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Legends for Supplementary Figures 1-2, Tables 1-6 from BMS-754807, a small molecule inhibitor of insulin-like growth factor-1R/IR

Supplementary Tables 1-6 from BMS-754807, a small molecule inhibitor of insulin-like growth factor-1R/IR

Supplementary Figure 1 from BMS-754807, a small molecule inhibitor of insulin-like growth factor-1R/IR

BMS-754807 is a potent and reversible inhibitor of the insulin-like growth factor 1 receptor/insulin receptor family kinases (Ki, in vitro, including mesenchymal (Ewing's, rhabdomyosarcoma, neuroblastoma, and liposarcoma), epithelial (breast, lung, pancreatic, colon, gastric), and hematopoietic (multiple myeloma and leukemia) tumor cell lines (IC50, 5–365 nmol/L); the compound caused apoptosis in a human rhabdomyosarcoma cell line, Rh41, as shown by an accumulation of the sub-G1 fraction, as well as by an increase in poly ADP ribose polymerase and Caspase 3 cleavage. BMS-754807 is active in vivo in multiple (epithelial, mesenchymal, and hematopoietic) xenograft tumor models with tumor growth inhibition ranging from 53% to 115% and at a minimum effective dose of as low as 6.25 mg/kg dosed orally daily. Combination studies with BMS-754807 have been done on multiple human tumor cell types and showed in vitro synergies (combination index, in vivo, at multiple dose levels, resulted in improved clinical outcome over single agent treatment. These data show that BMS-754807 is an efficacious, orally active growth factor 1 receptor/insulin receptor family–targeted kinase inhibitor that may act in combination with a wide array of established anticancer agents. [Mol Cancer Ther 2009;8(12):3341–9]

Supplementary Figure 2 from BMS-754807, a small molecule inhibitor of insulin-like growth factor-1R/IR

Myeloperoxidase (MPO) is a heme peroxidase found in neutrophils, monocytes and macrophages that efficiently catalyzes the oxidation of endogenous chloride into hypochlorous acid for antimicrobial activity. Chronic MPO activation can lead to indiscriminate protein modification causing tissue damage, and has been associated with chronic inflammatory diseases, atherosclerosis, and acute cardiovascular events. Triazolopyrimidine 5 is a reversible MPO inhibitor; however it suffers from poor stability in acid, and is an irreversible inhibitor of the DNA repair protein methyl guanine methyl transferase (MGMT). Structure-based drug design was employed to discover benzyl triazolopyridines with improved MPO potency, as well as acid stability, no reactivity with MGMT, and selectivity against thyroid peroxidase (TPO). Structure-activity relationships, a crystal structure of the MPO-inhibitor complex, and acute in vivo pharmacodynamic data are described herein.

The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRβ-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area.

C-terminal Src kinase (Csk) is one of the critical negative regulators of the Src family of kinases. The Src family of kinases are nonreceptor tyrosine kinases that regulate inflammation, cell proliferation, motility, and adhesion. To investigate potential histologic lesions associated with systemic loss of Csk gene activity in adult mice, conditional Csk-knockout mice were examined. Cre-mediated systemic excision of Csk induced by tamoxifen treatment resulted in multiorgan inflammation. Specifically, induction of Csk gene excision with three days of tamoxifen treatment resulted in greater than 90% gene excision. Strikingly, these mice developed enteritis that ranged from minimal and suppurative to severe, fibrinonecrosuppurative and hemorrhagic. Other inflammatory lesions included suppurative pneumonia, gastritis, and myocarditis, and increased numbers of inflammatory cells within the hepatic parenchyma. When tamoxifen treatment was reduced from three days to one day in an effort to lower the level of Csk gene excision and limit lesion development, the mice developed severe suppurative to pyogranulomatous pneumonia and minimal to mild suppurative enteritis. Lesions observed secondary to Csk gene excision suggest important roles for Csk in downregulating the proinflammatory activity of the Src family of kinases and limiting neutrophil-mediated inflammation.

The biphenyl derivatives 2 and 3 are prototypes of a novel class of NS5A replication complex inhibitors that demonstrate high inhibitory potency toward a panel of clinically relevant HCV strains encompassing genotypes 1-6. However, these compounds exhibit poor systemic exposure in rat pharmacokinetic studies after oral dosing. The structure-activity relationship investigations that improved the exposure properties of the parent bis-phenylimidazole chemotype, culminating in the identification of the highly potent NS5A replication complex inhibitor daclatasvir (33) are described. An element critical to success was the realization that the arylglycine cap of 2 could be replaced with an alkylglycine derivative and still maintain the high inhibitory potency of the series if accompanied with a stereoinversion, a finding that enabled a rapid optimization of exposure properties. Compound 33 had EC50 values of 50 and 9 pM toward genotype-1a and -1b replicons, respectively, and oral bioavailabilities of 38-108% in preclinical species. Compound 33 provided clinical proof-of-concept for the NS5A replication complex inhibitor class, and regulatory approval to market it with the NS3/4A protease inhibitor asunaprevir for the treatment of HCV genotype-1b infection has recently been sought in Japan.

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown to be efficacious as abortive migraine therapeutics with the absence of cardiovascular liabilities that are associated with triptans. Herein, we report the discovery of a highly potent CGRP receptor antagonist, BMS-742413, with the potential to provide rapid onset of action through intranasal delivery. The compound displays excellent aqueous solubility, oxidative stability, and toxicological profile. BMS-742413 has good intranasal bioavailability in the rabbit and shows a robust, dose-dependent inhibition of CGRP-induced increases in marmoset facial blood flow.

Introducing a uniquely substituted phenyl sulfone into a series of biphenyl imidazole liver X receptor (LXR) agonists afforded a dramatic potency improvement for induction of ATP binding cassette transporters, ABCA1 and ABCG1, in human whole blood. The agonist series demonstrated robust LXRβ activity (>70%) with low partial LXRα agonist activity (

The biology of IGF-IR/IR signaling was studied in normal mouse embryonic fibroblasts (MEFs) that were either wild type (wt), heterozygous (het), or null for the IGF-IR. The ability of IGF-I, IGF-II, or insulin to stimulate serum-starved MEFs was characterized by gene expression profiling and biochemical analyses for activation of downstream signals. Each genotypic group of MEFs exhibited distinct patterns of expression both while resting and in response to stimulation. The insulin receptor (IR) pathway in IGF-IR null MEFs was hypersensitive to insulin ligand stimulation resulting in greater AKT phosphorylation than in wt or het MEFs stimulated with the same ligand. Interestingly, the IR pathway hypersensitivity in IGF-IR null MEFs occurred with no observed changes in the levels of IR isoforms A or B. A new small molecule IGF-IR inhibitor (BMS-754807), having equipotent activity against both IGF-IR and IR, proved effective in suppressing both AKT and ERK phosphorylation from both the IGF-IR and IR pathways by all three ligands tested in wt, het, and null MEFs. The use of a dual IGF-IR/IR inhibitor addresses concerns about the use of growth inhibiting therapies directed against the IGF-IR receptor in certain cancers. Lastly, comparison of the antiproliferative effects (IC50s) of various compounds in wt vs. null MEFs demonstrates that genetically characterized MEFs provide a simple and inexpensive tool with which to define compounds as having mostly on-target or off-target IGF-IR activities because off-target compounds affect both wt and null MEFs equally.

The amino sugar galactosamine (galN) induces alterations in the hepatic uridine nucleotide pool and has been widely used as a model of human viral hepatitis. Histopathological and clinical chemistry analyses of a cohort of rats following administration of galN revealed extreme interindividual variability in the extent of the toxic response which enabled classification of 'responder' and 'non-responder' phenotypes. An integrative metabolic profiling approach was applied to characterize biomarkers of exposure to galN in urine, serum, feces and liver from responders and non-responders. The presence of N-acetylglucosamine and galN in the urine correlated with the occurrence and extent of toxic response. Conversely, the novel identification of galN-pyrazines in the feces of non-responders and their virtual absence in the feces of responders suggests an alternative means of distribution and metabolism of galN in non-responders. The absence of the UDP-hexosamines in the liver of non-responders further supports differential metabolism of galN and suggests an ability of non-responders to avoid UDP-glucose depletion. An observed disturbance of gut microbial derived metabolites in the urine and feces of non-responders may suggest a role of the microflora in reducing the effective dose of galN. This systems level metabonomic approach has provided new mechanistic insights into differential response to galN and is widely applicable to the study of interindividual variation in metabolism for any xenobiotic intervention.

The time-related metabolic effects of 1-cyano-2-hydroxy-3-butene (CHB, crambene), a naturally occurring nitrile and experimental model toxin causing exocrine pancreatitis, have been investigated in rats using high-resolution NMR spectroscopy of urine and serum in combination with pattern recognition analysis. Rats were administered CHB subcutaneously in two doses, 15 mg/kg dose (n = 10) and 150 mg/kg (n = 10), and conventional histopathology and clinical chemistry assessments were performed. Urine samples were collected at − 16 and 0, 8, 24, 48, 72, 96, 120, 144 and 168 h postdosing and serum samples were collected at 48 and 168 h postdosing; these were analyzed using a range of 1D and 2D NMR spectroscopic methods. The metabolic profile perturbations seen throughout the time-course of the study are described, and the application of the spectral correlation technique Statistical TOtal Correlation SpectroscopY (STOCSY) to detect both structural and novel toxicological connectivities between xenobiotic and endogenous metabolite signals is illustrated for the first time. As a result, it is suggested that the STOCSY approach may be of wider application in the identification of toxic versus nontoxic metabolites in drug metabolism studies. Copyright © 2009 John Wiley & Sons, Ltd.

Separation of concentrated bile acids from hepatic parenchymal cells is a key function of the bile duct epithelial cells (BDECs) that form intrahepatic bile ducts. Using coimmunostaining, we found that tissue factor (TF), the principal activator of coagulation, colocalized with cytokeratin 19, a marker of BDECs in the adult mouse liver. BDEC injury induced by xenobiotics such as α-naphthylisothiocyanate (ANIT) causes cholestasis, inflammation, and hepatocellular injury. We tested the hypothesis that acute ANIT-induced cholestatic hepatitis is associated with TF-dependent activation of coagulation and determined the role of TF in ANIT hepatotoxicity. Treatment of mice with ANIT (60 mg/kg) caused multifocal hepatic necrosis and significantly increased serum biomarkers of cholestasis and hepatic parenchymal cell injury. ANIT treatment also significantly increased liver TF expression and activity. ANIT-induced activation of the coagulation cascade was shown by increased plasma thrombin-antithrombin levels and significant deposition of fibrin within the necrotic foci. ANIT-induced coagulation and liver injury were reduced in low-TF mice, which express 1% of normal TF levels. The results indicate that ANIT-induced liver injury is accompanied by TF-dependent activation of the coagulation cascade and that TF contributes to the progression of injury during acute cholestatic hepatitis.

Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S -adenosyl- l -methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.

Objective—To describe the character and frequency of causes of death and associated lesions in long-distance racing sled dogs. Design—Retrospective case series. Animals—23 dogs. Procedures—Medical records of dogs that died during or soon after competition in the Iditarod Trail sled dog races (1994 through 2006) were examined for findings of gross necropsy and histologic evaluation of tissue samples. From the data, descriptive and comparative statistics were obtained. Results—Recognized causes of death included aspiration of gastric contents (n = 4), aspiration pneumonia (4), acute blood loss secondary to gastric ulceration (3), and sled dog myopathy (2). A cause of death was not established for 7 dogs. Prevalent lesions among the study population included rhabdomyolysis (n = 15), enteritis (10), gastritis (10), aspiration pneumonia (8), and gastric ulceration (8). All dogs with aspiration pneumonia had concurrent gastric mucosal lesions. Subjective biventricular cardiac hypertrophy was evident in most dogs; other lesions detected frequently included centrilobular hepatic fibrosis, gastric dilatation, and mild cardiac myodegeneration and necrosis. Conclusions and Clinical Relevance—Unexpected death is a rare event among conditioned sled dogs during competition in endurance races. Potentially life-threatening conditions of dogs that are associated with periods of long-distance physical exertion include aspiration pneumonia, gastric mucosal lesions, and severe rhabdomyolysis. Dogs that develop clinical signs suggestive of these conditions should be excluded from strenuous activities. Epidemiologic investigations are required to clarify the risk for death associated with these lesions in dogs competing in endurance races.

Idiosyncratic liver injury occurs in a small fraction of people on certain drug regimens. The cause of idiosyncratic hepatotoxicity is not known; however, it has been proposed that environmental factors such as concurrent inflammation initiated by bacterial lipopolysaccharide (LPS) increase an individual's susceptibility to drug toxicity. Ranitidine (RAN), a histamine-2 receptor antagonist, causes idiosyncratic liver injury in humans. In a previous report, idiosyncrasy-like liver toxicity was created in rats by cotreating them with LPS and RAN. In the present study, the ability of metabonomic techniques to distinguish animals cotreated with LPS and RAN from those treated with each agent individually was investigated. Rats were treated with LPS or its vehicle and with RAN or its vehicle, and urine was collected for nuclear magnetic resonance (NMR)- and mass spectroscopy-based metabonomic analyses. Blood and liver samples were also collected to compare metabonomic results with clinical chemistry and histopathology. NMR metabonomic analysis indicated changes in the pattern of metabolites consistent with liver damage that occurred only in the LPS/RAN cotreated group. Principal component analysis of urine spectra by either NMR or mass spectroscopy produced a clear separation of the rats treated with LPS/RAN from the other three groups. Clinical chemistry (serum alanine aminotransferase and aspartate aminotransferase activities) and histopathology corroborated these results. These findings support the potential use of a noninvasive metabonomic approach to identify drug candidates with potential to cause idiosyncratic liver toxicity with inflammagen coexposure.

Rats cotreated with lipopolysaccharide (LPS) and ranitidine (RAN) but not LPS and famotidine (FAM) develop hepatocellular injury in an animal model of idiosyncratic drug reactions. Evaluation of liver gene expression in rats given LPS and/or RAN led to confirmation that the hemostatic system, hypoxia, and neutrophils (PMNs) are critical mediators in LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression changes distinguish LPS/RAN-treated rats from rats given LPS or RAN alone and from those cotreated with LPS/FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 x 10(6) endotoxin units/kg, iv) or its vehicle. Two hours thereafter they were given RAN (30 mg/kg, iv), FAM (either 6 mg/kg, a pharmacologically equi-efficacious dose, or 28.8 mg/kg, an equimolar dose, iv), or vehicle. They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity (2 and 6 h) and liver gene expression (2 h only). At a time before the onset of hepatocellular injury, hierarchical clustering distinguished rats treated with LPS/RAN from those given LPS alone. 205 probesets were expressed differentially to a greater or lesser degree only in LPS/RAN-treated rats compared to LPS/FAM or LPS alone, which did not develop liver injury. These included VEGF, EGLN3, MAPKAPK-2, BNIP3, MIP-2, COX-2, EGR-1, PAI-1, IFN-gamma, and IL-6. Expression of these genes was confirmed by real-time PCR. Serum concentrations of MIP-2, PAI-1, IFN-gamma, and IL-6 correlated with their respective gene expression patterns. Overall, the expression of several gene products capable of controlling requisite mediators of injury (i.e., hemostasis, hypoxia, PMNs) in this model were enhanced in livers of LPS/RAN-treated rats. Furthermore, enhanced expression of MAPKAPK-2 in RAN-treated rats and its target genes in LPS/RAN-treated rats suggests that p38/MAPKAPK-2 signaling is a regulation point for enhancement of LPS-induced gene expression by RAN.

In an animal model of drug idiosyncrasy, rats cotreated with nonhepatotoxic doses of lipopolysaccharide (LPS) and ranitidine (RAN) develop hepatocellular injury, whereas rats treated with LPS and famotidine (FAM) do not. The coagulation system and neutrophils (PMNs) are requisite mediators of LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression in LPS/RAN-treated rats requires coagulation system activation and that these changes are absent in rats given LPS and FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 x 10(6) endotoxin units/kg i.v.) or its vehicle, and then 1 h later, they were treated with heparin (3000 U/kg) or its vehicle. One hour thereafter, they were given RAN (30 mg/kg), FAM (6 mg/kg, a pharmacologically equiefficacious dose, or 28.8 mg/kg, an equimolar dose), or vehicle (i.v.). They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity, coagulation system activation, and liver gene expression (2 h only). Statistical filtering of gene array results and real-time polymerase chain reaction identified groups of genes expressed in LPS/RAN-treated rats but not LPS/FAM-treated rats that were either changed or unchanged by heparin administration. For example, LPS/RAN-induced mRNA expression of the inflammatory mediators interleukin-6, cyclooxygenase-2, and macrophage inflammatory protein-2 (MIP-2) was reduced by anticoagulation. Enhancement of serum MIP-2 and plasminogen activator inhibitor-1 concentrations in LPS/RAN-treated rats was prevented by anticoagulation. The results suggest cross-talk between hemostasis-induced gene expression and inflammation (e.g., PMN function) in the genesis of hepatocellular injury in LPS/RAN-treated rats. In contrast, neither the expression of such genes nor hepatocellular necrosis occurred in rats treated with LPS/FAM.

Bovine leukemia virus (BLV) infection causes a significant polyclonal expansion of CD5+, IgM+ B lymphocytes known as persistent lymphocytosis (PL) in approximately 30% of infected cattle. There is evidence that this expanded B cell population has altered signaling, and resistance to apoptosis has been proposed as one mechanism of B cell expansion. In human and murine B cells, antigen binding initiates movement of the B cell receptor (BCR) into membrane microdomains enriched in sphingolipids and cholesterol, termed lipid rafts. Lipid rafts include members of the Src-family kinases and exclude certain phosphatases. Inclusion of the BCR into lipid rafts plays an important role in regulation of early signaling events and subsequent antigen internalization. Viral proteins may also influence signaling events in lipid rafts. Here we demonstrate that the largely CD5+ B cell population in PL cattle has different mobilization and internalization of the BCR when compared to the largely CD5-negative B cells in BLV-negative cattle. Unlike B cells from BLV-negative cattle, the BCR in B cells of BLV-infected, PL cattle resists movement into lipid rafts upon stimulation and is only weakly internalized. Expression of viral proteins as determined by detection of the BLV transmembrane (TM) envelope glycoprotein gp30 did not alter these events in cells from PL cattle. This exclusion of the BCR from lipid rafts may, in part, explain signaling differences seen between B cells of BLV-infected, PL, and BLV-negative cattle and the resistance to apoptosis speculated to contribute to persistent lymphocytosis.

Questions about pathogenesis and therapy for Helicobacter infections in dogs could be answered with a simple, noninvasive, sensitive, and specific diagnostic test. We hypothesized that a fecal polymerase chain reaction (PCR) assay would detect Helicobacter and could be useful for assessing therapeutic responses. Paired gastric biopsies and fecal samples were obtained from 39 random source dogs (group 1). Gastric biopsies from each of these dogs had histologic evidence of gastric spiral bacteria, and paired gastric tissue and fecal samples produced a 375-base pair (bp) product when amplified by PCR with Helicobacter-specific primers. Specificity of the PCR product was confirmed by detection of expected 60-, 119-, and 196-bp products following Hinfl digestion. Direct sequencing of amplicons from paired PCR products from gastric biopsy and fecal samples from 8 group I dogs showed that gastric products had the highest homologies with known gastric Helicobacter species, whereas fecal products had the highest homologies with intestinal species. Healthy mixed-breed dogs (group II; n = 8) with histologically confirmed spiral bacteria infection were treated with a 21-day course of metronidazole, amoxicillin, and famotidine. Fecal samples were collected from group II dogs twice before and within 3 days of completion of treatment. The PCR results correctly identified 15/16 pretreatment samples as positive: 1 pretreatment sample was negative. PCR results identified 8/8 posttreatment samples as Helicobacter negative. Fecal PCR is a useful test for detecting Helicobacter infection in dogs. This assay may be useful as a screening test for infection and could be used to address questions relevant to pathogenesis and therapy.

Liver X Receptors (LXRs) α and β are nuclear hormone receptors that regulate multiple genes involved in reverse cholesterol transport (RCT) and are potential drug targets for atherosclerosis. However, full pan agonists also activate lipogenic genes, resulting in elevated plasma and hepatic lipids. We report the pharmacology of BMS-779788 [2-(2-(1-(2-chlorophenyl)-1-methylethyl)-1-(3'-(methylsulfonyl)-4-biphenylyl)-1H-imidazol-4-yl)-2-propanol], a potent partial LXR agonist with LXRβ selectivity, which has an improved therapeutic window in the cynomolgus monkey compared with a full pan agonist. BMS-779788 induced LXR target genes in blood in vivo with an EC50 = 610 nM, a value similar to its in vitro blood gene induction potency. BMS-779788 was 29- and 12-fold less potent than the full agonist T0901317 in elevating plasma triglyceride and LDL cholesterol, respectively, with similar results for plasma cholesteryl ester transfer protein and apolipoprotein B. However, ABCA1 and ABCG1 mRNA inductions in blood, which are critical for RCT, were comparable. Increased liver triglyceride was observed after 7-day treatment with BMS-779788 at the highest dose tested and was nearly identical to the dose response for plasma triglyceride, consistent with the central role of liver LXR in these lipogenic effects. Dose-dependent increases in biliary cholesterol and decreases in phospholipid and bile acid occurred in BMS-779788-treated animals, similar to LXR agonist effects reported in mouse. In summary, BMS-779788, a partial LXRβ selective agonist, has decreased lipogenic potential compared with a full pan agonist in cynomolgus monkeys, with similar potency in the induction of genes known to stimulate RCT. This provides support in nonhuman primates for improving LXR agonist therapeutic windows by limiting LXRα activity.

A 9-year-old cat with hyperthyroidism was referred for radioactive iodine treatment. The cat also had a ventral cervical mass that the owners reported had been present for several years and had increased in size during the past few weeks. On physical examination, the mass was found to have caused lateral displacement of the trachea, esophagus, jugular vein, and common carotid artery. The mass was aspirated and was determined to be cystic in nature. Concentrations of thyroid hormones in the cystic fluid were similar to serum concentrations, and nuclear scintigraphy revealed thyroactive tissue lining the cyst wall. Magnetic resonance imaging suggested that the cyst originated from the right lobe of the thyroid gland. The cat was treated with sodium iodide I 131 but died 4 days later, presumably as a result of aspiration of gastric or esophageal contents. A necropsy was not performed, but histologic examination of a biopsy specimen of the mass indicated that it was a cystic thyroid adenoma.

Bovine leukemia virus (BLV), a retrovirus related to human T-cell leukemia virus types 1 and 2, can induce persistent nonneoplastic expansion of the CD5+B-cell population, termed persistent lymphocytosis (PL). As in human CD5+B cells, we report here that CD5 was physically associated with the B-cell receptor (BCR) in normal bovine CD5+B cells. In contrast, in CD5+B cells from BLV-infected PL cattle, CD5 was dissociated from the BCR. In B cells from PL cattle, apoptosis decreased when cells were stimulated with antibody to surface immunoglobulin M (sIgM), while in B cells from uninfected cattle, apoptosis increased after sIgM stimulation. The functional significance of the CD5-BCR association was suggested by experimental dissociation of the CD5-BCR interaction by cross-linking of CD5. This caused CD5+B cells from uninfected animals to decrease apoptosis when stimulated with anti-sIgM. In contrast, in CD5+B cells from PL animals, in which CD5 was already dissociated from the BCR, there was no statistically significant change in apoptosis when CD5 was cross-linked and the cells were stimulated with anti-sIgM. Disruption of CD5-BCR interactions and subsequent decreased apoptosis and increased survival in antigenically stimulated B cells may be a mechanism of BLV-induced PL.

Using serial section examination, we studied the conduction system in five Alaskan sled dogs that died suddenly: four during the Iditarod race and one during training. We compared our findings with the conduction system of three sled dogs of similar age that died of natural causes unrelated to the cardiovascular system. The conduction system of sudden death dogs revealed marked fibrosis of the sinoatrial (SA) node and/or its approaches and narrowing of the SA nodal artery in 3 dogs, fibrosis and marked fatty infiltration in and around the AV node in all 5, total isolation and/or tenuous connection of the AV node with its approaches in 4, fat and fibrosis in the A V bundle and bundle branches to a varying degree in all, and focal fibrotic scars in the left ventricle with fat and/or some disarray in 3. The control group revealed mild fibro-fatty changes in the conduction system without fibrotic scar areas in the heart. These findings are similar to the pathological findings in and around the conduction system in cases of sudden death in humans, especially trained athletes. These changes may form an anatomical substrate for an arrhythmic event in susceptible dogs during an altered physiological state.

Pathologists play a key role in discovery pathology and discovery toxicology in the pharmaceutical industry. Discovery pathologists function as well-rounded whole-body biologists. Their unique skill set provides critical perspectives on a wide variety of issues for the successful development of a new pharmaceutical candidate. These include identifying, characterizing, and validating animal models of disease and efficacy, phenotyping genetically engineered rodents, designing experiments to assess whether exaggerated pharmacology results in toxicity, and determining whether target expression in animals is representative of what is known in humans. These skills can be practiced as part of a discovery toxicology group, in which classical toxicologists work together with pathologists, in vitro biologists, cell and molecular biologists, genotoxicologists, cardiologists, electrophysiologists, bioinformaticians and genomicists, reproductive biologists, immunologists, and others. This multidisciplinary approach can often identify toxicologic liabilities at an early stage of drug discovery or development and provide insight into how to avoid or manage these liabilities.

Patterns of change of endogenous metabolites may closely reflect systemic and organ-specific toxic changes. The authors examined the metabolic effects of the cyanobacterial (blue-green algal) toxin microcystin-LR by 1H-nuclear magnetic resonance (NMR) analysis of urinary endogenous metabolites. Rats were treated with a single sublethal dose, either 20 or 80 µg/kg intraperitoneally, and sacrificed at 2 or 7 days post dosing. Changes in the high-dose, 2-day sacrifice group included centrilobular hepatic necrosis and congestion, accompanied in some animals by regeneration and neovascularization. By 7 days, animals had recovered, the necrotizing process had ended, and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. There was considerable interanimal variation in the histologic process and severity, which correlated with the changes in patterns of endogenous metabolites in the urine, thus providing additional validation of the biomarker and biochemical changes. Similarity of the shape of the metabolic trajectories suggests that the mechanisms of toxic effects and recovery are similar among the individual animals, albeit that the magnitude and timing are different for the individual animals. Initial decreases in urinary citrate, 2-oxoglutarate, succinate, and hippurate concentrations were accompanied by a temporary increase in betaine and taurine, then creatine from 24 to 48 hours. Further changes were an increase in guanidinoacetate, dimethylglycine, urocanic acid, and bile acids. As a tool, urine can be repeatedly and noninvasively sampled and metabonomics utilized to study the onset and recovery after toxicity, thus identifying time points of maximal effect. This can help to employ histopathological examination in a guided and effective fashion.

Chronic cholestatic liver injury induced by cholestasis in rodents is associated with hepatic fibrin deposition, and we found evidence of fibrin deposition in livers of patients with cholestasis. Key components of the fibrinolytic pathway modulate cholestatic liver injury by regulating activation of hepatocyte growth factor. However, the exact role of hepatic fibrin deposition in chronic cholestasis is not known. We tested the hypothesis that fibrinogen (Fbg) deficiency worsens liver injury induced by cholestasis. Fbg-deficient mice (Fbgα(-/-) mice) and heterozygous control mice (Fbgα(+/-) mice) were fed either the control diet or a diet containing 0.025% α-naphthylisothiocyanate (ANIT), which selectively injures bile duct epithelial cells in the liver, for 2 weeks. Hepatic fibrin and collagen deposits were evident in livers of heterozygous control mice fed the ANIT diet. Complete Fbg deficiency was associated with elevated serum bile acids, periportal necrosis, and increased serum alanine aminotransferase activity in mice fed the ANIT diet. Fbg deficiency was associated with enhanced hepatic expression of the transcription factor early growth response-1 (Egr-1) and enhanced induction of genes encoding the Egr-1-regulated proinflammatory chemokines monocyte chemotactic protein-1, KC growth-regulated protein, and macrophage inflammatory protein-2. Interestingly, peribiliary collagen deposition was not evident near necrotic areas in Fbg-deficient mice. The results suggest that in this model of chronic cholestasis, fibrin constrains the release of bile constituents from injured intrahepatic bile ducts, thereby limiting the progression of hepatic inflammation and hepatocellular injury.

Antibody from cattle immunized with purified major surface protein-1 (MSP-1) was demonstrated to significantly enhance phagocytosis of Florida strain Anaplasma marginale by bovine macrophages in vitro. Serum immunoglobulin from individual MSP-1 immunized, protected cattle varied in ability to promote phagocytosis, however all sera were significantly opsonic as compared with sera from sham immunized control cattle.

Cholestatic liver injury induced by alpha-naphthylisothiocyanate (ANIT) is provoked by injury to intrahepatic bile ducts and the progression of hepatic necrosis requires the procoagulant protein tissue factor (TF) and extrahepatic cells including neutrophils. Recent studies have shown that myeloid cell TF contributes to neutrophil activation. We tested the hypothesis that myeloid cell TF contributes to neutrophil activation in ANIT-treated mice. TF activity in liver homogenates increased significantly in TFflox/flox mice treated with ANIT, but not in TFflox/flox/LysMCre mice (TFΔMyeloid mice), which have reduced TF expression in monocytes/macrophages and neutrophils. Myeloid cell-specific TF deficiency did not alter expression of the chemokines KC or MIP-2, but reduced hepatic neutrophil accumulation in ANIT-treated mice at 48 hours as indicated by tissue myeloperoxidase (MPO) activity. Myeloid cell TF deficiency significantly reduced CD11b expression by blood neutrophils in ANIT-treated mice and this was associated with reduced plasma MPO protein levels, an index of neutrophil degranulation. However, myeloid cell-specific TF deficiency had no effect on ANIT-induced coagulation cascade activation. The increase in serum ALT and ALP activities in ANIT-treated mice was reduced by myeloid cell TF deficiency (p

2-Bromoethanamine (BEA) causes renal papillary necrosis (RPN) in rats after a single dose and has been widely used as a model compound for studying the lesion. Although the metabolism of BEA may be an important determinant of toxicity, the metabolic fate of the compound has not been fully elucidated. To date, the only identified BEA metabolites are aziridine, 2-oxazolidone and 5-hydroxy-2-oxazolidone. In this study, stable isotope labelling (SIL) of BEA analogs ((¹³C and ²H) were used to differentiate generated BEA metabolites from endogenous molecules which enabled the accurate liquid chromatography mass spectrometry detection of more than 180 novel metabolites. BEA metabolism was evaluated in rats after acute administration of a non-toxic dose (50 mg/kg) and a toxic dose (250 mg/kg) that caused frank RPN and polyuria. Newly identified metabolites include three carbamoylation products, two mercapturic acids and a group of amino acid conjugates. Overall, the results indicate that BEA metabolism is very complex, suggest the potential formation of reactive intermediates and establish that BEA is subject to conjugation with glutathione. The results also demonstrate the utility and sensitivity of the SIL approach for identification of metabolites from small, reactive compounds.

Chronic analgesic abuse has been shown to induce severe renal injury characterized by renal papillary necrosis (RPN), an injury detectable at late stage. While direct toxicity of the drug may exist, the molecular mechanisms underlying analgesics induction of RPN remain unknown. A major limitation to study the pathogenesis of RPN is the required chronic exposure before detection of injury. Here, we employed 2-bromoethanamine (BEA) to simulate rapid papillary toxicity using inner medullary collecting duct (IMCD3) cells. Although exposure to 10μM BEA had no effect on cellular viability under isotonic conditions, a 50% loss in cell viability was observed in the first 24 h when cells were subjected to sublethal hypertonic stress and nearly complete cell death after 48 h suggesting that BEA exerts cytotoxicity only under hypertonic conditions. Because TonEBP is a transcription factor critical for cell survival during hypertonic conditions, we undertook experiments to examine the effect of BEA on TonEBP expression and activity. Exposure of cells to 10μM BEA resulted in a substantial reduction in TonEBP protein expression after 24 h. In addition, TonEBP was not translocated to the nucleus in BEA-treated IMCD3 cells under acute hypertonic stress for transcription of target genes essential for osmolyte accumulation. Finally, we found a substantial decrease in TonEBP expression in medullary kidney tissues of mice injected with a single ip dose of BEA. Our data suggest that TonEBP is a potential target for BEA leading to the process of papillary necrosis in the settings of hypertonic stress.

Tax, the trans-activating protein of bovine leukemia virus, stimulates the long terminal repeat to promote viral transcription and also activates cellular genes that may be involved in tumorigenesis. To study Tax regulation, we identified antisense oligodeoxynucleotides that inhibit tax translation in rabbit reticulocyte lysate. Two antisense oligonucleotides directed toward the 5' end of tax RNA inhibited translation by 59% and 45%, when compared to the effect of a random sequence oligonucleotide. This inhibitory effect was independent of RNase H. In contrast, antisense directed at the middle of the tax RNA inhibited by only 12%, but, in the presence of RNase H, inhibited 38%. An antisense oligonucleotide directed at the 3' portion of tax RNA was not inhibitory and, in fact, stimulated translation. Identification of these inhibitory antisense sequences may allow elucidation of the biological role of Tax in BLV-persistent lymphocytosis and tumorigenesis.

BMS-754807 is a potent and reversible inhibitor of the insulin-like growth factor 1 receptor/insulin receptor family kinases (Ki

Acetaminophen (APAP)-induced hepatotoxicity accounts for nearly half of acute liver failure cases in the United States. The doses that produce hepatotoxicity vary considerably and many risk factors have been proposed, including liver inflammation from viral hepatitis. Interestingly, inflammatory stress from another stimulus, bacterial endotoxin (lipopolysaccharide, LPS), renders the liver more sensitive to hepatotoxicity from numerous xenobiotic agents. The purpose of these studies was to test the hypothesis that inflammation induced by LPS or infection with reovirus increases sensitivity to APAP-induced liver injury. For LPS-induced inflammation, C57BL/6J mice were treated with either saline or LPS (44 x 10(6) EU/kg, ip) 2 h before treatment with APAP (100-400 mg/kg, ip) or saline. No elevation in serum alanine aminotransferase (ALT) activity was observed in mice that received vehicle or LPS alone. LPS co-treatment produced a leftward shift of the dose-response curve for APAP-induced hepatotoxicity and led to significantly greater tumor necrosis factor-alpha (TNF) production than APAP alone. Reovirus serotype 1 (10(8) PFU, iv) induced inflammation in Balb/c mice as evidenced by increases in hepatic mRNAs for macrophage inhibitory protein-2, interleukin-6, and TNF. Co-administration of reovirus and APAP at doses of 450 and 700 mg/kg (2 h after reovirus) led to increases in serum ALT activity, whereas neither reovirus nor APAP alone produced liver injury. Consistent with the increases in serum ALT activity, histopathologic examination revealed centrilobular necrosis with marked neutrophilic accumulation only in livers of mice treated with LPS/APAP or with reovirus/APAP. The results suggest that normally noninjurious doses of APAP are rendered hepatotoxic by modest inflammation, whether bacterial or viral in origin.

The time-related metabolic effects of 1-cyano-2-hydroxy-3-butene (CHB, crambene), a naturally occurring nitrile and experimental model toxin causing exocrine pancreatitis, have been investigated in rats using high-resolution NMR spectroscopy of urine and serum in combination with pattern recognition analysis. Rats were administered CHB subcutaneously in two doses, 15 mg/kg dose (n = 10) and 150 mg/kg (n = 10), and conventional histopathology and clinical chemistry assessments were performed. Urine samples were collected at - 16 and 0, 8, 24, 48, 72, 96, 120, 144 and 168 h postdosing and serum samples were collected at 48 and 168 h postdosing; these were analyzed using a range of 1D and 2D NMR spectroscopic methods. The metabolic profile perturbations seen throughout the time-course of the study are described, and the application of the spectral correlation technique Statistical TOtal Correlation SpectroscopY (STOCSY) to detect both structural and novel toxicological connectivities between xenobiotic and endogenous metabolite signals is illustrated for the first time. As a result, it is suggested that the STOCSY approach may be of wider application in the identification of toxic versus nontoxic metabolites in drug metabolism studies.

Tissue injury and repair are often overlapping consequences of disease or toxic exposure, but are not often considered as distinct processes in molecular studies. To establish the systemic metabolic response to liver regeneration, the partial hepatectomy (PH) model has been studied in the rat by an integrated metabonomics strategy, utilizing (1)H NMR spectroscopy of urine, liver and serum. Male Sprague-Dawley rats were subjected to either surgical removal of approximately two-thirds of the liver, sham operated (SO) surgery, or no treatment (n = 10/group) and samples collected over a 7 day period. A number of urinary metabolic perturbations were observed in PH rats compared with SO and control animals, including elevated levels of taurine, hypotaurine, creatine, guanidinoacetic acid, betaine, dimethylglycine and bile acids. Serum betaine and creatine were also elevated after PH, while levels of triglyceride were reduced. In the liver, triglycerides, cholesterol, alanine and betaine were elevated after PH, while choline and its derivatives were reduced. Upon examining the dynamic pattern of urinary response (the 'metabolic trajectory'), several metabolites could be categorized into groups likely to reflect perturbations to different processes such as dietary intake or hepatic 1-carbon metabolism. Several of the urinary perturbations observed during the regenerative phase of the PH model have also been observed after exposure to liver toxins, indicating that hepatic regeneration may make a contribution to the systemic alterations in metabolism associated with hepatotoxicity. The observed changes in 1-carbon and lipid metabolism are consistent with the proposed role of these pathways in the activation of a regenerative response and provide further evidence regarding the utility of urinary NMR profiles in the detection of liver-specific pathology. Biofluid (1)H NMR-based metabolic profiling provides new insight into the role of metabolism of liver regeneration, and suggests putative biomarkers for the noninvasive monitoring of the regeneration process.

Summary recommendations for standardization and reporting of metabolic analyses : The Standard Metabolic Reporting Structures (SMRS) working group outlines its vision for an open,community-driven specification for the standardization and reporting of metabolic studies

Interspecies variation between rats and mice has been studied for hydrazine toxicity using a novel metabonomics approach. Hydrazine hydrochloride was administered to male Sprague-Dawley rats (30 mg/kg, n = 10 and 90 mg/kg, n = 10) and male B6C3F mice (100 mg/kg, n = 8 and 250 mg/kg, n = 8) by oral gavage. In each species, the high dose was selected to produce the major histopathologic effect, hepatocellular lipid accumulation. Urine samples were collected at sequential time points up to 168 h post dose and analyzed by 1H NMR spectroscopy. The metabolites of hydrazine, namely diacetyl hydrazine and 1,4,5,6-tetrahydro-6-oxo-3-pyridazine carboxylic acid (THOPC), were detected in both the rat and mouse urine samples. Monoacetyl hydrazine was detected only in urine samples from the rat and its absence in the urine of the mouse was attributed to a higher activity of N-acetyl transferases in the mouse compared with the rat. Differential metabolic effects observed between the two species included elevated urinary beta-alanine, 3-D-hydroxybutyrate, citrulline, N-acetylcitrulline, and reduced trimethylamine-N-oxide excretion unique to the rat. Metabolic principal component (PC) trajectories highlighted the greater degree of toxic response in the rat. A data scaling method, scaled to maximum aligned and reduced trajectories (SMART) analysis, was used to remove the differences between the metabolic starting positions of the rat and mouse and varying magnitudes of effect, to facilitate comparison of the response geometries between the rat and mouse. Mice followed "biphasic" open PC trajectories, with incomplete recovery 7 days after dosing, whereas rats followed closed "hairpin" time profiles, indicating functional reversibility. The greater magnitude of metabolic effects observed in the rat was supported by the more pronounced effect on liver pathology in the rat when compared with the mouse.

The role that metabonomics has in the evaluation of xenobiotic toxicity studies is presented here together with a brief summary of published studies. To provide a comprehensive assessment of this approach, the Consortium for Metabonomic Toxicology (COMET) has been formed between six pharmaceutical companies and Imperial College of Science, Technology and Medicine (IC), London, UK. The objective of this group is to define methodologies and to apply metabonomic data generated using H-1 NMR spectroscopy of urine and blood serum for preclinical toxicological screening of candidate drugs. This is being achieved by generating databases of results for a wide range of model toxins which serve as the raw material for computer-based expert systems for toxicity prediction. The project progress on the generation of comprehensive metabonomic databases and multivariate statistical models for prediction of toxicity, initially for Ever and kidney toxicity in the rat and mouse, is reported. Additionally, both the analytical and biological variation which might arise through the use of metabonomics has been evaluated. An evaluation of intersite NMR analytical reproducibility has revealed a high degree of robustness. Second, a detailed comparison has been made of the ability of the six companies to provide consistent urine and serum samples using a study of the toxicity of hydrazine at two doses in the male rat, this study showing a high degree of consistency between samples from the various companies in terms of spectral patterns and biochemical composition. Differences between samples from the various companies were small compared to the biochemical effects of the toxin. A metabonomic model has been constructed for urine from control rats, enabling identification of outlier samples and the metabolic reasons for the deviation. Building on this success, and with the completion of studies on approximately 80 model toxins, first expert systems for prediction of liver and kidney toxicity have been generated. (C) 2003 Elsevier Science (USA). All rights reserved.

Bovine leukemia virus (BLV) causes persistent lymphocytosis, a preneoplastic, polyclonal expansion of B lymphocytes. The expansion increases viral transmission to new hosts, but the mechanisms of this expansion have not been determined. We hypothesized that BLV infection contributes to B-cell expansion by signaling initiated via viral transmembrane protein motifs undergoing tyrosine phosphorylation. Viral mimicry of host cell proteins is a well-demonstrated mechanism by which viruses may increase propagation or decrease recognition by the host immune system. The cytoplasmic tail of BLV transmembrane protein gp30 (TM) has multiple areas of homology to motifs of host cell signaling proteins, including two immunoreceptor tyrosine-based activation motifs (ITAMs) and two immunoreceptor tyrosine-based inhibition motifs (ITIMs), which are homologous to B-cell receptor and inhibitory co-receptor motifs. Signaling by these motifs in B cells typically relies on tyrosine phosphorylation, followed by interactions with Src-homology-2 (SH2) domains of nonreceptor protein tyrosine kinases or phosphatases. Phosphorylation of tyrosine residues in the cytoplasmic tail of TM was tested in four systems including ex vivo cultured peripheral blood mononuclear cells from BLV infected cows, BLV-expressing fetal lamb kidney cell and bat lung cell lines, and DT40 B cells transfected with a fusion of mouse extracellular CD8alpha and cytoplasmic TM. No phosphorylation of TM was detected in our experiments in any of the cell types utilized, or with various stimulation methods. Detection was attempted by immunoblotting for phosphotyrosines, or by metabolic labeling of cells. Thus BLV TM is not likely to modify host signal pathways through interactions between phosphorylated tyrosines of the ITAM or ITIM motifs and host-cell tyrosine kinases or phosphatases.

A 9-year-old spayed female Golden Retriever was examined because of progressive hind limb lameness. Magnetic resonance imaging of the thoracic and lumbar portions of the vertebral column revealed a focal, contrast-enhancing, intramedullary spinal cord mass. The history, signalment, and magnetic resonance findings were suggestive of spinal cord neoplasia. A hemilaminectomy, durotomy, and longitudinal myelotomy were performed, and a 1 X 1-cm mass that contained numerous blood vessels was removed with blunt dissection. Results of histologic examination and immunohistochemical staining of the mass suggested that it was a hamartoma. The dog improved after surgery, with no evidence of a recurrence of clinical signs 14 months after surgery. Vascular malformations of the CNS in dogs include hamartomas, hemangiomas, angiomas, hemangioblastomas, meningocerebral hemangiomatosis, and arteriovenous malformations. A hamartoma is a non-neoplastic overgrowth of cells or an improper proportion of cells that are normally in the involved tissue. Although magnetic resonance imaging may be helpful in determining the extent of the lesion in dogs with vascular malforrmations, it cannot be used to distinguish neoplastic from non-neoplastic formations. Excision may result in a good outcome for dogs with an intramedullary spinal cord hamartoma.