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[Image: see text] Two novel compounds were identified as Naa50 binders/inhibitors using DNA-encoded technology screening. Biophysical and biochemical data as well as cocrystal structures were obtained for both compounds (3a and 4a) to understand their mechanism of action. These data were also used to rationalize the binding affinity differences observed between the two compounds and a MLGP peptide-containing substrate. Cellular target engagement experiments further confirm the Naa50 binding of 4a and demonstrate its selectivity toward related enzymes (Naa10 and Naa60). Additional analogs of inhibitor 4a were also evaluated to study the binding mode observed in the cocrystal structures.

Previous research has shown that postpartum administration of the nonsteroidal antiinflammatory drug (NSAID) sodium salicylate can increase 305-d milk yield in older dairy cattle (parity 3 and greater). However, in this prior work, sodium salicylate was delivered to cows via the drinking water, a method that does not align well with current grouping strategies on commercial dairy farms. The objective of the current study was to replicate these results on a commercial dairy farm with a simplified treatment protocol and to compare sodium salicylate with another NSAID, meloxicam. Dairy cattle in their second lactation and greater (n=51/treatment) were alternately assigned to 1 of 3 treatments at parturition, with treatments lasting for 3d. Experimental treatments began 12 to 36 h after parturition and were (1) 1 placebo bolus on the first day and 3 consecutive daily drenches of sodium salicylate (125 g/cow per day; SAL); (2) 1 bolus of meloxicam (675 mg/cow) and 3 drenches of an equal volume of water (MEL); or (3) 1 placebo bolus and 3 drenches of water (CON). Blood samples were collected on the first day of treatment, immediately following the last day of treatment, and 7d after the last day of treatment; plasma was analyzed for glucose, β-hydroxybutyrate (BHB), free fatty acids, haptoglobin, and paraoxonase. Milk production, body condition score, reproductive status, and retention in the herd were monitored for 365 d posttreatment, and effects of treatment, parity, days in milk, and interactions were evaluated in mixed effects models. Significance was declared at P

The objective of this study was to determine the pharmacokinetics of intravenous and oral firocoxib in 10 healthy preweaned calves. Firocoxib (0.5 mg/kg) was initially administered i.v. to calves, and following a 14-day washout period, animals received firocoxib orally prior to cautery dehorning. Firocoxib concentrations were determined by liquid chromatography-tandem mass spectrometry. Changes in hematology and plasma chemistry were determined using automated methods. Computer software was used to estimate pharmacokinetic parameters best described with a two-compartment model for i.v. administration and a one-compartment model for p.o. administration. Following i.v. dosing, the geometric mean (range) T1/2K10 and T1/2β were 6.7 (4.6-9.7) and 37.2 (23.5-160.4) h, respectively, Vss was 3.10 (2.10-7.22) L/kg, and CL was 121.7 (100.1-156.7) mL/h/kg. Following oral administration, geometric mean (range) Cmax was 127.9 (102.5-151.3) ng/mL, Tmax was 4.0 (2.6-5.6) h, and T1/2K10 was 18.8 (14.2-25.5) h. Bioavailability of oral firocoxib was calculated using the AUC derived from both study populations to be 98.4% (83.1-117.6%). No adverse clinical effects were evident following firocoxib administration. Pharmacokinetic analysis of i.v. and p.o. firocoxib indicates high bioavailability and a prolonged terminal half-life in preweaned calves.

Management of neuropathic pain in dairy cattle could be achieved by combination therapy of gabapentin, a GABA analog and meloxicam, an nonsteroidal anti-inflammatory drug. This study was designed to determine specifically the depletion of these drugs into milk. Six animals received meloxicam at 1 mg/kg and gabapentin at 10 mg/kg, while another group (n=6) received meloxicam at 1 mg/kg and gabapentin at 20 mg/kg. Plasma and milk drug concentrations were determined over 7 days postadministration by HPLC/MS followed by noncompartmental pharmacokinetic analyses. The mean (±SD) plasma C(max) and T(max) for meloxicam (2.89±0.48 μg/mL and 11.33±4.12 h) were not much different from gabapentin at 10 mg/kg (2.87±0.2 μg/mL and 8±0 h). The mean (±SD) milk C(max) for meloxicam (0.41±80.16 μg/mL) was comparable to gabapentin at 10 mg/kg (0.63±0.13 μg/mL and 12±6.69 h). The mean plasma and milk C(max) for gabapentin at 20 mg/kg p.o. were almost double the values at 10 mg/kg. The mean (±SD) milk to plasma ratio for meloxicam (0.14±0.04) was lower than for gabapentin (0.23±0.06). The results of this study suggest that milk from treated cows will have low drug residue concentration soon after plasma drug concentrations have fallen below effective levels.

Al-Bataineh, M. M., van der Merwe, D., Schultz, B. D., Gehring, R. Molecular and functional identification of organic anion transporter isoforms in cultured bovine mammary epithelial cells (BME-UV). J. vet. Pharmacol. Therap. 35, 209–215. Mammary epithelial cells express a diversity of membrane transporters including members of organic cation and organic anion (OAT) transporter subfamilies. Four mammal OAT isoforms have been identified: OAT-1, OAT-2, OAT-3, and OAT-4. The pharmacological significance of OAT isoforms has been emphasized because of their role in the movement of a wide variety of substrates across epithelial barriers. The present study identified (molecularly and functionally) bovine OAT isoforms in bovine mammary epithelial (BME-UV) cells. mRNA expression levels of all tested transporters in BME-UV cells were less than expression levels of the corresponding transporters in bovine kidney. Directionality in the flux of P-aminohippuric acid and acetylsalicylate, compounds known to interact with OAT-1 and OAT-2, respectively, across BME-UV monolayers was not observed at the concentrations used in this study. Directionality was, however, observed in the flux of estrone sulfate (EsS). Adding probenecid, penicillin G or nonradiolabeled EsS to the apical donor compartment significantly increased the apical-to-basolateral flux of EsS across the BME-UV monolayer. These results suggest that BME-UV cells express an organic anion transport system, making it a potentially useful model to study the role of this transport system in the mammary epithelial barrier.

A novel class of heat shock protein 90 (Hsp90) inhibitors was discovered by high-throughput screening and was subsequently optimized using a combination of structure-based design, parallel synthesis, and the application of medicinal chemistry principles. Through this process, the biochemical and cell-based potency of the original HTS lead were substantially improved along with the corresponding metabolic stability properties. These efforts culminated with the identification of a development candidate (compound 42) which displayed desired PK/PD relationships, significant efficacy in a melanoma A2058 xenograft tumor model, and attractive DMPK profiles.

Bergamasco, L., Coetzee, J. F., Gehring, R., Murray, L., Song, T., Mosher, R. A. Effect of intravenous sodium salicylate administration prior to castration on plasma cortisol and electroencephalography parameters in calves. J. vet. Pharmacol. Therap. 34, 565–576. Nociception is an unavoidable consequence of many routine management procedures such as castration in cattle. This study investigated electroencephalography (EEG) parameters and cortisol levels in calves receiving intravenous sodium salicylate in response to a castration model. Twelve Holstein calves were randomly assigned to the following groups: (i) castrated, untreated controls, (ii) 50 mg/kg sodium salicylate IV precastration, were blood sampled at 0, 5, 10, 20, 30, 45, 60, 90, 120, 150, 180, 240, 360, and 480 min postcastration. The EEG recording included baseline, castration, immediate recovery (0–5 min after castration), middle recovery (5–10 min after castration), and late recovery (10–20 min after castration). Samples were analyzed by competitive chemiluminescent immunoassay and fluorescence polarization immunoassay for cortisol and salicylate, respectively. EEG visual inspection and spectral analysis were performed. Statistical analyses included anova repeated measures and correlations between response variable. No treatment effect was noted between the two groups for cortisol and EEG measurements, namely an attenuation of acute cortisol response and EEG desynchronization in sodium salicylate group. Time effects were noted for EEG measurements, cortisol and salicylates levels. Significant correlations between cortisol and EEG parameters were noted. These findings have implications for designing effective analgesic regimens, and they suggest that EEG can be useful to monitor pain attributable to castration.

In order to determine the absolute scale of the neutrino mass with a sensitivity of 0.2 (90% Confidence Level), the Karlsruhe Tritium Neutrino experiment (KATRIN) operates a series of superconducting magnet systems, which guide the electrons adiabatically from the source of tritium beta-decay to the detector within a magnetic flux of 191 . The 7 m long Cryogenic Pumping Section (CPS) is designed as the final barrier of tritium circulation. It has to reduce the tritium partial pressure below Pa in order to limit the background count rate in the measurement. To achieve this, the tritium entering the CPS must be adsorbed onto a pre-condensed argon layer on the inner surface of the beam tube at a temperature of 3 K. The zigzag arrangement of the magnet modules increases the efficiency of tritium retention, but makes the transition of the magnetic flux rather complicated. The solenoids are operated in persistent mode with a central magnetic flux density of 5.6 T. The field drop of the magnet has to be less than 0.1% over one month. This report describes the design of the CPS and the current status of the project.

Reinbold, J. B., Coetzee, J. F., Gehring, R., Havel, J. A., Hollis, L. C., Olson, K. C., Apley, M. D. Plasma pharmacokinetics of oral chlortetracycline in group fed, ruminating, Holstein steers in a feedlot setting. J. vet. Pharmacol. Therap. 33, 76–83. Chlortetracycline HCl (CTC) has impacted profitable livestock production since 1945. However, pharmacokinetic parameters for CTC in ruminating cattle are unavailable in peer-reviewed literature. A total of 18 steers were randomized to 4.4, 11, or 22 mg/kg/day p.o. CTC treatment groups (n = 6). Chlortetracycline treatment was offered as one-half of the daily dose b.i.d. (160 total doses/group) for 80 days. Blood samples were collected at selected time points throughout an 83-day study and analyzed with a solid phase extraction technique and novel ultrahigh performance liquid chromatography-mass spectroscopy/mass spectroscopy analytical method. Noncompartmental analysis (NCA) determined individual pharmacokinetic parameters by treatment group with coefficient of variation (CV %) estimates. A one-compartment open model with first order absorption and elimination, where absorption rate constant was equal to elimination rate constant, was fitted using nonlinear mixed effects modeling (NLMEM). NLMEM determined the primary pharmacokinetic parameters: volume of distribution (V/F, 40.9 L/kg) and rate constant (k, 0.0478 h−1), and the secondary parameters: dose-normalized area under the curve (AUC/D, 0.29 h·μg/L), clearance (Cl/F, 1.8 L/kg/h), elimination half-life (t1/2, 16.2 h), Cmax/Dose (4.5 ng/mL), and time of Cmax (Tmax, 23.3 h) with improved CV estimates over NCA. Dose linearity was confirmed by anova of parameters derived from NCA by treatment group. Further studies are necessary for determining absolute bioavailability and pharmacokinetic–pharmacodynamic relationships of CTC in group fed, ruminating cattle.

The discovery and optimization of potency and metabolic stability of a novel class of dihyroxyphenylisoindoline amides as Hsp90 inhibitors are presented. Optimization of a screening hit using structure-based design and modification of log D and chemical structural features led to the identification of a class of orally bioavailable non-quinone-containing Hsp90 inhibitors. This class is exemplified by 14 and 15, which possess improved cell potency and pharmacokinetic profiles compared with the original screening hit.

As part of an oncology chemistry program directed toward discovery of orally bioavailable inhibitors of the 90 kDa heat shock protein (Hsp90), several solution-phase libraries were designed and prepared. A 2 x 89 library of racemic resorcinol amides was prepared affording 131 purified compounds. After evaluation in a binding assay, followed by an AKT-Luminex cellular assay, three potent analogs had functional activity between 0.1 and 0.3 microM. Resolution by preparative chiral SFC chromatography led to (+)-15, (+)-16, and (+)-17 having functional IC(50) = 27, 43, and 190 nM, respectively. (+)-15 exhibited high clearance in human hepatocytes driven primarily by glucuronidation as confirmed by metabolite identification. A second 8 x 14 exploratory library was designed to investigate heterocyclic replacements of the resorcinol ring. The second library highlights the use of the (-)-sparteine-mediated enantioselective Pd-catalyzed alpha-arylation of N-Boc-pyrrolidine to prepare chiral 2-arylpyrrolidines in parallel.

The rate and extent of dermal absorption are important in the analysis of risk from dermal exposure to toxic chemicals and for the development of topically applied drugs, barriers, insect repellents, and cosmetics. In vitro flow-through cells offer a convenient method for the study of dermal absorption that is relevant to the initial processes of dermal absorption. This study describes a physiologically based pharmacokinetic (PBPK) model developed to simulate the absorption of organophosphate pesticides, such as parathion, fenthion, and methyl parathion through porcine skin with flow-through cells. Parameters related to the structure of the stratum corneum and solvent evaporation rates were independently estimated. Three parameters were optimized based on experimental dermal absorption data, including solvent evaporation rate, diffusivity, and a mass transfer factor. Diffusion cell studies were conducted to validate the model under a variety of conditions, including different dose ranges (6.3-106.9 microg/cm2 for parathion; 0.8-23.6 microg/cm2 for fenthion; 1.6-39.3 microg/cm2 for methyl parathion), different solvents (ethanol, 2-propanol and acetone), different solvent volumes (5-120 microl for ethanol; 20-80 microl for 2-propanol and acetone), occlusion versus open to atmosphere dosing, and corneocyte removal by tape-stripping. The study demonstrated the utility of PBPK models for studying dermal absorption, which can be useful as explanatory and predictive tools that may be used for in silico hypotheses generation and limited hypotheses testing. The similarity between the overall shapes of the experimental and model-predicted flux/time curves and the successful simulation of altered system conditions for this series of small, lipophilic compounds indicated that the absorption processes that were described in the model successfully simulated important aspects of dermal absorption in flow-through cells. These data have direct relevance to topical organophosphate pesticide risk assessments.

In this paper, we described the optimization of a generic binding assay to measure ligand–receptor interactions for peroxisome proliferator-activated receptors (PPARs). The assay is based on scintillation proximity assay, in which a protein is coated on scintillant-incorporated beads, and a radiolabeled ligand stimulates the beads to emit a signal by binding to the immobilized protein. An intrinsic binding affinity of unlabeled ligands is determined by competitive displacement of the radioligand. The protein coating and ligand binding are achieved in one step by simply mixing ligands, protein and beads in sequence. No additional steps of pre-coating and washing of beads are required. Protein is captured on beads effectively by electrostatic interactions, thus no affinity labeling of protein is required. In data analysis, ligands are grouped into two classes based on their binding affinities. For tight binding ligands, an equation is derived to accurately determine the binding affinity. Otherwise a general equation applies. This quantitative and high throughput assay provides a tool to screen a large library of molecules in search of potent ligands.

Objective—To investigate the feasibility of using multivariate cluster analysis to meta-analyze pharmacokinetic data obtained from studies of pharmacokinetics of ampicillin trihydrate in cattle and identify factors that could account for variability in pharmacokinetic parameters among studies.Sample Population—Data from original studies of the pharmacokinetics of ampicillin trihydrate in cattle in the database of the Food Animal Residue Avoidance Databank.Procedure—Mean plasma or serum ampicillin concentration versus time data and potential factors that may have affected the pharmacokinetics of ampicillin trihydrate were obtained from each study. Noncompartmental pharmacokinetic analyses were performed, and values of pharmacokinetic parameters were clustered by use of multivariate cluster analysis. Practical importance of the clusters was evaluated by comparing the frequency of factors that may have affected the pharmacokinetics of ampicillin trihydrate among clusters.Results—A single cluster with lower mean values for clearance and volume of distribution of ampicillin trihydrate administered PO, compared with other clusters, was identified. This cluster included studies that used preruminant calves in which feeding was withheld overnight and calves to which probenecid had been administered concurrently.Conclusions and Clinical Relevance—Meta-analysis was successful in detecting a potential subpopulation of cattle for which factors that explained differences in pharmacokinetic parameters could be identified. Accurate estimates of pharmacokinetic parameters are important for the calculation of dosages and extended withdrawal intervals after extralabel drug administration. (Am J Vet Res2005;66:108–112)

Perioperative analgesic effects of oral firocoxib following cautery disbudding were investigated in preweaned calves. Twenty Holstein calves approximately 4 to 6wk old received a single oral dose of firocoxib, a nonsteroidal antiinflammatory, at 0.5mg/kg (n=10) or placebo (n=10) in a randomized controlled clinical trial. Responses, including ocular temperature determined by infrared thermography, pressure algometry measuring mechanical nociception threshold, and heart rate, were evaluated at 2, 4, 7, 8, and 24h after cornual nerve block and cautery disbudding. Blood samples were collected over 96h and analyzed for plasma cortisol and substance P concentrations by RIA. Additionally, ex vivo prostaglandin E2 concentrations were determined over a 72-h study period using an enzyme immunoassay. Data were analyzed using a linear mixed effects model with repeated measures. An inhibition of ex vivo prostaglandin E2 synthesis was observed from 12 to 48h following disbudding in calves treated with firocoxib. Cautery disbudding was associated with an increased nociception for the duration of sampling (24h). During the initial 24-h period following disbudding, no difference in response between treatment groups was noted. Following 24h, mean cortisol concentrations diverged between the 2 study groups with placebo-treated calves having increased cortisol concentrations at approximately 48h after disbudding. Furthermore, the overall integrated cortisol response as calculated as area under the effect curve tended to be reduced in firocoxib-treated calves. The prolonged effects of cautery dehorning require further investigation. Moreover, the effect of firocoxib on cortisol reduction observed in this study requires additional exploration.

Allometric scaling is widely used for the determination of first dosage regimen and the interpolation or extrapolation of pharmacokinetic parameters across many animal species during drug development. In this article, 85 drugs used in veterinary medicine obtained from the Food Animal Residue Avoidance Databank database were selected for allometric scaling analysis. Outlier species were identified by statistical methods. The results showed that 77% and 88% of drugs displayed significant correlations between total systemic clearance (CL) and volume of distribution at steady status (Vss) vs. body weight (P

Human pro-matrix metalloproteinase 3 (proMMP-3) lacking the N-terminal 34 amino acids and the C-terminal hemopexin-like domain was expressed in E. coli and used to investigate the process of proenzyme activation and its interaction with an endogenous inhibitor TIMP-1 during activation. The truncated precursor was purified from the E. coli extract in the presence of 5mM EGTA. The active 23.5 kDa form was generated simply by exposure to Ca2+ and Zn2+ but not either by Ca2+ alone or by Zn2+ alone. The rate of MMP-3(deltaC) formation was concentration dependent, indicating that autoactivation is a bimolecular reaction. The truncated precursor was able to interact with the N-terminal domain of TIMP-1 without losing the 48 residue-long propeptide. However, upon a longer incubation, the propeptide was slowly processed, indicating that the association of the N-terminally truncated proMMP-3 with TIMP-1 is weaker than that of the fully activated MMP-3 and TIMP-1. These results indicate that the expression of MMP activities is regulated by endogenous inhibitor TIMPs during their activation processes which provide an additional control mechanism of extracellular matrix breakdown.

Dynamic nuclear polarization (DNP) measurements have been performed in butanol and deuterated butanol. Both target materials have been chemically prepared for the DNP process with the highly stable free nitroxyl radical 2,2,6,6-tetramethyl-piperidine-1-oxyl, also known as TEMPO. Maximum proton polarization values of 89.2% and maximum deuteron polarization values of 38.4% have been obtained in a magnetic field of 2.5 T and at a temperature of about 200 mK. Polarization relaxation times of >200 h (protons) and >100 h (deuterons) have been measured at 0.35 T and 60 mK. The results together with an easy preparation procedure make TEMPO-doped butanol attractive for the frozen spin operation mode of polarized solid-state targets.

Human promatrilysin (matrix metalloproteinase-7) has been produced in Escherichia coli as an N-terminal fusion protein with ubiquitin. The insoluble product was solubilized, refolded, and activated with amino-phenylmercuric acetate. Activation of the fusion protein demonstrated kinetics and intermediates that were very similar to those observed during activation of promatrilysin produced in Chinese Hamster Ovary (CHO) cells. Following activation, matrilysin was purified to > 95% homogeneity using a Sepharose-Pro-Leu-Gly-NHOH affinity column. The matrilysin purified by this procedure is indistinguishable from the enzyme purified from CHO cells with respect to the kinetic parameters for hydrolysis of a peptide substrate and the ability to obtain diffraction quality crystals in the presence of an inhibitor of the enzyme. Additionally, to facilitate detailed kinetic analyses of matrilysin, a new fluorogenic peptide substrate with the optimized sequence Dnp-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (Dnp, dinitrophenyl) has been synthesized. This peptide is the best substrate developed for matrilysin thus far with Km and kcat values of 26 microM and 5.0 s-1, respectively.

Objective To describe the pharmacokinetics, cortisol response and behavioral changes associated with administration of sub-anesthetic xylazine and ketamine prior to castration. Study design Prospective, randomized experiment. Animals Twenty-two male beef calves (260-310 kg). Methods Calves were randomly assigned to receive the following treatment immediately prior to surgical or simulated castration; 1) uncastrated, placebo-treated control (CONT) (n = 4), 2) Castrated, placebo treated control (CAST) (n = 6), 3) castrated with intravenous xylazine (X) (0.05 mg kg−1) (n = 6), and 4) castrated with IV xylazine (X) (0.05 mg kg−1) combined with ketamine (K) (0.1 mg kg−1) (n = 6). Blood samples collected over 10 hours post-castration were analyzed by LC-MS-MS for drug concentrations and chemiluminescent immunoassay for cortisol determination. Results Drug concentrations during the first 60 minutes post-castration fit a one-compartment open model with first-order elimination. The harmonic mean elimination half-lives (± pseudo SD) for X, X with K and K were 12.9 ± 1.2, 11.2 ± 3.1 and 10.6 ± 2.8 minutes, respectively. The proportion of the total area under the effect curve (AUEC) for cortisol during this period was significantly lower in the X group (13 ± 3%; p = 0.006) and the X+K group (14 ± 2%; p = 0.016) compared with the CAST calves (21 ± 2%). However, after 300 minutes the AUEC in the X group was higher than CAST. Significantly more calves demonstrated attitude that was unchanged from pre-manipulation behavior in the CONT (p = 0.021) and X+K treated calves (p = 0.0051) compared with the CAST calves. Conclusions Behavioral changes and lower serum cortisol concentrations during the first 60 minutes post-castration were associated with quantifiable xylazine and ketamine concentrations. Clinical relevance Low doses of xylazine and ketamine administered immediately prior to castration may offer a safe, efficacious and cost-effective systemically administered alternative or adjunct to local anesthesia.

There is ongoing concern about the potential adverse effects of xenobiotic residues in cows' milk to the human consumer. Although drugs that are intentionally administered to lactating dairy cattle are rigorously regulated to prevent harmful residues, there are numerous other potential sources of exposure that are not as easily controlled. For example, cattle may be exposed to mycotoxins, pesticides and/or persistent organic pollutants through feed, water and inhalation of polluted air. Accurate estimates of the rate and extent of excretion of these compounds into milk is important to assess the risk of exposure through cows' milk. In the present study, the expression of carrier mediated transport processes in cultured mono layers of an immortalized bovine mammary epithelial cell line (BME-UV*) was determined using a flow-through diffusion cell system, selective substrates and inhibitors of organic cation transporters (OCT†) and organic anion transporters (OAT‡). The basal to apical (BL-to-Ap§) flux of tetraethylammonium (TEA**) and estrone sulfate (ES††) significantly exceeded their flux in the opposite direction. The addition of selective inhibitors to the donor compartment significantly decreased the BL-to-Ap flux of either selective substrate. These results suggest that both OCT and OAT are functionally expressed by BME-UV cells.

This study examined the efficacy of sodium salicylate for providing analgesia in an amphotericin B-induced bovine synovitis-arthritis model using 10 male Holstein calves, 4 to 6 mo old and weighing approximately 250 kg. The study used a repeated measures partial crossover design with 2 phases, consisting of 3 treatment periods within each phase. Calves were blocked by body weight and randomly assigned to the sodium salicylate (50 mg/kg i.v.) or placebo group for phase 1. In period 1, lameness induction was simulated with a needle prick of the coronary band, followed by drug or placebo administration. At predetermined time points, serial blood samples for cortisol and salicylate concentrations, electrodermal activity measurements, heart rates, and pressure mat data were collected. Visual lameness scores were recorded by an observer blinded to treatments. In period 2, lameness was induced with injection of amphotericin B into the distal interphalangeal joint, followed by drug or placebo administration, with sample collection as described previously. In period 3, the drug or placebo was administered to the respective calves with sample collection. After a 10-d washout period, phase 2 was conducted with treatments crossed over between groups. Cortisol and salicylate samples were analyzed by competitive chemiluminescent immunoassay and fluorescence polarization immunoassay, respectively. The pharmacokinetic data were analyzed using compartmental analysis. Mean intravenous salicylate apparent volume of distribution was 0.2 +/- 0.005 L/kg, total body clearance was 4.3 +/- 0.2 mL/min.kg, and elimination half-life was 36.9 +/- 1.2 min. The repeated measures data were analyzed based on a univariate split-plot approach with a random effects-mixed model. Differences in stance phase duration and serum cortisol concentration values were seen both between periods and between treatment group x periods; differences in heart rate, contact surface area, and contact pressure values were seen between periods, suggesting that our lameness model was effective. No differences were seen between treatment groups. When analyzed by visual lameness score, differences were seen in heart rate, contact surface area, contact pressure, and cortisol concentrations. Area under the time-effect curves, determined by using the trapezoidal rule, had results similar to the repeated measures data, except for a difference in period for electrodermal activity. This amphotericin B-induced synovitis-arthritis model is a useful tool for studying changes associated with lameness in cattle. Sodium salicylate was not effective in providing analgesia after lameness.

The objective of this study was to evaluate the plasma pharmacokinetics of ketamine and its active metabolite norketamine administered intravenously at a dose of 0.1 mg/kg together with xylazine (0.05 mg/kg) to control the pain associated with castration in calves. A two-compartment model with an additional metabolite compartment linked to the central compartment was used to simultaneously describe the time-concentration profiles of both ketamine and its major metabolite norketamine. Parameter values estimated from the time-concentration profiles observed in this study were volume of the central compartment (V(c) = 132.82 +/- 68.23 mL/kg), distribution clearance (CL(D) = 15.49 +/- 2.56 mL/min/kg), volume of the peripheral compartment (V(T) = 257.05 +/- 41.65 mL/kg), ketamine clearance by the formation of the norketamine metabolite (CL(2M) = 8.56 +/- 7.37 mL/kg/min) and ketamine clearance by other routes (CL(o) = 16.41 +/- 3.42 mL/kg/min). Previously published data from rats suggest that the metabolite norketamine contributes to the analgesic effect of ketamine, with a potency that is one-third of the parent drug. An understanding of the time-concentration relationships and the disposition of the parent drug and its metabolite is therefore important for a better understanding of the analgesic potential of ketamine in cattle.

The 1996 data taking of the SMC experiment used polarized protons to measure the spin-dependent structure function g(1) of the proton. Three liters of solid granular ammonia were irradiated at the Bonn electron linac in order to create the paramagnetic radicals which are needed for polarizing the protons. Proton polarizations of +/- (90 +/- 2.5)% were routinely reached. An analysis based on a theoretical line shape for spin-1. systems with large quadrupolar broadening was developed which allowed the nitrogen polarization in the ammonia to be determined with a 10% relative error. The measured quadrupolar coupling constant of N-14 agrees well with earlier extrapolated values. The polarization of the nitrogen nuclei was measured as a function of the proton polarization in order to provide a test of the equal spin temperature (EST) hypothesis. It was found to be closely valid under the dynamic nuclear polarization conditions with which the protons are polarized. Large deviations from EST could be induced by cross relaxing the proton and nitrogen spin systems at low fields. Nitrogen polarizations up to 40% were reached by these means. (C) 1998 Elsevier Science B.V. All rights reserved.

Information from X-ray crystal structures were used to optimize the potency of a HTS hit in a Hsp90 competitive binding assay. A class of novel and potent small molecule Hsp90 inhibitors were thereby identified. Enantio-pure compounds 31 and 33 were potent in PGA-based competitive binding assay and inhibited proliferation of various human cancer cell lines in vitro, with IC 50 values averaging 20 nM.

Objective—To evaluate plasma concentrations of substance P (SP) and cortisol in calves after castration or simulated castration. Animals—10 Angus-crossbred calves. Procedures—Calves were acclimated for 5 days, assigned to a block on the basis of scrotal circumference, and randomly assigned to a castrated or simulated-castrated (control) group. Blood samples were collected twice before, at the time of (0 hours), and at several times points after castration or simulated castration. Vocalization and attitude scores were determined at time of castration or simulated castration. Plasma concentrations of SP and cortisol were determined by use of competitive and chemiluminescent enzyme immunoassays, respectively. Data were analyzed by use of repeated-measures analysis with a mixed model. Results—Mean ± SEM cortisol concentration in castrated calves (78.88 ± 10.07 nmol/L) was similar to that in uncastrated control calves (73.01 ± 10.07 nmol/L). However, mean SP concentration in castrated calves (506.43 ± 38.11 pg/mL) was significantly higher than the concentration in control calves (386.42 ± 40.09 pg/mL). Mean cortisol concentration in calves with vocalization scores of 0 was not significantly different from the concentration in calves with vocalization scores of 3. However, calves with vocalization scores of 3 had significantly higher SP concentrations, compared with SP concentrations for calves with vocalization scores of 0. Conclusions and Clinical Relevance—Similar cortisol concentrations were measured in castrated and control calves. A significant increase in plasma concentrations of SP after castration suggested a likely association with nociception. These results may affect assessment of animal well-being in livestock production systems.

An analytic model of the deuteron absorption function has been developed and is compared to experimental NMR signals of deuterated butanol obtained at the SMC experiment in order to determine the deuteron polarization. The absorption function model includes dipolar broadening and a frequency-dependent treatment of the intensity factors. The high-precision TE signal data available are used to adjust the model for Q-meter distortions and dispersion effects. Once the Q-meter adjustment is made, the enhanced polarizations determined by the asymmetry and TE-calibration methods compare well within the accuracy of each method. In analyzing the NMR signals, the quadrupolar coupling constants could be determined for both the CD and the OD bonds of deuterated butanol.

Pain associated with castration in cattle is an animal welfare concern in beef production. This study examined the effect of oral aspirin and intravenous (i.v.) sodium salicylate on acute plasma cortisol response following surgical castration. Twenty bulls, randomly assigned to the following groups, (i) uncastrated, untreated controls, (ii) castrated, untreated controls, (iii) 50 mg/kg sodium salicylate i.v. precastration and (iv) 50 mg/kg aspirin (acetylsalicylic acid) per os precastration, were blood sampled at 3, 10, 20, 30, 40, 50 min and 1, 1.5, 2, 4, 6, 8, 10 and 12 h postcastration. Samples were analyzed by competitive chemiluminescent immunoassay and fluorescence polarization immunoassay for cortisol and salicylate, respectively. Data were analyzed using noncompartmental analysis, a simple cosine model, anova and t-tests. Intravenous salicylate V(d(ss)) was 0.18 L/kg, Cl(B) was 3.36 mL/min/kg and t(1/2 lambda) was 0.63 h. Plasma salicylate concentrations above 25 microg/mL coincided with significant attenuation in peak cortisol concentrations (P = 0.029). Peak salicylate concentrations following oral aspirin administration was

The purpose of the study was to compare the disposition of pharmacologic markers for cytochrome P-450 (CYP) metabolism, glomerular filtration rate (GFR), and extracellular (ECFV) and total body fluid volumes (TBFV) of Greyhounds and Beagles. Six healthy Greyhound and six healthy Beagle dogs were studied. Antipyrine, a marker for CYP metabolism and TBFV, and inulin, a marker for the GFR and ECFV, were administered i.v. Samples were collected at predetermined times and plasma was analyzed by validated high-pressure liquid chromatography (HPLC) methods. There were no differences in the disposition or pharmacokinetic parameters for inulin between the dog breeds. However, the clearance of antipyrine (mean = 8.33 mL/min/kg) in Greyhounds was significantly slower than Beagles (13.42 mL/min/kg, P = 0.004). The volume of distribution of antipyrine was significantly larger in Greyhounds (0.789 L/kg) than in Beagles (0.644 L/kg, P = 0.01). The half-life of antipyrine was significantly longer in Greyhounds (1.09 h) compared with Beagles (0.55 h, P = 0.002). The in vitro plasma protein binding of antipyrine was significantly less in Greyhounds (28%) compared with Beagles (40.3%, P = 0.008). Greyhounds exhibited significantly slower CYP metabolism, higher TBFV, and lower in vitro protein binding of antipyrine compared with Beagles. No differences in GFR or ECFV were found.

The efficacy of a molybdate formulation and a zinc oxide bolus as prophylactic agents for enzootic icterus was evaluated in sheep. Before copper loading, liver biopsies were performed on 12 male, 6-month-old, Mutton Merino sheep to determine hepatic copper (Cu) and zinc (Zn) concentrations. The animals were restrictively randomised according to liver copper concentrations to 3 treatment groups (n = 4) to achieve similar mean liver copper concentrations per group. All sheep received 4 m /kg of a 0.5 %aqueous solution of CuSO4·5H2O intraruminally 7 days per week for 10 weeks. On Day 0 the sheep in the Mo-group were injected subcutaneously with 42 mg molybdenum (Mo) contained in a commercial molybdate formulation. The animals in the Zn-group each received a zinc oxide bolus, containing 43 g zinc oxide, via a rumen cannula. Treatment was repeated on Day 42. Four animals served as untreated controls. Urinary copper excretion, plasma copper concentration, haematocrit and glutamate dehydrogenase (GLDH) activity were determined throughout the trial. The animals were sacrificed after 10 weeks and liver samples were submitted for histopathological examination. Liver and kidney copper and zinc concentrations were determined. Neither the molybdate treatment nor the zinc oxide boluses prevented hepatic copper accumulation. The urinary copper excretion, plasma copper concentration, haematocrit and GLDH activity were not significantly different (P 0.05) from the controls.

A plot of the pH dependence of kcat/KM for human stromelysin-1 (HS) exhibits a narrow range of maximal activity extending from pH 5.75 to 6.25 and a broad shoulder in the pH range of 7.5-8.5. In contrast, the pH profiles that have been reported for other members of the matrix metalloproteinase (MMP) family are bell-shaped and exhibit neutral pH optima. We hypothesized that the anomalous pH dependence of HS reflects the ionization of His-224, a residue located in a flexible loop that contributes to the S1' binding pocket of the enzyme. HS is the only known MMP that has a histidine in this position. To test this hypothesis, the H224Q mutant of the short form (lacking the C-terminal hemopexin-like domain) of HS (sHS) has been prepared and studied. The pH profile of H224Q sHS is bell-shaped and similar to those reported for other MMPs. Although H224Q and wild-type sHS possess similar activities at pH6, the kcat/KM of H224Q sHS is more than 5-fold greater than that of the wild-type enzyme at pH7. These data strongly suggest that the deprotonation of His-224 attenuates the activity of HS, thereby accounting for its low pH optimum and the characteristic shoulder in its pH profile. This attenuation of activity appears to be predominantly a KM effect, reflecting a decrease in the affinity of the enzyme for the peptide substrate.

Matrix metalloproteinases (MMPs), also termed “matrixins”, constitute a family of zinc metalloendopeptidases that participate in breakdown of extracellular matrix macromolecules (Woessner, 1991). These enzymes are considered to play an important role in many biological processes such as in reproduction, embryogenesis, tissue resorption, and in the control of cell behavior. Overproduction of matrixins is associated with various connective tissue diseases such as arthritis, periodontitis, glomerulonephritis, tissue ulceration as well as being connected with tumor cell invasion and metastasis (Woessner, 1991; Birkedal-Hansen et al., 1993).

The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays crucial roles in cell growth, proliferation and survival. Genomic aberrations in the PI3K pathway, such as mutational activation of PI3Kα or loss of function of tumor suppressor PTEN, have been closely linked to the development and progression of a wide range of cancers. Hence, inhibition of the key targets in the pathway, e.g. PI3K, AKT, mTOR, offers great potential for the treatment of cancer. In an effort to discover compounds that inhibit PI3Kα, a high throughput screen was carried out, and 4-methyl-pyrido-pyrimidine (MPP) derivatives were identified as potent and selective inhibitors of PI3Kα. For example, PF-00271897, 8-cyclopentyl-6-[3-(hydroxymethyl)phenyl]-4-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one demonstrated PI3Kα Ki of 2.2 nM. Multiple crystal structures of inhibitors bound to PI3K gamma were determined to inform design and optimization of the ADMET properties of this lead series. Crystallographic studies with PI3K gamma protein indicated that the aminopyrimidine moiety forms two hydrogen bonds to the kinase backbone, and the aromatic moiety at the 6 position binds in a hydrophobic pocket. The X-ray structure suggested that the 4-methyl group on the MPP core structure conferred the excellent overall kinase selectivity to the series. The structure and SAR suggested optimization could come from keeping N-R group at 2 position very small and maintaining aromatic moiety at 6 position for hydrophobic interaction. Introduction of polar groups to the 8N side chains that are located in the ribose binding pocket increased both metabolic stability and solubility. Based on the overall properties, PF-04691502, 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one, was selected as a clinical candidate. PF-04691502 demonstrated Ki's of 1.2-2.2 nM against PI3K α, β, γ and δ isoforms, and Ki of 9.1 nM against recombinant mTOR. PF-04691502 inhibited AKT phosphorylation at S473 in BT20 breast cancer line with IC50 of 12 nM. PF-04691502 is highly selective for inhibition of PI3K family kinases as shown by lack of activity against a panel of >75 protein kinases, including the class III PI3K hVps34. In the in vivo rat PK studies, PF-04691502 demonstrated the following properties: Clearance = 5.2 ml/min/kg, Vdss = 1.4 L/kg, T1/2 = 3.1 h, F% = 63%. The design, synthesis, in vitro potency SAR, selectivity, ADMET of the MPP derivatives will be discussed. The crystal structure of PF-04691502 in PI3K gamma will also be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5779.

We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently in Escherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved in de novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed in E. coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure. Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins. The active monofunctional GART protein produced in E. coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein.

Diphenylphosphinsaurecyanid und Diphenylthiophosphinsaurecyanid reagieren selektiv mit der Mercaptogruppe—auch bei Anwesenheit von Verbindungen mit primaren Aminogruppen. Die Thioloesterbindung in Thiophosphin-, Thiophosphon- und Thiophosphorsaure-S-estern wird durch nicht solvatisierte Fluoridionen ([R4N]F°3 H2O (R = C4H9) und CsF) unter schonenden Bedingungen in Chloroform, Methylenchlorid oder THF gespalten. (C6H5)2P(O)CN und (C6H5)2P(S)CN sind als Schutzgruppen-Reagentien fur Mercaptane geeignet. Diphenylphosphinsaurechlorid und Diphenylthiophosphinsaurechlorid sind in Konkurrenzversuchen mit Butylamin/Butylmercaptan N-selektiv. Bei Cysteamin und Cysteinmethylester wird aber im Gegensatz zu den entsprechenden S-selektiven Cyaniden keine Selektivitat beobachtet. Diphenylphosphinicacidcyanide and diphenylthiophosphinicacidcyanide react selectively with the mercaptogroup even in presence of primary aminogroups. The thioloesterbond in thiophosphinic-, thiophosphonic- and thiophosphoricacid-S-este...

A silver ore concentrate (≈50 mg/kg Ag) was studied to determine whether electrolytic treatment could be used to enhance silver extraction from the ore. The hydrometallurgical leach was carried out in a batch mode using hydrochloric acid. The ore was mechanically slurried in an electrochemical cell with graphite rod electrodes. A comparison of the extractions using acid, acid + chlorine or electrolytic means was made by measuring silver, iron and sulphur dissolved as a function of time, concentration and sparging gas. The results demonstrated that the electrolytic leach improved both the rate and extent of silver extraction over non-electrolytic leaches. Up to 57% silver extraction was obtained in a 3 hour period compared with 15 to 25% with acid or acid + chlorine leaches respectively. The rate of silver extraction at the end of the 3 hour period was also substantially higher. Under the same conditions only 9% of the iron was extracted from the ore.

Six alpha 2-macroglobulin cDNA clones were isolated from two liver cDNA libraries produced from rats undergoing acute inflammation. The coding sequence for rat alpha 2-macroglobulin including its 27-residue signal peptide and the 3' - and part of the 5' nontranslated regions were determined. The mature protein consisting of 1445 amino acids is coded for by a 4790 +/- 40 nucleotide messenger RNA. It contains a typical internal thiol ester region and 25 cysteine residues which are conserved between rat and human alpha 2-macroglobulin. Although the amino acid sequences of rat and human alpha 2-macroglobulin share 73% identity, two small divergent areas of 17 and 38 residues were found, corresponding to 29 and 11% identity, respectively. These areas are located in the bait region and, therefore, may confer specific proteinase recognition capabilities on rat alpha 2-macroglobulin. Following an inflammatory stimulation, rat alpha 2-macroglobulin mRNA levels increased 214-fold over control values and reached a maximum at 18 h. By 24 h the levels had decreased to less than 30% of the maximum value. Transcription rates from the alpha 2-macroglobulin gene as measured in nuclear run-on experiments showed a less than 3-fold increase in nuclei from acutely inflamed rats as compared to controls. These results suggest that the accummulation of alpha 2M mRNA is due to the combined effects of increased transcription rates and post-transcriptional processing.

The mechanism of the reaction of phosphoryl fluoride ( P(O)F) with alcohols in the presence of an amine is fundamentally different from the reaction of phosphoryl chlorides ( P(O)Cl) with primary or secondary amines. The following observations strongly support this proposal: 1. 1H-NMR-, 31P-NMR- and 19F-NMR-spectroscopic investigations show that methyl-phenyl-phosphinicacid-fluoride and n-butylamine form a thermolabile adduct, which yields the methyl-phenyl-phosphinicacid-amide only very slowly. 2. The rate of the reaction of methyl-phenyl-phosphinicacid-fluoride with ethanol is independent of the basicity of the amine but very sensitive to steric factors. Imidazole (which is only a weak base) is surprisingly active. 3. The application of optically active amines as bases in the reaction of racemic methyl-phenyl-phosphinicacid-fluoride with methanol leads to partially optically active methyl-phenyl-phosphinicacid-methylester. This observation proves that the amines are incorporated in the intermed...

Phosphin-, Phosphon- und Phosphorsaurederivate vom Typ R1R2P(O)X konnen in Abhangigkeit von der austretenden Gruppe X (X = Cl, F, CN, N3, OC6H4NO2(p) und der Base B selektiv mit Nukleophilen RYH (R = nC4H9; Y = O, NR, S) nach (1) abreagieren. Die Liganden R1 und R2 uben auf den Reaktionsablauf einen vergleichsweise nur geringen Einflus aus. Methodik: (a) In Konkurrenzversuchen last man die Phosphylierungsmittel R1R2P(O)X mit zwei Nukleophilen: RYH und RY'H im Verhaltnis 1:1:1 nach (2) abreagieren und bestimmt die Reaktionsprodukte R1R2P(O)YR und R1R2P(O)Y'R. (b) Verbindungen HY—CH2—CH2—Y'H (bzw. Serin-n-butylamid und L-Cysteinmethylester) werden mit den Phosphylierungsmitteln R1R2P(O)X im Verhaltnis 1:1 nach (3) bzw. (4) umgesetzt, die Reaktionsprodukte isoliert, identifiziert und quantitativ bestimmt. Ergebnisse: Mit X = F, CN, OC6H4NO2(p) entstehen praktisch nur die O-Ester, mit X = Cl nur die Amide. Azide (X = N3) diskriminieren nicht. In der Konkurrenz: n-Butylamin/n-Butylmercaptan sind die P...

The carbonyl groups of saturated and unsaturated aliphatic and aromatic aldehydes and ketones add diphenylphosphinic acid nitrile and dimethylphosphinic acid nitrile according to (1) and (2) forming O-(phosphinoyl) cyanohydrins. O-(diphenylphosphinoyl) acrolein cyanohydrin isomerizes according to (3) to (2-diphenylphosphinoyloxy)-but-2-en-nitrile following a shift of the double bond (a) at 200°C or (b) on interaction with KOBut. Aminonitriles are formed according to (4) by a modified Strecker reaction on reacting O-(diphenylphosphinoyl) aldehyde (ketone) cyanohydrins with secondary amines. The anionic form of O-(diphenylphosphinoyl) aldehyde cyanohydrins reacts (a) with alkyl halides forming O-(diphenylphosphinoyl) ketone cyanohydrins (5) and (b) with aldehydes (6) to yield O-(diphenylphosphinoyl) acyloins after a transacylation. Phosphinic acid esters are cleaved quantitatively by fluorolysis using tetrabutylammonium fluoride at room temperature (7). Diphenylphosphinsaurecyanid und Dimethylphosp...

The nucleotide sequence coding for the ninth component of human complement (C9) has been determined and the corresponding amino acid sequence has been derived. A human liver cDNA library was screened by the colony-hybridization technique using two radiolabeled oligonucleotide probes that correspond to known regions of the C9 amino acid sequence. Two recombinant plasmids were isolated and their cDNA inserts were sequenced. The derived protein sequence consists of 537 amino acids in a single polypeptide chain. A profile of the hydropathic index versus sequence number indicates that the amino-terminal half of C9 is predominantly hydrophilic in character whereas the carboxyl-terminal section of this protein is more hydrophobic. The amphipathic organization of the primary structure of C9 is consistent with the known potential of polymerized C9 to penetrate lipid bilayers, causing the formation of transmembrane channels.