Early cathepsin K degradation of type II collagen in vitro and in vivo in articular cartilage.

OBJECTIVE: To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage. DESIGN: Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, in vitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage. RESULTS: Six cathepsin K cleavage sites distributed throughout the triple helical region were identified in equine type II collagen. Most of the cleavages occurred following a hydroxyproline residue. The most N-terminal site was within three residues of the previously identified site in bovine type II collagen. Western blotting using anti-neoepitope antibodies showed that the initial cleavages occurred at the N-terminal sites and this was followed by more extensive degradation resulting in products too small to be resolved by SDS gel electrophoresis. Immunohistochemical staining of cartilage sections from equine or human osteoarthritic joints showed staining in lesional areas which was not observed in non-arthritic sites. CONCLUSIONS: Cathepsin K cleaves triple helical collagen by erosion from the N-terminus and with subsequent progressive cleavages. The liberated fragments can be detected in osteoarthritic cartilage and may represent useful biomarkers for disease activity.

Assessing experimental visceral pain in dairy cattle: A pilot, prospective, blinded, randomized, and controlled study focusing on spinal pain proteomics.

Few studies have verified the validity of behavioral and physiological methods of pain assessment in cattle. This prospective, blinded, randomized controlled experimental study aimed to validate different methods of pain assessment during acute and chronic (up to 21 d postintervention) conditions in dairy cattle, in response to 3 analgesic treatments for traumatic reticuloperitonitis. Cerebrospinal fluid (CSF) biomarkers and mechanical sensitization were measured as indicators of centralized pain. Proteomics in the CSF were examined to detect specific (to pain intensity) and sensitive (responsive to analgesia) markers. Recordings of spontaneous behavior with video analysis, telemetered motor activity, pain scales, electrodermal activity, and plasma cortisol concentration were quantified at regular intervals. Cows were assigned to group 1 (n=4, standard control receiving aspirin), group 2 (n=5, test group receiving preemptive tolfenamic acid), or group 3 (n=3, positive control receiving preemptive multimodal analgesia composed of epidural morphine, plus tolfenamic acid and butorphanol). Rescue analgesia was administered as needed. Generalized estimating equations tested group differences and the influence of rescue analgesia on the measurements. All 3 groups demonstrated a long-term decrease in a CSF protein identified as transthyretin. The decrease in transthyretin expression inversely correlated with the expected level of analgesia (group 1<2<3). Moreover, in group 1, CSF noradrenaline decreased long term, cows were hypersensitive to mechanical stimulation, and they demonstrated signs of discomfort with higher motor activity and "agitation while lying" recorded from video analysis. Decreased "feeding behavior," observer-reported pain scales, electrodermal activity, and plasma cortisol concentration were inconsistent to differentiate pain intensity between groups. In summary, changes in CSF biomarkers and mechanical sensitization reflected modulation of central pain in dairy cows. The spontaneous behavior "agitation while lying" was the only behavioral outcome validated for assessing acute and chronic pain in this visceral pain model.

Economic-based projections of future land use in the conterminous United States under alternative policy scenarios.

Land-use change significantly contributes to biodiversity loss, invasive species spread, changes in biogeochemical cycles, and the loss of ecosystem services. Planning for a sustainable future requires a thorough understanding of expected land use at the fine spatial scales relevant for modeling many ecological processes and at dimensions appropriate for regional or national-level policy making. Our goal was to construct and parameterize an econometric model of land-use change to project future land use to the year 2051 at a fine spatial scale across the conterminous United States under several alternative land-use policy scenarios. We parameterized the econometric model of land-use change with the National Resource Inventory (NRI) 1992 and 1997 land-use data for 844 000 sample points. Land-use transitions were estimated for five land-use classes (cropland, pasture, range, forest, and urban). We predicted land-use change under four scenarios: business-as-usual, afforestation, removal of agricultural subsidies, and increased urban rents. Our results for the business-as-usual scenario showed widespread changes in land use, affecting 36% of the land area of the conterminous United States, with large increases in urban land (79%) and forest (7%), and declines in cropland (-16%) and pasture (-13%). Areas with particularly high rates of land-use change included the larger Chicago area, parts of the Pacific Northwest, and the Central Valley of California. However, while land-use change was substantial, differences in results among the four scenarios were relatively minor. The only scenario that was markedly different was the afforestation scenario, which resulted in an increase of forest area that was twice as high as the business-as-usual scenario. Land-use policies can affect trends, but only so much. The basic economic and demographic factors shaping land-use changes in the United States are powerful, and even fairly dramatic policy changes, showed only moderate deviations from the business-as-usual scenario. Given the magnitude of predicted land-use change, any attempts to identify a sustainable future or to predict the effects of climate change will have to take likely land-use changes into account. Econometric models that can simulate land-use change for broad areas with fine resolution are necessary to predict trends in ecosystem service provision and biodiversity persistence.

Gait analysis and pain response of two rodent models of osteoarthritis.

The purpose of this study was to compare the gait parameters recorded on the CatWalk and the mechanical sensitivity with von Frey filaments of two putative models of osteoarthritis over a one month period, and to evaluate the effect of celecoxib on these parameters. Animals underwent either a surgical sectioning of the anterior cruciate ligament with partial medial menisectomy (ACLT+pMMx) to create a joint instability model or received an intra-articular injection of monoiodoacetate (MIA) as a putative inflammatory joint pain model. Animals were assessed for four consecutive weeks and knee joints were then evaluated histologically. Spinal cord lumbar enlargements were harvested for selected neuropeptide analysis (substance P (SP) and calcitonin gene related peptide (CGRP)). With the MIA model, significant changes persisted in selected dynamic gait parameters throughout the study in the injured limb as well as with the von Frey filaments. The ACLT+pMMx model in contrast showed no clear differential response between both hind limb for both gait parameters and pain-related behavior with von Frey filaments occurred only on the last day of the study. Neuropeptide analysis of spinal cord lumbar enlargements revealed a significant increase in CGRP concentration in both models and an increase in SP concentration only in the MIA model. Histological evaluation confirmed the presence of articular cartilage lesions in both models, but they were much more severe in the MIA model. Celecoxib had an effect on all selected gait parameters at the very beginning of the study and had an important alleviating effect on mechanical allodynia. These results suggest that the MIA model may be more appropriate for the evaluation of short term pain studies and that celecoxib may modulate mechanical allodynia through central sensitization mechanisms.

Joint inflammation increases glucosamine levels attained in synovial fluid following oral administration of glucosamine hydrochloride.

OBJECTIVE: To compare synovial glucosamine levels in normal and inflamed equine joints following oral glucosamine administration and to determine whether single dose administration alters standard synovial parameters of inflammation. METHODS: Eight adult horses were studied. On weeks 1 and 2, all horses received 20mg/kg glucosamine hydrochloride by nasogastric (NG) intubation or intravenous injection. On weeks 3 and 4, 12h after injection of both radiocarpal joints with 0.25 ng Escherichia coli lipopolysaccharide (LPS) to induce inflammation, glucosamine hydrochloride or a placebo was administered by NG intubation. Plasma samples were collected at baseline and 5, 15, 30, 60, 120, 360, 480 and 720 min after dosing. Synovial fluid (SF) samples were collected within 48 h before dosing and 1, 6 and 12h post-dosing. Glucosamine was analyzed by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI/MS/MS). Clinicopathological evaluation of SF parameters included white blood cell (WBC) count and total protein (TP) analyses. RESULTS: No significant differences between groups were observed in SF baseline levels of WBC and TP at any stage of the study. SF WBC and TP significantly increased following IA LPS. The mean (+/-SD) maximal SF glucosamine levels (422.3+/-244.8 ng/mL) were significantly higher (>fourfold) in inflamed joints when compared to healthy joints (92.7+/-34.9 ng/mL). Glucosamine did not have any effect on standard SF parameters of inflammation. CONCLUSION: Synovial inflammation leads to significantly higher synovial glucosamine concentrations compared to levels attained in healthy joints following oral administration of glucosamine hydrochloride. Whether these higher levels are translated into a therapeutic effect on the joint tissues remains to be elucidated.

Pharmacokinetics of morphine and its metabolites in freshwater rainbow trout (Oncorhynchus mykiss).

In this study, we injected morphine sulfate IP into rainbow trout and measured the concentration of morphine and all potential metabolites in plasma using LC-MS/MS at a series of times after the injection. The pharmacokinetics of morphine were similar to those previously reported for seawater-acclimated rainbow trout, i.e. they were about one order of magnitude slower than in similarly sized mammals. The only metabolite of morphine present in the plasma was morphine-3-beta-D-glucuronide (M3G); morphine-6-beta-D-glucuronide (M6G) was not detected. M3G gradually increased after the morphine injection, peaked about 2 days later, then gradually decreased. In mammals, M3G plasma levels exceed morphine levels extremely rapidly, i.e. in less than an hour, regardless of dose, route of administration, or species. In trout, it took 2 days for M3G levels to exceed morphine levels. This is the first study of the metabolites of morphine in any ectotherm. We conclude that trout can metabolize morphine, but at a rate much slower than in mammals.

Comparison of pharmacokinetics of glucosamine and synovial fluid levels following administration of glucosamine sulphate or glucosamine hydrochloride.

OBJECTIVE: To compare the pharmacokinetics of glucosamine and the synovial fluid levels attained following treatment with glucosamine sulphate or glucosamine hydrochloride in a large animal model at clinically relevant doses. METHODS: Eight adult female horses were used. Crystalline glucosamine sulphate (Dona) or glucosamine hydrochloride was administered at a dose of 20 mg/kg by either intravenous (i.v.) injection or nasogastric (n.g.) intubation. Plasma samples were collected before dosing and at 5, 15, 30, 60, 120, 360, 480 and 720 min after dosing. Synovial fluid samples were collected from the radiocarpal joints within 48 h before dosing and at 1, 6 and 12 h post-dosing. Glucosamine was assayed by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI/MS/MS). RESULTS: Plasma concentrations reached approximately 50 microg/mL after i.v. injection and approximately 1 microg/mL after n.g. administration of both types of glucosamine. The median oral bioavailability was 9.4% for glucosamine sulphate and 6.1% for glucosamine hydrochloride. Synovial fluid concentrations were significantly higher at 1 and 6 h following oral treatment with glucosamine sulphate compared to glucosamine hydrochloride. Twelve hours following oral administration, glucosamine levels in the plasma and the synovial fluid were still significantly higher than baseline for the glucosamine sulphate preparation, but not for the hydrochloride preparation. CONCLUSION: Following oral administration of a clinically recommended dose of glucosamine sulphate (Dona), significantly higher synovial fluid concentrations of glucosamine are attained, when compared to an equivalent dose of glucosamine hydrochloride. Whether this difference is translated into a therapeutic effect on the joint tissues remains to be elucidated.

Comparative oxidative metabolic profiles of clomipramine in cats, rats and dogs: preliminary results from an in vitro study.

The objectives of this in vitro study were to describe cytochrome-dependent metabolism of clomipramine in canine and feline microsomes, compare metabolic profiles between cats, rats and dogs, and investigate a potential gender-related difference in metabolic activity between male and female cats. Pooled liver microsomes were incubated with clomipramine, where species and gender-specific reactions were initiated by the addition of a nicotinamide adenine dinucleotide phosphate regenerating system and quenched with methanol at 0, 5, 15, 30, 45 and 60 min, and 0, 30, 60, 90, 120, 180, 240 and 360 min respectively. Liquid chromatography tandem mass spectrometry was used to measure clomipramine and its metabolites. Preliminary results showed that cat microsomes biotransformed clomipramine slower and less efficiently than rat and dog microsomes. Moreover, gender differences in metabolic profiles suggested that male cat microsomes may be less efficient demethylators and hydroxylators than female cat microsomes. As gender metabolic differences may carry clinical significance for this antidepressant, further studies are warranted.

Effects of physiological covariables on pharmacokinetic parameters of clomipramine in a large population of cats after a single oral administration.

This study was conducted to confirm an interindividual variability in pharmacokinetic parameters of clomipramine in a large population of cats and to identify potential covariables that would explain the presence of such pharmacokinetic variability after a single dose of Clomicalm. Clomipramine hydrochloride was administered orally according to a weight-dose chart from 0.32 to 0.61 mg/kg, to 76 cats and five blood samples were then taken by direct venipuncture at 1, 3, 6, 12, and 24 h. Plasma concentrations of clomipramine and desmethylclomipramine (DCMP) were measured by LC-MS/MS. The Standard Two-Stage technique was used to assess differences and detect correlations between pharmacokinetic parameter estimates and individual covariables. A large interindividual variability in all pharmacokinetic parameters (CV% 64-124) was detected. Statistically significant gender-related differences were detected in MR and Cl/F, where female cats had a higher mean MR (0.53) and faster Cl/F (0.36 L/h.kg) than males (0.36 and 0.21 L/h.kg, respectively). No correlation could be found between clomipramine AUC0-24 h or DCMP AUC0-24 h and sedation scores. Further feline studies are required to assess these findings after multiple dosing of clomipramine and DCMP to allow clinical extrapolation.

Pharmacokinetics and anesthetic activity of eugenol in male Sprague-Dawley rats.

Eugenol, the principle chemical constituent of clove oil, has recently been evaluated for its anesthetic and analgesic properties in fish and amphibians. The objective of this study was to determine the pharmacokinetic (PK) and anesthetic activity of eugenol in rats. Male Sprague-Dawley rats received single i.v. doses of eugenol (0, 5, 10, 20, 40 and 60 mg/kg) and anesthetic level was evaluated with the withdrawal reflex. For the 20 mg/kg dose level, blood and urinary samples were collected over 1 h for the PK assessment. Plasma and blood concentrations of eugenol, as well as metabolite identification in urine, were determined using a novel dansyl chloride derivatization method with liquid chromatography mass spectrometry (LC/MS/MS). PK parameters were calculated using noncompartmental methods. Eugenol-induced loss of consciousness in a dose-dependent manner, with mean (+/-SEM) recovery in reflex time of 167 +/- 42 sec observed at the highest dose level. Mean systemic clearance (Cl) in plasma and blood were 157 and 204 mL/min/kg, respectively. Glucuronide and sulfate conjugates were identified in urine. Overall, eugenol produced a reversible, dose-dependent anesthesia in male Sprague-Dawley rats.

A pharmacokinetic study of amoxycillin in febrile beagle dogs following repeated administrations of endotoxin.

The pharmacokinetics of amoxycillin was studied in nine male beagle dogs under healthy and febrile conditions. In Period 1, dogs received 20 mg/kg of an oral suspension of amoxycillin. Intravenous doses of saline, 2 and 20 microg/kg of endotoxin (LPS from Escherichia coli serotype) were administered to dogs (three per group) prior to administration of 20 mg/kg of amoxycillin in Period 2. Rectal temperature and behavioral changes were recorded and blood samples were collected over 12 h for pharmacokinetic analysis. Amoxycillin was assessed in plasma using liquid chromatography coupled with mass spectrometry. Plasma concentrations were analysed using a one-compartment model with lag-time for absorption using an iterative two-stage method. As compared with control groups, amoxycillin clearance decreased significantly with preliminary treatments of 2 microg/kg endotoxin (0.209 vs. 0.140 L/h kg, P < 0.05) and 20 microg/kg endotoxin (0.214 vs. 0.075 L/h kg, P < 0.05). As a result of this, the area under curve for the 2 and 20 microg/kg endotoxin groups increased significantly 100.4 vs. 149.4 microg h/mL (P < 0.05) and 99.2 vs. 277.7 microg h/mL (P < 0.05), respectively. Other drugs currently used for the treatment of fever and septic shock should be re-evaluated using a febrile animal model to avoid improper dose administration.

Determination of drug-plasma protein binding using human serum albumin chromatographic column and multiple linear regression model.

Reversible attachment to serum proteins plays a significant role in pharmacokinetics and pharmacodynamics, and a clear understanding of this process is fundamental in the development of the rational use of many therapeutics agents. Over the last few years, it has been demonstrated that immobilized human serum albumin (HSA) could be used to estimate plasma protein binding. A series of 40 structurally unrelated pharmaceutical compounds were chromatographed on an immobilized HSA column in order to construct a protein binding 'calibration curve' and multiple linear regression system. When studying the relationship between the chromatographic retention and the percentage of binding determined in vitro, a good correlation can be observed (r(2) = 0.799) using a wide variety of compounds with different binding affinities (from 0 to 99% binding). Using a quantitative structure-retention relationships (QSRR) approach to analysing chromatographic data, the correlation was improved compared to the traditional approach (r(2) = 0.824).

Metabolite profiling study of propranolol in rat using LC/MS/MS analysis.

Metabolite profiling is one of the most challenging fields in applied mass spectrometry. Mass spectrometry was used to characterize the metabolites of propranolol, a beta-adrenergic receptor antagonist containing numerous oxidation sites. Propranolol is extensively metabolized, with most metabolites appearing in urine. Urine samples were collected from young adult male Sprague-Dawley rats. Structural identification of various metabolites was performed by LC/MS/MS, using a PE SCIEX triple quadrupole instrument (PE SCIEX API 3000). Metabolites were itemized using several LC/MS/MS techniques, including Q3 full scan and precursor and constant neutral loss experiments. A looped experiment technique revealed the presence of mono- and di-hydroxylated metabolites as well as regio isomers of hydroxy- and dihydroxy-propranolol glucuronides and propranolol glucuronic acid. Propranolol glucuronide was not observed, while the presence of dealkylated metabolites was suggested but not confirmed.

In vivo pharmacokinetic screening in cassette dosing experiments; the use of on-line Pprospekt liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry technology in drug discovery.

Drug discovery is a fast growing field and the number of compounds generated daily by the pharmecutical industry is enormous. The necessity of developing new experimental strategies and analytical methods to rapidly screen the pharmacokinetics (PK) behavior of these compounds becomes a real challenge. A novel strategy to support in vivo PK screening in cassette doing experiments, using a fully automated system capable of analyzing between 320 to 960 samples a day by instrument in n-in-one experiment ( n = 64 in this work), has been developed. Using an on-line extraction technique, the average observed recovery was 64% using a single C18 procedure. A weighted (1/x) linear equation was used to perform standard calibration (0.5 to 500 ng/microL) and the average R value obtained was 0.994 (R2 = 0.997) for 63 analytes. The limit of detection, defined as a signal-to-noise ratio of 3 or greater, was found to be 25 pg for 41 of the 63 analytes (65%) and 250 pg for 57 of the 63 analytes (90%). The complete automation procedure using the Prospekt-LC-APCI/MS/MS system has substantially improved throughput in the area of drug discovery and bioanalysis.