it selectively modulated an invertebrate specific glutamate-gated ion channel for which no mammalian homolog exists. Consistent with these mechanistic conclusions, dogs treated orally with either NsA A or closely related amide analogs (15 mg/kg dosages) showed no apparent toxicity, and measurement of systemic flea efficacy in these animals demonstrated that prolonged antiparasitic activity was attained, up to 14 days subsequent to treatment with a single p.o. dose for fleas or up to 4 weeks for ticks. The extended flea efficacy was correlated to both the in vivo pharmacokinetic profile of a given analog and its intrinsic in vitro potency against fleas. In addition, studies directed towards the total synthesis of NsA A have been reported.

Afoxolaner is an isoxazoline compound characterized by a good safety profile and extended effectiveness against fleas and ticks on dogs following a single oral administration. In vitro membrane feeding assay data and in vivo pharmacokinetic studies in dogs established an afoxolaner blood concentration of 0.1–0.2 μg/ml to be effective against both fleas ( Ctenocephalides felis ) and ticks ( Dermacentor variabilis ). Pharmacokinetic profiles in dogs following a 2.5 mg/kg oral dosage demonstrated uniform and predictable afoxolaner plasma concentrations above threshold levels required for efficacy for more than one month. Dose ranging and a 5-month multi-dose experimental study in dogs, established that the 2.5 mg/kg oral dosage was highly effective against fleas and ticks, and produced predictable and reproducible pharmacokinetics following repeated dosing. Mode of action studies showed that afoxolaner blocked native and expressed insect GABA-gated chloride channels with nanomolar potency. Afoxolaner has comparable potency between wild type channels and channels possessing the A302S (resistance-to-dieldrin) mutation. Lack of cyclodiene cross-resistance for afoxolaner was confirmed in comparative Drosophila toxicity studies, and it is concluded that afoxolaner blocked GABA-gated chloride channels via a site distinct from the cyclodienes.

[reaction--see text] The semisynthetic conversion of nodulisporic acid A (1) into a set of three heterocyclic side chain derivatives provided compounds, highlighted by 6, with an improved spectrum of ectoparasiticidal activity and pharmacokinetic profile relative to the natural product.

Medicinal chemistry efforts were initiated to identify the key constituents of the nodulisporic acid A (1) pharmacophore that are integral to its potent insecticidal activity. New semisynthetic derivatives delineated 1 into 'permissive' and 'nonpermissive' regions and led to the discovery of new nodulisporamides with significantly improved flea efficacy.

Larvae of the cat flea, Ctenocephalides felis (Bouché), are the target of numerous growth regulators. This study explores the development of an assay that tests the susceptibility of cat flea larvae to a wide range of compounds. Different rearing media and containment units were tested that would facilitate optimization. Larvae of various ages were compared, and 7-d-old larvae were found to be optimal because they were the most uniform in size and age and exhibited a need to feed. The assay could be used to distinguish insecticides from growth inhibitors. The insecticides chlorpyrifos and carbaryl caused 100% larval mortality in 24 h at 10 ppm, and cythioate and fipronil killed the larvae at concentrations ofor = 100 ppm within 24 h. The insect growth regulators methoprene and pyriproxifen caused molt delay at concentrations of 100 ppm and bioallethrin delayed molt at 1,000 ppm. This assay can be used to identify compounds that are specific to cat flea larvae that may be useful in the control of cat flea infestations.

Three isolates of Haemonchus contortus selected for avermectin resistance in sheep were compared in three in vitro pharmacological tests previously shown to discriminate between field isolates of H. contortus resistant and susceptible to the avermectins. Two isolates, F7-A and IVC, were selected for avermectin resistance in the laboratory from a reference susceptible isolate using suboptimal doses of ivermectin (LD95) for 7 and 16 generations, respectively. In these isolates avermectin resistance was not associated with a decreased sensitivity to avermectin inhibition of larval development or L3 motility but was associated with an increased sensitivity to paraherquamide. The third isolate, Warren, was derived from an overwhelmingly avermectin-susceptible, mixed species field isolate in a single generation by propagating the small number of survivors of a 0.2 mg/kg ivermectin treatment (i.e. 10×LD95). This isolate, like previously characterised avermectin-resistant H. contortus isolates derived from the field in South Africa and Australia, showed a markedly reduced sensitivity to avermectin inhibition of larval development and L3 motility, as well as an increased sensitivity to paraherquamide. These results suggest that avermectin resistance can manifest itself in different ways and that the two selection protocols used to generate the F7-A, IVC and Warren isolates have resulted in the selection of different resistance phenotypes.

The potent insecticidal agent nodulisporic acid A (1a), representative of a new class of indole terpenes, was isolated from fermentations of a Nodulisporium sp. Nodulisporic acid A was active against the larvae of the blowfly and mosquito at sub-part-per-million levels. The structure followed mainly from a consideration of spectroscopic evidence, including Dunkel's computerized 2D INADEQUATE analysis. The relative stereochemistry of the eastern and western hemispheres of 1a and its methyl ester, 1b, were independently determined on the basis of ROESY, NOESY, NOEDS, and Jvic evidence. By employing the same methods, the complete relative stereochemistry was determined by analysis of suitable transformation products, using the reduced ‚-ketodihydropyrrole ring as a stereochemical bridge between the two zones. These results were confirmed by X-ray analysis of the 7-p-bromobenzoate methyl ester derivative, 1c. The absolute stereochemistry was established by application of the advanced Mosher method to methyl ester 1b. Of biogenetic interest is the presence of a unique isoprenylated indole moiety not previously found in other indole mycotoxins. The development of resistance to existing insecticides and the desire to have compounds with less mammalian and environmental toxicity continues to spur the search for novel insecticides. Screening culture extracts using the larvae of the mosquito, Aedes aegypti, has been well documented as a viable approach to discovering new insecticidal activities. 1a A unique macrocyclic lactone, spinosyns, now under development as a crop protection agent, was detected using the mosquito larvae assay and was isolated from Saccharopolyspora spinosa. 1c Another very useful assay involves the larvae of the blowfly, Lucilia seracata. Either of the assays can be used to measure insecticidal activities of agents such as avermectins or milbemycins. 1b,d In our ongoing screening program for biologi- cally active natural products, we have isolated a novel indole terpene designated nodulisporic acid A (1a) from a Nodulispo- rium sp. as a potent insecticide. We describe herein the isolation and structure elucidation of 1a, including the relative and absolute stereochemistry, solution conformation, and biological activity. A number of chemical transformations are also described.

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 ≥1.0, whereas those unsaturated at this bond were generally ≤1.0. The milk to plasma ratio of eprinomectin was ≤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 (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.

Reductive amination of 4″-oxo-5-O-tert-butyldimethylsilyl-avermectins with sodium cyanoborohydride and ammonium acetate gave an epimeric mixture of 4″-deoxy-4″-amino analogs with the epimeric, axial 4″-β-amino derivative as the major component. Acylation of the amino substituent gave highly active broad spectrum antiparasitic compounds, as determined in a sheep anthelmintic assay. 4″-Epi-acetylamino-4″-deoxyavermectin B 1 ( 12 ) was selected for further antiparasitic studies and is currently under development as a novel avermectin endectocide.

Allylic oxidation of avermectin B 1 and C 4″ -substituted averinectin B 1 analogues at C 4a followed by derivatization of the C 4a oxygen provides compounds that have an improved safety profile, as judged by mouse LD 50 data, and are substantially more potent than avermectin B 1 against flies

Comb-counting and thumb-counting were compared in a cross-over study to determine which was more accurate for quantifying flea infestation levels on dogs. Twenty beagle dogs were used in the study and infested with either 50 or 100 adult fleas (Ctenocephalides felis). Two groups of five dogs each were infested with either 50 or 100 fleas per dog, and then comb-counted with a fine-toothed flea comb for 8 min periods. An additional two groups of five dogs each were also given 50 or 100 fleas, and then thumb-counted. The counting time for this technique is both lower and more variable because the fleas are only observed and not captured; thus, the speed at which the dog is covered must be increased in order to prevent counting the same fleas more than once. The mean time of thumb-counting per dog was 3.2 min. Fleas removed during comb-counting were placed back on the dog they were taken from after the count was concluded. At the cross-over point, the ten dogs that had been comb-counted were then thumb-counted and the ten dogs that had been thumb-counted were comb-counted. The results showed that comb-counting recovered significantly (Por = 0.05) more fleas than did thumb-counting. On dogs given 50 and 100 fleas, comb-counting gave mean percentage recoveries of 67.6% and 75.4%, respectively, whereas thumb-counting found means of 8.8% and 7.7%, respectively. The order in which the counting methods were employed produced no significant effect (P0.05) on the number of fleas counted.

Sulfonylation of the 4″-α(or β)-hydroxyl of 5-OTBDMS-avermectin B1a with trifluoromethanesulfonic anhydride yield triflates which were displaced stereospecifically with diverse sulfur nucleophiles. This sulfonylation/substitution protocol also was performed on the 4′-α(or β)-hydroxyl of the corresponding avermectin monosaccharide. The sulfides, sulfoxides and sulfones thus obtained exhibited potent, broad spectrum anthelmintic and acaricidal activity.

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.

The preparation of the methyl imidate of the antiparasitic oxindole alkaloid paraherquamide, its utilization in the synthesis of a variety of 14-O-substituted parherquamide analogs and their biological activity are described.

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.

Paraherquamide, an oxindole alkaloid metabolite of Penicillium paraherquei and Penicillium charlesii, was tested against the adult stages of nine common gastrointestinal and lung nematodes of calves at single, oral dosages of 0.5, 1.0, 2.0 or 4.0 mg kg-1. At dosages 1.0-4.0 mg kg-1 there was 95% or more removal of Haemonchus placei, Ostertagia ostertagi, Trichostrongylus axei, Trichostrongylus colubriformis, Cooperia oncophora, Nematodirus helvetianus, Oesophagostomum radiatum, and Dictyocaulus viviparus. Cooperia punctata, the dosage-limiting species, was virtually unaffected by any dosage except the highest, which produced an efficacy of 89%. The 0.5 mg kg-1 dosage was 95% or more efficacious against H. placei, O. ostertagi, C. oncophora, and D. viviparus, but weaknesses were evident against the other five species. No adverse reaction was observe in any calf.

Treatment of 4″-oxo-5-OTBDMS-Avermectin B 1 ( 2 ) with trimethylsilyldiazomethane or diazomethane resulted in the stereoselective formation of the 4″,4″a-oxide ( 3 ) in addition to the ring-expanded oxepinyl epoxide ( 4 ). The resultant epoxides were opened regiospecifically with sulfur, amine and halogen nucleophiles. The new 4″-substituted avermectins thus formed exhibited potent, broad-spectrum anthelmintic activity.

The disposition and metabolism of paraherquamide (PHQ), a potent and broad-spectrum anthelminthic, were examined in sheep, dogs, and gerbils. The metabolism of PHQ in these species was extensive and marked by significant species differences both in vitro and in vivo. In sheep and gerbils, PHQ metabolism occurs mainly at the pyrrolidine moiety, generating several metabolites that, for the most part, retained nematodicidal activity in vitro. In dogs, the dioxepene group was also extensively metabolized, ultimately resulting in formation of a catechol and loss of pharmacological activity. After oral administration of [3H]PHQ to intact sheep, gerbils, and dogs, the majority of the administered radioactivity was recovered in feces. Intact PHQ accounted for 0% (dogs) to approximately 30% (sheep and gerbils) of drug-related material in feces. A detailed investigation of the composition of the intestinal content of sheep indicated that a significant amount of the dose was still present in the rumen 24 h after dose and that PHQ underwent significant dehydration in the cecum. The oral pharmacokinetic parameters of PHQ in sheep and dogs suggest that its absorption is rapid in both species but that its apparent elimination rate is significantly higher in the dog (t(1/2) approximately 1.5 h) than it is in sheep (t(1/2) approximately 8.5 h). The short elimination half-life and the absence of PHQ or other active components in the dog gastrointestinal tract provide a potential explanation of the lack of efficacy of PHQ in this species.

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]- N -hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in ( i ) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, ( ii ) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, ( iii ) ≈50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and ( iv ) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit “point of no return” model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax.

The objective of this study was to determine if there is evidence of parthenogenesis or autogenous reproduction in the cat flea, Ctenocephalides felis (Bouché). To examine parthenogenesis, 400 newly eclosed virgin female fleas were collected from a laboratory colony and 100 were placed into each of 4 feeding cages and fed bovine blood through a Parafilm membrane. Three of the feeding cages were monitored for egg production for 7 d and each group of 100 virgin female fleas produced an average 1,119 eggs per cage, but none was viable. Fifty male fleas were added to those 3 feeding cages on day 7, and within 24 h the female fleas began ovipositing fertile eggs and nearly quadrupled their egg output. The other cage in which no males were introduced served as a control and did not produce a single viable egg in the 14-d experimental period. A similar experiment examined the continuance of virgin females to lay nonviable eggs and it was found that they continued to lay nonviable eggs for at least 58 d. Egg production was also studied in unfed fleas and it was found that unfed fleas did not produce eggs. These results suggest that neither parthenogenetic reproduction nor autogeny are exhibited by the cat flea.

Nodulisporic acids D, E, and F are the newest members of a family of nontremorogenic indole-diterpenoids that are potent, orally bioavailable, antiflea agents derived from a fungus belonging to the genus Nodulisporium. The four members of the D series are each devoid of an isoprene residue that is present at C-26 in the three nodulisporic acids described originally (the A series). Nodulisporic acid E (11a) has a simpler structure, which lacks not only the isoprene residue at C-26 but also two that form the A/B rings. Nodulisporic acid F is the simplest of all nodulisporic acids and is devoid of all three isoprene residues of the indole unit; as such, it represents the earliest biosynthetic intermediate in this series. A biogenetic grid based on mutation studies is proposed that encompasses all the known nodulisporic acids. Structure-activity relationships of the known natural nodulisporic acids have been elucidated. Within a series the most active compound possesses a dienoic acid chain, and overall, the end product of the biogenetic grid, i.e., nodulisporic acid A, exhibits the most potent antiflea activity. Additionally, the stereochemistries of C-3' ' and C-4' ' of nodulisporic acid D(2) and therefore of nodulisporic acids A(2), B(2), and C(2) have been assigned.

Four new congeners, rediocides B-E (2-5), of the previously reported rediocide A (1) were isolated from a methanol extract of the roots of the plant Trigonostemon reidioides. The structures of these minor analogues were elucidated by comparison of their NMR and mass spectral data with those of rediocide A and confirmed by extensive 2D NMR spectral analysis. They all possess potent activity against fleas (Ctenocephalides felis) in an artificial membrane feeding system and exhibited LD(90) values ranging from 0.25 to 0.5 ppm.

Nodulisporic acids C (4a), C1 (5a), and C2 (6a), a series of D-ring-opened nodulisporic acids, were isolated from fermentations of a mutant culture of Nodulisporium sp. MF5954. Nodulisporic acid C, the most potent of the three, showed good activity against fleas with an LD90 of 10 micro g/mL. These compounds tend to be unstable in the free acid form and were isolated as stable sodium salts.

Imidazo[1,2-a]pyridyl N-arylpyridazinones were hybridized from the classic pyridinylimidazoles and the more recent dual hydrogen bond acceptors, resulting in a new structural class of selective p38 MAP kinase inhibitors.

Mellamide, a novel indole amide, was isolated from a fermentation of Aspergillus melleus using silica gel and high-performance liquid chromatographic methods. This allowed its separation from three known antiparasitic compounds (ochratoxin A, viomellin and xanthomegnin) also present in the potent extract. The structure was elucidated by (1)H, (13)C, COSY, DEPT, HMQC and HMBC NMR experiments. HR-FTMS aided in the molecular weight and formula determination. Mellamide showed in vitro insecticidal activity in bioassays against larvae of Lucilia sericata and Aedes egypti with LD(90) of 1,000 and 50 micro g/ml, respectively.

Efficient routes to access the 2", 3", 4", and 6" registers of the nodulisporic acid (NsA) side chain are disclosed. A mild one-carbon, Ph(2)CdoublebondNCH(2)CtriplebondN mediated homologation of NsA's 3"-aldehyde permitted access to the 4"-register. Curtius reaction of NsA's 3"-acid yielded the corresponding 2"-aldehyde 4 from which the unnatural Delta(2",3")-olefin isomer 2b was obtained. In addition, Arndt-Eistert reactions of the parent NsA permitted a one-carbon homologation to the 6" register. These efforts identified new analogues with significant flea activity and illustrated the biological significance of unsaturation at the 1",2" register.

Vertical transmission of larvae is a major pathway in the life cycle of several species of Strongyloides, but evidence for it occurring in humans or dogs with Strongyloides stercoralis is absent. In an effort to determine if vertical transmission could occur with S. stercoralis, each of 3 female dogs was infected with filariform larvae at a different stage of the reproductive cycle, i.e., preconception, gestation, or postpartum. Results showed that none of 6 pups born to a female infected before conception or any of 6 pups born to another female infected during gestation harbored any stage of S. stercoralis when necropsied at parturition. Conversely, all 5 pups that nursed from the female infected immediately postpartum became infected with adult S. stercoralis in their small intestines (range, 56-129 adult worms). Significantly, live filariform larvae of S. stercoralis were observed on 2 different occasions from milk samples taken from the lactating female. Because arrested development of larvae is not known in S. stercoralis, there is no reservoir of larvae in the parenteral tissues of females to queue for passage to the pups and, thus, it is not surprising that only timely infections, perhaps very late in gestation and during lactation, can be successful. These data support previous work in dogs with S. stercoralis, which concluded that vertical transmission through prenatal pathways does not occur, but they are the first from the dog to indicate that vertical transmission of this parasite through transmammary routes is possible. Whether transmammary transmission of S. stercoralis occurs in humans remains unknown but given its immense pathological potential, it should not be overlooked.

A medicinal chemistry program on the nodulisporic acid chemical class, guided by an artificial membrane flea-feeding assay, has recently identified permissive and nonpermissive regions of the pharmacophore for exploitation against fleas. This pathway was validated when several promising compounds from this program were administered orally to dogs at 15.0 mg/kg and found to have90% flea activity for 2 wk. To determine if a surrogate insect assay would have provided the same guidance, a nodulisporic acid analog series was examined in both a Lucilia sericata larval assay and an artificial membrane flea-feeding assay using Ctenocephalides felis. Results from both insect assays were concordant in that even subtle chemical modification or substitution to the left-hand side of the nodulisporic acid pharmacophore resulted in substantial loss of insecticidal activity. Both assays were also in general agreement that the only modifications to the pharmacophore that did not result in loss of activity occurred to the C-8 side chain on the right-hand side of the molecule. Although there was good agreement between the 2 assays on the general regions of the pharmacophore, there was variance on individual compounds in the mono- and disubstituted amide series from the C-8 side chain. For example, the L. sericata assay showed several analogs from this subclass to possess similar activity to the parent acid, whereas the membrane assay indicated superior activity against fleas relative to the same parent. Consequently, although there was substantial general agreement between the assays, it was concluded that finer optimization of a lead compound should be done against the target parasite, even if it is ex vivo, as early as possible in a medicinal chemistry program.

Nodulisporic acid A (NSA) has been shown previously to be safe in dogs and to deliver90% flea control for 4 days following a single oral administration. Three newly prepared nodulisporamide derivatives were subsequently identified from an artificial membrane flea feeding system as exhibiting potency substantially greater than NSA. To determine if they have superior in vivo activity, these 3 nodulisporamides, as well as NSA, were evaluated in dogs at 15 mg/kg/os. Parasite challenges were made by placing 100 live Ctenocephalides felis fleas onto the dorsum of dogs every 48 hr and examining efficacy at each of those intervals over a 22-day period. Results showed that NSA produced90% efficacy at day 2 and 81% efficacy at day 4, and its residual flea killing fell to approximately 50% by day 6 posttreatment. All dogs treated with the 3 new experimental nodulisporamides were 100% protected from flea challenges to day 8 posttreatment, and 2 of the compounds continued to produce90% residual activity to 2 wk posttreatment. Pharmacokinetic analysis showed that plasma profiles and half-lives of NSA and these 3 new compounds correlated closely with flea efficacy. These results demonstrate that specific substitutions to the pharmacophore of NSA can substantially increase the duration of activity against fleas.

Ivermectin has potent systemic activity against numerous species of nematodes and arthropods, but there are some important species in these two groups, such as the cat flea, Ctenocephalides felis (Bouche), that appear to be refractory to it. In an effort to determine if the lack of systemic activity against C. felis is specific to ivermectin, or if it is a class-wide phenomenon, 20 avermectin derivatives were tested in an artificial membrane flea feeding system at concentrations of 20, 10, and 1 microg/ml. Results showed that ivermectin had LC90 and LC50 values against fleas of 19.1 and 9.9 microg/ml, respectively. Only four of the other 19 compounds evaluated possessed both LC90 and LC50 values more potent than ivermectin and even then the advantage was modest. Among those four compounds was a two-fold increase in potency relative to ivermectin when the LC90 values were considered (range, 9.2-10.3 microg/ml) and a two- to eight-fold increase when the LC50 values were examined (range, 1.23-5.26 microg/ml). Neither the possession nor the number of oleandrosyl sugars on the macrocyclic backbone were relevant for additional flea activity because among these four compounds were two disaccharides, a monosaccharide and an aglycone. Also, bond disposition between C-22 and 23 did not contribute to increase in activity because these molecules comprise members with either single or double bonds. One of these avermectin analogs was scaled-up and tested subcutaneously in a dog at >100 times the commercial ivermectin dosage and zero efficacy was observed against the flea. We conclude that even the best in vitro avermectin does not have the in vivo potential to become a commercial oral or subcutaneous flea treatment for companion animals.

Paraherquamide, an oxindole alkaloid metabolite of Penicillium paraherquei and Penicillium charlesii , was tested against the common gastrointestinal nematodes of dogs at a single oral dosage of 0.5, 1.0, or 2.0 mg kg −1 . Efficacy was poor (less than 85%) against Ancylostoma caninum, Uncinaria stenocephala, Toxascaris leonina, Trichuris vulpis , and Strongyloides stercoralis at the low- and mid-dosage levels. At the high dosage level, good efficacy (91%) was observed only against S. stercoralis . Adverse reactions were observed in all dogs at every dosage level and included depression, ataxia, and protrusion of the nictitating membrane.

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.

SUMMARY A chewable tablet incorporating ivermectin and pyrantel was tested in 12 Beagle dogs for efficacy against the adult hookworm, Ancylostoma braziliense. The dogs were administered infective larvae of A braziliense orally. Twenty-one days after infection the dogs were weighed and allocated randomly to receive either an oral treatment with ivermectin and pyrantel in a beef-based chewable tablet or no treatment. The chewable tablet was a commercially available product, which was made to deliver ivermectin at 6 μg/kg and pyrantel at 5.0 mg/kg to each dog. Seven days after treatment the dogs were euthanased, necropsied, and examined for adult hookworms. At necropsy, no adult A braziliense was observed in any of the 6 treated dogs and all 6 dogs that had been left untreated were infected with adult A braziliense (range, 48 to 161). It was concluded that this combination product is 100% efficacious against adult A braziliense.

Combing the haircoat to count fleas has been used to determine the efficacy of insecticides against fleas on dogs, but no standardization of method has been reported. In this study, the effect of combing time on flea recovery from dogs was examined. Six beagle dogs were infested with 100 unfed, adult Ctenocephalides felis (Bouche) on each of three consecutive days. A crossover design, balanced for carryover effects, was used to evaluate flea removal rates from each dog by comb-counting for three different time intervals; i.e. 5, 10 and 15 min. Each dog was combed once at each time interval on a different day, over three consecutive days. The results showed that the majority of fleas were recovered in the first 5 min of combing and there were no significant differences (P ⩾ 0.19) in the total number of fleas recovered between the 5, 10 or 15 min protocols. Moreover, the standard deviation and coefficient of variation increased with an increase in the amount of time spent combing, resulting in a decrease in precision for the longer protocols. Therefore, the comb time of 5 min provided a precise and accurate representation of the number of fleas present on an animal and could be useful as a standard measure of flea infestation levels in efficacy trials.

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.

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.