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Drosophila suzukii Matsumura (Diptera: Drosophilidae) has caused significant economic damage to berry and stone fruit production regions. Markers that are systemic in plants and easily transferred to target organisms are needed to track D. suzukii exploitation of host resources and trophic interactions. High and low concentrations of the trace element, rubidium (Rb), and the stable isotope, 15N, were tested to mark D. suzukii larvae feeding on fruits of enriched strawberry plants grown in containers under greenhouse conditions. Fly marker content and proportion of flies marked 1, 7, and 14 d after emergence from enriched fruits and fly dry mass were analyzed. Nearly 100% of the flies analyzed 14 d after emerging from 15N-enriched plants were marked, whereas only 30-75% and 0-3% were marked 14 d after emerging from high and low Rb concentration plants, respectively. Rapid Rb decay, strong 15N persistence, and the economics of using these markers in the field to elucidate D. suzukii pest ecology are discussed.

Background Urinalysis data in preclinical toxicology studies can be influenced by preanalytic and analytic factors which have the potential to confound interpretation. There is a paucity of information regarding positive reagent strip urinary blood reactions in healthy nonhuman primates (NHP) and Beagle dogs used in preclinical toxicology studies. Objectives The objectives were (1) to establish historical control data for reagent strip urinary blood reactions in healthy NHP and Beagle dogs, (2) to determine the incidence of positive urinary blood reactions during predose and dosing phases, and (3) to determine if collection practice was a relevant parameter. Methods Historical control data from 2 institutions in the biopharmaceutical industry were retrospectively analyzed for reagent strip urinary blood reactions in healthy NHP and Beagles. The incidence of positive results between the 2 institutions with different urine collection practices and between males and females was compared. Results The incidence of positive urinary blood reactions in NHP was comparable between institutions (≤ 14% in males; ≤ 33% in females), while the incidence of positive urinary blood reactions in Beagles was more variable (≤ 77% in males; ≤ 69% in females), and higher in females during the dosing phase. Conclusions Positive urinary blood results that could potentially be misinterpreted as toxicologically relevant were identified in healthy NHP and Beagles during predose and dosing phases. Different incidences of positive results between the 2 institutions were likely related to collection practices. Strategies to reduce feces and food contamination of collected urine samples should help minimize false-positive urinary blood reactions.

Root weevils in the genus Otiorhynchus are cited as one of the most important pests in the major nursery and small fruit production areas throughout the United States, western Canada, and northern Europe. A major problem in combating weevil attack is monitoring and timing of control measures. Because of the night-activity of the adult weevils growers do not observe the emerging weevils in a timely manner and oviposition often starts before effective control measures are taken. Several vine weevil electroantennogram-active plant volatiles were identified from a preferred host plant, Euonymus fortunei. Main compounds evoking antennal responses on the weevils' antennae were (Z)-2-pentenol, (E)-2-hexenol, (Z)-3-hexenol, methyl benzoate, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene, methyl eugenol, and (E, E)-alpha-farnesene. Several of these compounds were tested alone and in mixtures on attractiveness for the vine weevil Otiorhynchus sulcatus (F.) in field-grown strawberry in Oregon. O. sulcatus were attracted to (Z)-2-pentenol (approximately 3 x more than control) and a 1:1 ratio mixture of (Z)-2-pentenol and methyl eugenol (4.5 x more than control). This is the first report of field-active attractants for O. sulcatus which holds promise for the development of new monitoring strategies for growers in the near future.

Summary Pseudomonas fluorescens CHA0 and the related strain Pf-5 are well-characterized representatives of rhizosphere bacteria that have the capacity to protect crop plants from fungal root diseases, mainly by releasing a variety of exoproducts that are toxic to plant pathogenic fungi. Here, we report that the two plant-beneficial pseudomonads also exhibit potent insecticidal activity. Anti-insect activity is linked to a novel genomic locus encoding a large protein toxin termed Fit (for P. fluorescens insecticidal toxin) that is related to the insect toxin Mcf (Makes caterpillars floppy) of the entomopathogen Photorhabdus lumine- scens, a mutualist of insect-invading nematodes. When injected into the haemocoel, even low doses of P. fluorescens CHA0 or Pf-5 killed larvae of the tobacco hornworm Manduca sexta and the greater wax moth Galleria mellonella. In contrast, mutants of CHA0 or Pf-5 with deletions in the Fit toxin gene were significantly less virulent to the larvae. When expressed from an inducible promoter in a non-toxic Escherichia coli host, the Fit toxin gene was sufficient to render the bacterium toxic to both insect hosts. Our findings establish the Fit gene products of P. fluorescens CHA0 and Pf-5 as potent insect toxins that define previously unappreciated anti-insect properties of these plant-colonizing bacteria.

The objective of this study was to determine the susceptibility of the two primary direct insect pests of hazelnuts in Oregon to three species of entomopathogenic nematodes. The entomopathogenic nematodes (Heterorhabditis marelatus Pt. Reyes, Steinernema carpocapsae All and Steinernema kraussei L137) were used in laboratory soil bioassays to determine their virulence against filbertworm, Cydia latiferreana (Walsingham) (Lepidoptera: Tortricidae) and filbert weevil, Curculio occidentalis (Casey) (Coleoptera: Curculionidae). All three nematode species were infective in laboratory bioassays. Infectivity ranged from 73-100% and 23-85% for filbertworm and filbert weevil, respectively. Field results were similar to those found in the laboratory with filbertworm larvae being more susceptible to nematode infection.

Laboratory soil bioassays were performed at economic field rates for in-furrow (3.85 × 106 spores/g dry soil) and broadcast (3.85 × 105 spores/g dry soil) applications with three isolates of Metarhizium anisopliae (F52, ATCC62176, and ARSEF5520) and one isolate of Beauveria bassiana (GHA). All isolates tested were infective to second instar Delia radicum (L.). The conditionally registered M. anisopliae isolate (F52) performed best killing an average of 85 and 72% of D. radicum larvae at the high and low concentration, respectively. The mean LC50 and LC95 of F52 against second instar D. radicum was 2.7 × 106 and 1.8 × 108 spores/g dry soil, respectively. The use of F52 in an integrated management program is discussed.

In August 2008, the first detection of the spotted wing drosophila, Drosophila suzukii, to the North America mainland in California caused great concern, as the fly was found infesting a variety of commercial fruits. Subsequent detections followed in Oregon, Washington, Florida and British Columbia in 2009; in Utah, North Carolina, South Carolina, Michigan, and Louisiana in 2010; and in Virginia, Montana, Wisconsin, Pennsylvania, New Jersey, Maryland and Mexico in 2011. In Europe, it has been detected in Italy and Spain in 2009 and in France in 2010. Economic costs to the grower from D. suzukii include the increased cost of production (increased labor and materials for chemical inputs, monitoring and other management tools) and crop loss. An effective response to the invasion of D. suzukii requires proper taxonomic identification at the initial phase, understanding basic biology and phenology, developing management tools, transferring information and technology quickly to user groups, andevaluatingtheimpactoftheresearchandextensionprogramonaneconomic,social,andenvironmentallevel.AsD.suzukii continues to expand its range, steps mustbe initiated in each new region to educate and inform the public as well as formulate managementtactics suitable for the crops and growing conditions in each. c � 2011 Society of Chemical Industry

Nosema pyrausta is an obligate pathogen causing reduced fecundity and longevity of Ostrinia nubilalis. This study was conducted to determine the combined effects of N. pyrausta infection and temperature on O. nubilalis egg production and hatch. N. pyrausta-infected and noninfected O. nubilalis were maintained in two different temperature regimes. The first regime allowed females to oviposit under optimum conditions (27 degrees C, 65% RH, 16:8 (L:D)), while females in the second regime were held initially under the same humidity and light conditions, but a constant temperature of 16 degrees C for 1 week after which they were transferred to optimum ovipositional conditions. Studies were performed initially with O. nubilalis populations and later with individual mating pairs. In studies with O. nubilalis populations, the mean number of eggs laid per female under optimum conditions was 660, while N. pyrausta-infected females held initially at 16 degrees C laid 116 eggs per female. In studies with individual mating pairs, N. pyrausta infection reduced egg production per female 53 and 11% in the 16 and 27 degrees C temperature regimes, respectively, compared to noninfected females under optimum conditions. Exposure to 16 degrees C temperatures early in the ovipositional period had a more profound impact on reducing egg production in N. pyrausta-infected than noninfected O. nubilalis.

Drosophila suzukii (Matsumura), an invasive pest of small and stone fruits, has been recently detected in 39 states of the United States, Canada, Mexico, and Europe. This pest attacks ripening fruit, causing economic losses including increased management costs and crop rejection. Ongoing research aims to improve the efficacy of monitoring traps. Studies were conducted to evaluate how physical trap features affect captures of D. suzukii. We evaluated five colors, two bait surface areas, and a top and side position for the fly entry point. Studies were conducted at 16 sites spanning seven states and provinces of North America and nine crop types. Apple cider vinegar was the standard bait in all trap types. In the overall analysis, yellow-colored traps caught significantly more flies than clear, white, and black traps; and red traps caught more than clear traps. Results by color may be influenced by crop type. Overall, the trap with a greater bait surface area caught slightly more D. suzukii than the trap with smaller area (90 vs. 40 cm(2)). Overall, the two traps with a side-mesh entry, with or without a protective rain tent, caught more D. suzukii than the trap with a top-mesh entry and tent.

Background The serum activities of ALT and AST are key indicators of liver toxicity in the drug safety evaluation of laboratory animals and patients. To ensure that the full aminotransferase activity is measured, exogenous Pyridoxyl-5-Phosphate (P5P) cofactor is included in the assay reagent. Clinical pathology laboratories make a choice to use aminotransferase assays with or without the added P5P cofactor, and the impact of assay selection on safety assessment is not well understood. Objectives The objective of this report was to investigate the effect of aminotransferase assay selection on the detection of liver toxicity based on a literature review. Methods Literature in public databases was searched using combinations of the search terms alanine aminotransferase, aspartate aminotransferase, pyridoxyl-5-phosphate, holoenzyme, apoenzyme, enzyme inhibition, artifact, clinical pathology, toxicology, and safety assessment. Regulations or guidance documents published by health authorities specifying clinical pathology evaluation in nonclinical and clinical safety studies of biopharmaceuticals, chemicals, and devices were also reviewed. Results Aminotransferase testing is not standardized in safety assessment studies and consequently, laboratories use aminotransferase assays with or without P5P cofactor. Individual studies have demonstrated mean differences of approximately 10–20% in serum ALT activity in animal and human populations. The impact of aminotransferase testing without P5P on detection of toxicity and decision-making in drug development has not been systematically evaluated. Conclusions The use of different assays for measuring aminotransferase activity contributes to the variability in data between laboratories and studies. Standardizing aminotransferase assays is an avenue for improving the diagnostic performance in drug safety evaluation.

Ten strains representing four lineages of the Pseudomonas fluorescens group (P. chlororaphis, P. corrugata, P. koreensis, and P. fluorescens subgroups) were evaluated for toxicity to the tobacco hornworm Manduca sexta and the common fruit fly Drosophila melanogaster. The three strains within the P. chlororaphis subgroup exhibited both oral and injectable toxicity to the lepidopteran M. sexta. All three strains have the gene cluster encoding the FitD insect toxin and a ΔfitD mutant of P. protegens strain Pf-5 exhibited diminished oral toxicity compared to the wildtype strain. Only one of the three strains, P. protegens Pf-5, exhibited substantial levels of oral toxicity against the dipteran D. melanogaster. Three strains in the P. fluorescens subgroup, which lack fitD, consistently showed significant levels of injectable toxicity against M. sexta. In contrast, the oral toxicity of these strains against D. melanogaster was variable between experiments, with only one strain, Pseudomonas sp. BG33R, causing significant levels of mortality in repeated experiments. Toxin complex (Tc) gene clusters, which encode insecticidal properties in Photorhabdus luminescens, were identified in the genomes of seven of the ten strains evaluated in this study. Within those seven genomes, six types of Tc gene clusters were identified, distinguished by gene content, organization and genomic location, but no correlation was observed between the presence of Tc genes and insect toxicity of the evaluated strains. Our results demonstrate that members of the P. fluorescens group have the capacity to kill insects by both FitD-dependent and independent mechanisms.

Drosophila suzukii Matsumura (Diptera: Drosophilidae), a recent invasive pest of small and stone fruits, has been detected in more than half of the U.S. states, and in Canada, Mexico, and Europe. Upon discovery, several different trap designs were recommended for monitoring. This study compared the trap designs across seven states/provinces in North America and nine crop types. Between May and November 2011, we compared a clear cup with 10 side holes (clear); a commercial trap with two side holes (commercial); a Rubbermaid container with mesh lid and rain tent (Haviland), and with 10 side holes and no tent (modified Haviland); a red cup with 10 side holes (red); and a white container with mesh lid and rain tent (Van Steenwyk). Although fly catches among traps varied per site, overall, the Haviland trap caught the most D. suzukii, followed by the red, Van Steenwyk, and clear trap. The modified Haviland and commercial trap had low captures. Among five crop types in Oregon, a clear cup with mesh sides (Dreves) also was tested and caught the most flies. Traps with greater entry areas, found in mesh traps, caught more flies than traps with smaller entry areas. In terms of sensitivity and selectivity, traps that caught more flies likewise caught flies earlier, and all traps caught 26-31% D. suzukii out of the total Drosophila captured. Future trap improvements should incorporate more entry points and focus on selective baits to improve efficiency and selectivity with regard to the seasonal behavior of D. suzukii.

The spotted wing Drosophila, Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae), is an invasive pest of small-fruit crops. Unlike most other Drosophila, this insect is able to oviposit into and damage ripe and ripening fruit, making it unmarketable. Because this is a new pest in the United States, it is necessary to identify registered insecticides to manage this insect effectively in conventional and organic production systems.The present laboratory bioassays and field trials identified a number of insecticides representing various modes of action that are effective in controlling D. suzukii. Products that performed well in the laboratory bioassay also performed well in the field, indicating that screening of new chemistries in the laboratory is a worthy exercise. Field application of pyrethoids, organophosphates or spinosyns provided 5-14 days of residual control of D. suzukii. The efficacy of the neonicotinoids as adulticides was not satisfactory compared with the other contact-mode-of-action chemistries. Based on the zero tolerance by the small-fruit industry and the individual effects mentioned above, neonicotinoids are not currently recommended for D. suzukii management.There are effective insecticides registered for controlling D. suzukii infestations in susceptible small-fruit crops.

BACKGROUND: The spotted-wing drosophila, Drosophila suzukii Matsumura, is native to Asia and was first detected in the North American mainland and Europe in 2008–2010. Drosophila suzukii is a serious economic pest to stone and small fruits because the female lays eggs within ripening fruit on a plant before harvest, which can lead to crop loss. The aim of this study was to evaluate the susceptibility of blackberries, blueberries, cherries, grapes, raspberries and strawberries to D. suzukii among various ripeness stages and cultivars. RESULTS: In 26 no-choice and choice replicated laboratory cage tests on ripeness stages, fruits were generally susceptible to D. suzukii once fruits started to color. Few D. suzukii developed on green fruit, wine grapes or overripe blueberries. In seven cultivar tests, D. suzukii preferences ranged from no differences to fourfold differences for specific cultivars of blackberries, blueberries, raspberries and wine grapes. As brix levels increased, more eggs were laid or more D. suzukii developed on blackberries, blueberries, cherries, raspberries and strawberries. In a choice test of various fruit types, strawberries, raspberries, blackberries, cherries and blueberries were more susceptible to D. suzukii than green table grapes ('Thompson'). CONCLUSION: The results suggest that fruits may become susceptible to D. suzukii as they start to turn color, and that specific varieties of grapes and overripe blueberries have low susceptibility to D. suzukii. Copyright © 2011 Society of Chemical Industry

Understanding habitat selection of fungal entomopathogens is critical to improve the efficacy, persistence and cost of these fungi as microbial insecticides. This study sought to determine the prevalence of Metarhizium and Beauveria spp. isolated from the rhizosphere of strawberry, blueberry, grape and Christmas tree crops in the Willamette Valley of Oregon. Entomopathogenic fungi were assigned to thirteen species based on molecular phylogenetic criteria. Four species of Metarhizium were isolated including Metarhizium brunneum, Metarhizium guizhouense, Metarhizium robertsii, and Metarhizium flavoviride var. pemphigi. Nine Beauveria species were isolated including, Beauveria brongniartii, an undescribed species referred to as Clade C and seven phylogenetic species of Beauveria bassiana. Strawberries and blueberries were significantly associated with M. brunneum and Christmas trees with M. guizhouense and M. robertsii. Grapes were significantly associated with B. bassiana phylogenetic species Bbas-16. All of the Metarhizium isolates screened were pathogenic to Otiorhynchus sulcatus larvae in laboratory bioassays but only M. brunneum and M. robertsii caused significant levels of infection. The study results suggest that certain species of Metarhizium and Beauveria are significantly associated with the strawberry, blueberry and Christmas tree rhizosphere and could potentially provide better control of O. sulcatus.

Filbertworm, Cydia latiferreana (Walsingham) (Lepidoptera: Tortricidae), is a key insect pest associated with hazelnuts, Corylus avellana L. (Fagales: Betulaceae), in North America. This study investigated the feasibility of entomopathogenic nematodes as an alternative strategy for filbertworm control. Laboratory and field experiments were conducted between October 2007 and May 2008 in Benton County, OR, to evaluate the ability of the nematode Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae) to infect filbertworm larvae and pupae. The susceptibility of larvae with and without hibernacula as well as pupae to S. carpocapsae was tested in laboratory bioassays using nematode concentrations between 40 and 200 infective juveniles (IJs) per cm2. Percentage of nematode infection was not significantly affected by the presence of hibernacula (infection range, 90-92%) compared with larvae without hibernacula (80-95%), or by pupal stage (50-75%) compared with larvae (65-75%). In additional field trials, the effect of nematode rate, water application rate, and orchard floor cover on nematode efficacy was determined in October 2007 and May/June 2008. Sentinel filbertworm larvae in plots with either bare soil or debris (leaves, twigs, husks, and blank nuts) were treated with S. carpocapsae at rates ranging from 40 to 150 IJs per cm2 applied in 75 or 190 ml/m2 water. Average filbertworm mortality ranged from 2 to 11% and from 50 to 78% in October and May, respectively. Larval mortality increased with increasing nematode concentration, but was not significantly affected by water application rate or orchard floor cover. Our experiments suggest that S. carpocapsae can play a role in the control of filbertworm larvae overwintering on the orchard floor.

The objective of this study was to determine the susceptibility of the strawberry crown moth, Synanthedon bibionipennis (Boisduval) (Lepidoptera: Sesiidae) larvae to two species of entomopathogenic nematodes. The entomopathogenic nematodes Steinernema carpocapsae (Weiser) strain Agriotos and Heterorhabditis bacteriophora (Steiner) strain Oswego were evaluated in laboratory soil bioassays and the field. Both nematode species were highly infective in the laboratory bioassays. Last instars were extremely susceptible to nematode infection in the laboratory, even in the protected environment inside the strawberry (Fragaria x ananassa Duch.) crown. Infectivity in the laboratory was 96 and 94% for S. carpocapsae and H. bacteriophora, respectively. Field applications in late fall (October) were less effective with S. carpocapsae and H. bacteriophora, resulting in 51 and 33% infection, respectively. Larval mortality in the field from both nematode treatments was significantly greater than the control, but treatments were substantially less efficacious than in the laboratory. Soil temperature after nematode applications in the field (11 degrees C mean daily temperature) was below minimum establishment temperatures for both nematode species for a majority of the post-application period. It is clear from laboratory data that strawberry crown moth larvae are extremely susceptible to nematode infection. Improved control in the field is likely if nematode applications are made in late summer to early fall when larvae are present in the soil and soil temperatures are more favorable for nematode infection.

A newly discovered microsporidium infecting the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae), provisionally placed in the genus Canningia, was studied to determine its impact on O. sulcatus. O. sulcatus populations from several locations were sampled and evaluated for microsporidiosis. A very low prevalence of the disease was observed in all locations surveyed (

The objective of this study was to determine the persistence of Metarhizium anisopliae (F52), measured as infectivity against black vine weevil larvae, in a soilless potting medium at six wholesale nursery locations across the Willamette Valley, Oregon. A granule formulation (0.30 and 0.60 kg/m3) was incorporated into media at planting and fungal persistence determined over two growing seasons. The fungus persisted in the potting media over the duration of the experiment with 50–60% of the larvae exposed to treated media becoming infected at the end of the experiment. The percentage of infected larvae gradually declined from ⩾90% on week 3 to 40–60% by week 19. Larval infection rebounded over the fall and winter months of 2004 to 75–80% followed again by a slow decline over the course of the second growing season.

Black vine weevil, Otiorhynchus sulcatus (F.), is a severe pest of small fruit and nursery crops around the world. These studies were conducted to determine the efficacy of three species of entomopathogenic nematodes (Heterorhabditis marelatus, Heterorhabditis bacteriophora, and Steinernema riobrave) applied in infected host cadavers or as aqueous applications for black vine weevil larval control. Experiments were conducted in the greenhouse and outdoors. Application of three infected host cadavers or 40 infective juvenile nematodes (IJs) /cm2 were made to pots of Impatiens walleriana 5-7 d after larval infestation. Efficacy was assessed at 14 d in the greenhouse and at 14 and 28 d after nematode application in outdoor trials. In the greenhouse, all treatments with the exception the S. riobrave (cadaver and aqueous applications) provided nearly 100% efficacy after 14 d. The S. riobrave applications, although significantly better than the control, only provided 40-70% control and were not included in the outdoor trials. Nematode efficacy was slowed in the outdoor trials particularly in the cadaver applications. In the initial outdoor trial (soil temperatures12 degrees C), there were no significant differences between any nematode treatment and the control after 14 d. The nematode efficacy in the initial outdoor trial after 28 d was improved from the 14-d evaluation but not to the level seen in the second trial. In the second outdoor trial, in which soil temperatures were higher (12 degrees C), the aqueous applications of H. marelatus and H. bacteriophora provided nearly complete control after 14 d. The cadaver applications also provided nearly complete control in the second outdoor trial after 28 d. Even though the potential total number of IJs estimated per pot was higher in the cadaver-applied treatments, cool soil temperatures apparently delayed or potentially reduced IJ emergence from cadavers resulting in delayed control.

A dog with severe cholestasis secondary to chronic hepatitis was treated with ursodeoxycholic acid (UDCA) PO. After 2 weeks of daily treatment, the dog was more active and had an improved appetite. Monthly serum biochemical determinations and analysis of individual bile acid profiles documented improvement in hepatobiliary tests and a marked reduction in the concentrations of potentially hepatotoxic endogenous bile acids. These effects were maintained for approximately 6 months. The findings in this dog are similar to those reported for human patients treated with UDCA and provide preliminary evidence in support of its continued evaluation in the treatment of cholestatic liver disease in the dog.

OSI-7836 (4'-thio-beta-D-arabinofuranosylcytosine) is a novel nucleoside analog in phase I clinical development for the treatment of cancer. As with other nucleoside analogs, the proposed mechanism of action involves phosphorylation to the triphosphate form followed by incorporation into cellular DNA, leading to cell death. This hypothesis has been examined by measuring and comparing the incorporation of ara-C, OSI-7836, and gemcitabine (dFdC) into DNA of cultured cells and by investigating the role of deoxycytidine kinase in OSI-7836 toxicity. We report here additional studies in which the role of cell cycling on OSI-7836 toxicity was investigated and incorporation of OSI-7836 into DNA of xenograft tumors measured. The role of the cell cycle was examined by comparing the toxicity of OSI-7836 in A549 NSCLC cells that were either in log phase growth or had reached confluence. A novel validated LC-MS/MS assay was developed to quantify the concentrations of OSI-7836 in DNA from Calu-6 and H460 human tumor xenografts in mice. Results showed that apoptosis induced by OSI-7836 was markedly greater in cycling cells than in confluent non-cycling cells despite only a modest increase in intracellular OSI-7836 triphosphate concentration. The LC-MS/MS assay developed to measure OSI-7836 incorporation into DNA had an on-column detection limit of 0.25 fmol, a quantification limit of 0.5 fmol, and a sensitivity of approximately 0.1 pmol OSI-7836/micromol dThy. Concentrations of OSI-7836 in splenic DNA (0.4 pmol OSI-7836/micromol dThy) averaged fivefold less than the average concentration in Calu-6 and H460 xenograft DNA (3.0 pmol OSI-7836/micromol dThy) following a 400 mg/kg dose of OSI-7836. Concentrations of OSI-7836 in Calu-6 tumor DNA isolated 24 h following a dose of 400, 1000, or 1600 mg OSI-7836/kg were approximately 1.3, 1 and 1.3 pmol OSI-7836/micromol dThy, respectively. Concentrations of OSI-7836 in DNA from H460 and Calu-6 xenografts did not appear to increase during repeated administration of 400 mg OSI-7836/kg on days 1, 4, 7, and 10. The majority of OSI-7836 in DNA from Calu-6 and H460 tumors of mice dosed with 1600 mg/kg was located at internal nucleotide linkages, similar to dFdC and ara-C. In conclusion, cell cycling studies supported the hypothesis that OSI-7836 cytotoxicity is dependent upon DNA synthesis. A validated LC-MS/MS assay was developed that could quantify OSI-7836 in DNA from tissues. The assay was used to show that OSI-7836 was incorporated in internal linkages in tumor DNA in a manner that was dose-independent at the doses tested and did not appear to accumulate during repeated dosing. The results suggest that if DNA incorporation is a toxic event, the relationships between administered dose, DNA incorporation, and toxicity are complex.

OSI-211 (liposomal lurtotecan), was evaluated using several different dose schedules (1mg/kg, d1-5, 1.75 mg/kg d1, 3, 5 and 6 mg/kg d1, 8) in severe combined immunodeficient (SCID) mouse models of acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) with early treatment (ET, days 6-8) or late treatment (LT, days 15-19), examining early and advanced disease, respectively. Due to the aggressive nature of the Molt-4 model, the ET and LT were accelerated to day 3 or 4 and day 8 post-implant, respectively. For each model, 2 x 10(7) (KBM-3B) or 1 x 10(7) (Molt-4, HL-60 and CEM) leukemia cells were injected intravenously into the tail vein. Each control and test group consisted of eight animals. All three schedules (1mg/kg qd1-5, 1.75 mg/kg d1, 3, 5 and 6 mg/kg d1, 8) increased the life span of OSI-211 treated animals in each model, with a tendency toward improved efficacy with the 6 mg/kg d1, 8 schedule. As a result, the activity of the 6 mg/kg d1, 8 schedule is detailed for each model. ET significantly (P0.005) increased survival in the KBM-3B model with 86% long-term survivors (LTS). Using PRC analysis, human beta-globin gene sequences in one or several tissues were amplified in all but 3 LTS, suggesting minimal residual disease in 26 of the 29 LTS. LT also significantly (P0.005) improved average life span in the KBM-3B model, with an average ILS=196+/-11% and one LTS. Treatment of HL-60 leukemia animals significantly (P0.005) increased life span, with an ILS=213+/-9% and two LTS for ET, and with an ILS=219+/-4% and no LTS for LT. Treatment of Molt-4 animals, the most aggressive leukemia model tested, significantly (P0.005) increased life span, with an average ILS=181+/-3% and no LTS for ET and an average ILS=172+/-1% with no LTS for LT. In the CEM model, ET resulted in a significantly (P0.005) improved ILS=244+/-24% with one LTS. In comparison to OSI-211, treatment with DaunoXome, the liposomal formulation of daunorubicin, a drug with clinical efficacy in AML and ALL, had no effect on survival in the KBM-3B, nor Molt-4 A4 leukemia models when administered at its maximum or near maximum tolerated doses of 3mg/kg d1, 8. These data demonstrate that OSI-211 has potent antileukemia activity in preclinical SCID mouse AML and ALL leukemia models, supporting the clinical investigation of OSI-211 for hematological malignancies.

This retrospective study summarizes 10 Dalmatians suspected of having hepatic copper toxicosis. Hepatic copper toxicosis can result from either a primary metabolic defect in hepatic copper metabolism or from altered hepatic biliary excretion of copper. An inherited copper-associated hepatopathy has been documented in Bedlington Terriers, and there is evidence for familial copper-associated liver disease in West Highland White (WHW) Terriers and Skye Terriers. Nine of the 10 Dalmatians in this study presented for gastrointestinal clinical signs, including anorexia and vomiting. All animals had increased alanine aminotransferase (ALT) enzyme activity, and 9 of 10 had increased alkaline phosphatase (ALP) enzyme activity. The relative increase in ALT activity was much greater than the relative increase in ALP activity, suggesting a predominantly hepatocellular rather than cholestatic liver disease. The mean hepatic copper concentration for 9 Dalmatians was 3,197 microg/g dry weight liver (dwl) (normal,450 microg/g). In 5 of these 9 dogs, hepatic copper concentrations exceeded 2,000 microg/g dwl. Necroinflammatory alterations associated with copper-laden parenchymal cells were the notable histopathologic finding. The inflammatory infiltrate was either primarily lymphocytic or neutrophilic. Morphologic features of cholestasis generally were not prominent except in those dogs with severe pathology. These findings lend support to the hypothesis that a primary metabolic defect in hepatic copper metabolism occurs in the Dalmatian breed. The mechanism and genetic basis of this condition require further study.

The toxicities of 2'-fluorouridine (2'-FU) and 2'-fluorocytidine-HCl (2'-FC) were separately evaluated in 2 species, male Fischer 344 (F334) rats and woodchucks. Particular attention was focused on the ability of these nucleosides to induce toxicities similar to those induced by the antiviral drug fialuridine (FIAU). 2'-FU or 2'-FC was administered to F344 male rats by intravenous injection at doses of 5, 50, and 500 mg/kg/day for 90 consecutive days and to male and female woodchucks at doses of 0.75 and 7.5 mg/kg/ day for 90 consecutive days. Clinical chemistry, hematology, and urinalysis (woodchuck only) profiles were assessed during and at the termination of the study. At necropsy, organs were weighed and tissues collected for routine histologic analysis. Cytochrome c oxidase activity, citrate synthase activity, and mitochondrial DNA content were measured, and micronucleus formation in the bone marrow (rats only) was evaluated. No adverse clinical effects were observed in either species. Rats treated with high doses of either 2'-FU or 2'-FC had body weights that were 90% of those of controls. 2'-FU and 2'-FC both induced a moderate decrease in the median lymphocyte count, and 2'-FC and 2'-FU induced a mild increase in mean corpuscular hemoglobin and mean corpuscular volume. Both compounds caused slight to moderate, reversible, histologic changes in the spleen and thymus. In the woodchuck, 2'-FC caused a slight increase in mean absolute lymphocytes, and 2'-FC and 2'-FU slightly increased hepatic periportal vacuolation and/ or mononuclear cell infiltration. In summary, neither compound showed evidence of the toxicity induced by fialuridine in either species. Although compound effects were observed, none of these effects were considered to be adverse, and the no-observed adverse effect level was determined to be 500 mg/kg/day for both compounds in the male F344 rat and 7.5 mg/kg/day in the woodchuck.