Pharmacokinetics of ceftiofur sodium in equine pregnancy.

Eleven pregnant pony mares (D270-326) were administered ceftiofur sodium intramuscularly at 2.2 mg/kg (n = 6) or 4.4 mg/kg (n = 5), once daily. Plasma was obtained prior to ceftiofur administration and at 0.5, 1, 2, 4, 8, 12, and 24 hr after administration. Eight pony mares were re-enrolled in the study at least 3 days from expected foaling to ensure steady-state concentrations of drug at the time of foaling. Mares were administered ceftiofur sodium (4.4 mg/kg, IM) daily until foaling. Parturition was induced using oxytocin 1 hr after ceftiofur sodium administration. Allantoic and amniotic fluid, plasma, and colostrum samples were collected at time of foaling. Serial foal plasma samples were obtained. Placental tissues were collected. Desfuroylceftiofur acetamide (DCA) concentrations were measured in samples by high-performance liquid chromatography (HPLC). Mean (±SD) peak serum concentrations of DCA were 3.97 ± 0.50 µg/ml (low dose) and 7.45 ± 1.05 µg/ml (high dose). Terminal half-life was significantly (p = .014) shorter after administration of the low dose (2.91 ± 0.59 hr) than after administration of the high dose (4.10 ± 0.72 hr). The mean serum concentration of DCA from mares at time of foaling was 7.96 ± 1.39 µg/ml. The mean DCA concentration in colostrum was 1.39 ± 0.70 µg/ml. DCA concentrations in allantoic fluid, amniotic fluid, placental tissues, and foal plasma were below the limit of quantification (<0.1 µg/ml) and below the minimum inhibitory concentration of ceftiofur against relevant pathogens. These results infer incomplete passage of DCA across fetal membranes after administration of ceftiofur sodium to normal pony mares.

Plasma pharmacokinetics and tissue depletion of cyromazine and its metabolite melamine following oral administration in laying chickens.

The study was designed to characterize the plasma pharmacokinetics and tissue depletion profiles (including eggs) of cyromazine (CYR) in chickens following oral administration alone or in combination with melamine (MEL). In order to assess the pharmacokinetic profile of CYR, chickens were administered 1 or 10 mg/kg (single oral doses), whereas residue studies were conducted in chickens fed CYR alone (5 or 10 mg/kg) or CYR (5 mg/kg) and MEL (5 mg/kg) for a period of 14 days. Estimates for the apparent volume of distribution (1.66 L/kg), clearance (7.17 mL/kg/min), and elimination half-life (2.82 h) were derived by noncompartmental analyses. The highest concentration of CYR occurred in liver but fell below detectable limits within 3 days following drug withdrawal from feed. Combined feeding of MEL with CYR did not significantly alter CYR tissue levels. CYR residues were detected only in egg white and were undetectable at the 2nd day postadministration. No MEL was found in eggs unless it had been added to the feed, and when present, it almost exclusively restricted to the egg white. Based upon the results of this initial study of CYR pharmacokinetics and residue depletion, it appears that use of CYR as a feed additive either alone (5 or 10 mg/kg) or in combination with MEL (both agents at 5 mg/kg) does not produce unsafe residue levels in edible products as long as appropriate withdrawal periods are followed for tissues (3 days) and eggs (2 days). However, our results indicate that adoption of a zero-day withdrawal period should be reconsidered in light of these results.

Disposition of ampicillin trihydrate in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle.

The objective of this study was to determine the disposition of ampicillin in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle. Ampicillin trihydrate was administered by intramuscular (i.m.) injection at a dose of 11 mg/kg of body weight every 24 h (n = 6, total of 3 doses) or every 12 h (n = 6, total of 5 doses) for 3 days. Concentrations of ampicillin were measured in plasma, uterine tissue, lochial fluid, and milk using HPLC with ultraviolet absorption. Quantifiable ampicillin concentrations were found in plasma, milk, and lochial fluid of all cattle within 30 min, 4 h, and 4 h of administration of ampicillin trihydrate, respectively. There was no significant effect of dosing interval (every 12 vs. every 24 h) and no significant interactions between dosing interval and sampling site on the pharmacokinetic variable measured or calculated. Median peak ampicillin concentration at steady-state was significantly higher in lochial fluid (5.27 µg/mL after q 24 h dosing) than other body fluids or tissues and significantly higher in plasma (3.11 µg/mL) compared to milk (0.49 µg/mL) or endometrial tissue (1.55 µg/mL). Ampicillin trihydrate administered once daily by the i.m. route at the label dose of 11 mg/kg of body weight achieves therapeutic concentrations in the milk, lochial fluid, and endometrial tissue of healthy postpartum dairy cattle.

Safety of fenbendazole in Chinese ring-necked pheasants (Phasianus colchicus).

Ring-necked pheasants raised on propagation farms can be severely parasitized with Syngamus trachea (gapeworm) and other parasitic worms. Fenbendazole is a highly effective benzimidazole-class anthelmintic that is not currently approved for game bird species in the United States. The objective of this work was to provide target animal safety data to support a label claim for fenbendazole in pheasants at 100 parts per million (ppm) in the feed for 7 consecutive days. Demonstration of safety in young pheasants and a separate demonstration of reproductive safety in adult birds were required. In the young bird study, 160 Chinese ring-necked pheasants (Phasianus colchicus, 80 males and 80 females) were fed a commercial game bird starter ration containing no antibiotics, growth promoters, or coccidiostats until day 0 of the study (approximately 21 days of age). On day 0 the birds were placed on their respective study diets containing fenbendazole at 0, 100, 300, and 500 ppm for 21 days (three times the normal treatment duration). Clinical observations were recorded twice daily. Feed consumption, feed conversion rate, and body weights were determined for each pen. Three birds from each pen were randomly selected for necropsy, histopathology, and clinical pathology. Birds were carefully examined for feathering abnormalities immediately following euthanasia. The remaining birds in each pen were submitted for drug concentration analysis so that concentrations (for low vs. high treatment levels) could be correlated with clinical observations, clinical pathology, and histologic findings. There no morbidities or mortalities after study day--1. There were no statistically significant treatment-related differences in feed consumption, feed conversion rates, body weights, serum biochemistry profiles, hematologic profiles, gross necropsy findings, histopathologic examination, and feathering. Allowable liver and muscle concentrations of fenbendazole sulfone in turkeys are 6 and 2 ppm, respectively, with a 6-hr feed withdrawal. Analysis of fenbendazole concentrations in kidney, liver, leg/thigh, and breast muscle and skin with associated fat revealed that, even at the highest dose level used and with no feed withdrawal, fenbendazole concentrations were relatively low in these tissues. These findings indicate that fenbendazole has a relatively wide margin of safety in young pheasants and that the proposed dose of 100 ppm in the feed for 7 consecutive days is well within the margin of safety. In the reproductive safety study, two large game bird farms fed fendbendazole at 100 ppm for 7 days and collected data on hatching percentage of pheasant eggs before and after treatment. Reproductive performance in hen pheasants was not adversely affected.

Levetiracetam rectal administration in healthy dogs.

BACKGROUND: Levetiracetam is used to manage status epilepticus (SE) and cluster seizures (CS) in humans. The drug might be absorbed after rectal administration and could offer a practical adjunct to rectal administration of diazepam in managing SE and CS. HYPOTHESIS: Levetiracetam is rapidly absorbed after rectal administration in dogs and maintains target serum concentrations for at least 9 hours. ANIMALS: Six healthy privately owned dogs between 2 and 6 years of age and weighing 10-20 kg. METHODS: Levetiracetam (40 mg/kg) was administered rectally and blood samples were obtained immediately before (time zero) and at 10, 20, 40, 60, 90, 180, 360, and 540 minutes after drug administration. Dogs were observed for signs of adverse effects over a 24-hour period after drug administration. RESULTS: CLEV at 10 minutes was 15.3 ± 5.5 µg/mL (mean, SD) with concentrations in the target range (5-40 µg/mL) for all dogs throughout the sampling period. Cmax (36.0 ± 10.7 µg/mL) and Tmax (103 ± 31 minutes) values were calculated and 2 disparate groups were appreciated. Dogs with feces in the rectum at the time of drug administration had lower mean Cmax values (26.7 ± 3.4 µg/mL) compared with those without (45.2 ± 4.4 µg/mL). Mild sedation was observed between 60 and 90 minutes without other adverse effects noted. CONCLUSIONS AND CLINICAL IMPORTANCE: This study supports the use of rectally administered levetiracetam in future studies of clinical effectiveness in the management of epileptic dogs.

Pharmacokinetics and pharmacodynamics of glycopyrrolate following a continuous-rate infusion in the horse.

Glycopyrrolate (GLY) is an antimuscarinic agent that is used in humans and domestic animals primarily to reduce respiratory tract secretions during anesthesia and to reverse intra-operative bradycardia. Although GLY is used routinely in veterinary patients, there is limited information regarding its pharmacokinetic (PK) and pharmacodynamic (PD) properties in domestic animals, and an improved understanding of the plasma concentration-effect relationship in racehorses is warranted. To accomplish this, we characterize the pharmacokinetic-pharmacodynamic (PK-PD) actions of GLY during and after a 2-h constant-rate intravenous infusion (4 µg/kg/h) and evaluate potential PK-PD models for cardiac stimulation in adult horses. Measurements of plasma GLY concentrations, heart and respiration rates, and frequency of bowel movements were performed in six Thoroughbred horses. The time course for GLY disposition in plasma followed a tri-exponential equation characterized by rapid disappearance of GLY from blood followed by a prolonged terminal phase. Physiological monitoring revealed significant (P < 0.01) increases in heart (>70 bpm) and respiratory rates accompanied by a marked and sustained delay in the frequency of bowel movements (1.1 ± 0.2 h [saline group] vs. 6.0 ± 2.0 h [GLY group]). Two of six horses showed signs of colic during the 8-h observation period after the end of the GLY infusion, but were treated and recovered without further complications. The relationship between plasma GLY concentration and heart rate exhibited counterclockwise hysteresis that was adequately described using an effect compartment.

Disposition of desfuroylceftiofur acetamide in serum, placental tissue, fetal fluids, and fetal tissues after administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis.

The objective of this study was to determine the pharmacokinetics of CCFA in mares with placentitis and evaluate the disposition of the drug in fetal fluids, fetal membranes, colostrum, and serum of foals. A secondary objective was to obtain pilot data regarding the efficacy of CCFA for improving foal survival in mares with placentitis. Twelve pregnant pony mares were enrolled in the study, inoculated with Streptococcus zooepidemicus, intracervically and assigned to one of three groups: CEFT (n = 3; administered CCFA only; 6.6 mg/kg, i.m., q96h); COMBO (n = 6; administered combination therapy of CCFA, altrenogest, and pentoxifylline); UNTREAT (n = 3, no treatment). Treatment was initiated at the onset of clinical signs. Concentrations of desfuroylceftiofur acetamide (DCA), the acetamide derivative of ceftiofur and desfuroylceftiofur metabolites, were measured using high-performance liquid chromatography. Maximum and minimum serum concentrations of DCA at steady state in treated mares were 2.40±0.40 µg/mL and 1.06±0.29 µg/mL, respectively. Concentration of DCA in colostrum was 1.51±0.60 µg/mL. DCA concentrations in placenta and fetal tissues were very low (median = 0.03 µg/mL) and below the minimum inhibitory concentration of relevant pathogens. DCA was not detected in amniotic fluid or foal serum. Treatment did not appear to improve foal survival (CEFT: 0/3; COMBO: 2/6; UNTREAT: 2/3). Bacteria were recovered from the uterus of most mares postpartum and from blood cultures of most foals regardless of treatment.

Treatment efficacy of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis.

The objective was to determine if long-term treatment with trimethoprim sulfamethoxazole (antimicrobial), pentoxifylline (anti-inflammatory/anti-cytokine) and altrenogest (synthetic progestin), would improve pregnancy outcome in mares with experimentally induced placentitis. Seventeen normal, pregnant pony mares were enrolled in the study at 280-295 d of pregnancy. Placentitis was induced in all mares by intra-cervical inoculation of Streptococcus equi subsp. zooepidemicus (10(7) CFU). Five mares served as infected, untreated control animals (Group UNTREAT). Twelve mares (Group TREAT) were infected and given trimethoprim sulfamethoxazole (30 mg/kg, PO, q 12h), pentoxifylline (8.5 mg/kg, PO, q 12h) and altrenogest (0.088 mg/kg, PO, q 24h) from the onset of clinical signs to delivery of a live foal or abortion. Blood samples were cultured from all foals at delivery and fetal stomach and thoracic contents were obtained for culture from dead fetuses. More mares in Group TREAT delivered viable foals (10/12; 83%; P < 0.05) than mares in Group UNTREAT (0/5; 0%). Ten of 12 foals (83%) in Group TREAT had negative blood cultures at birth. All foals in Group UNTREAT (5/5; 100%) had positive cultures from one or more samples (blood, stomach contents, and thoracic fluid). Bacteria were recovered from uterine culture samples in both groups. Streptococcus equi subsp. zooepidemicus was the predominant organism recovered from fetal/foal or mare culture samples. The authors inferred that administration of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest may improve the viability of foals from mares with experimentally induced placentitis.

Caffeine-induced hyperactivity in the horse: comparisons of drug and metabolite concentrations in blood and cerebrospinal fluid.

The goals of this study were to elucidate the temporal and quantitative relationships between caffeine and its major bioactive metabolites in blood and cerebrospinal fluid (CSF) and to characterize the pharmacokinetic-pharmacodynamic relationship for caffeine-induced changes in spontaneous locomotor activity in the horse. We hypothesized that caffeine and its metabolites distribute efficiently into the CSF to antagonize adenosine A1 and A2a receptors and that spontaneous locomotor activity correlates well with caffeine and/or metabolite concentrations in CSF and blood. A microdialysis system was developed to allow simultaneous monitoring of locomotor activity and collection of CSF and blood samples for pharmacokinetic analysis. CSF concentrations of caffeine and its metabolites were evaluated to determine the percentage of central adenosine receptor blockade by the established standard inhibition curves. Caffeine increased the spontaneous locomotor activity for up to 4 h in a dose-dependent manner. After 3 mg/kg caffeine administration, blood caffeine concentration as well as locomotor activity increased sharply to near peak level while CSF caffeine concentrations exhibited a slow rise to a steady-state 75 min later. High correlation coefficient was found between locomotor activity and caffeine concentrations in blood (R(2 )=0.95) and in CSF (R(2) = 0.93). At 3 mg/kg dosage, theophylline was the only detectable caffeine metabolite in the CSF. The concentrations reached in the CSF were sufficient to partially block central adenosine A1 (14% blockade) and A2a (11% blockade) receptors. There were no statistically significant differences between the pharmacokinetics of caffeine in the blood and CSF. This study provides novel evidence that locomotor stimulation in horses is closely correlated with caffeine concentrations in the blood and CSF and, furthermore, is consistent with blockade of central adenosine receptors.

The effect of codeine on the Urge-to-Cough response to inhaled capsaicin.

We have shown previously in normal subjects that a sensory measure, the Urge-to-Cough rating, increases at concentrations of inhaled capsaicin that are lower than those necessary to elicit reflex cough. This finding suggests that the Urge-to-Cough may represent an index of the cough response. Research on cough in the human has most often employed challenge with inhaled capsaicin to induce reflex cough. Current measures of cough sensitivity in the human provide no information regarding the intensity of cough. The influence of codeine on cough perceptual sensitivity and the relationship to cough intensity with capsaicin-induced cough in normal subjects has not been evaluated. This study determined the effect of codeine on capsaicin-induced cough perceptual sensitivity and motor response in normal subjects in a double-blind, placebo-controlled, crossover study. This approach investigated the relevance of cough sensitivity, intensity, and sensory modalities in the assessment of cough suppression in humans. This study consisted of three experimental trials: administration of placebo, 30 mg codeine and 60 mg codeine. The study was double-blinded. The order of the three trials was randomized. Respiratory motor pattern was recorded with EMGs from the rectus abdominis, lateral abdominal muscles and eighth intercostal space. The subjects leaned into a fume hood to inspire deeply for 2 s once through a mouthpiece connected to the nebulizer. A modified Borg scale was used to estimate their Urge-to-Cough. The experimental trial consisted of eight test solutions of 0-200 microM capsaicin. Each solution was presented three times in a randomized block order for a total of 24 presentations. The lowest capsaicin concentration to elicit a cough was determined. The lowest capsaicin concentration to elicit an Urge-to-Cough greater than zero was identified. The Urge-to-Cough sensitivity was determined from the log-log slope. For placebo, the Urge-to-Cough was zero with inhalation of the vehicle and no coughs were observed. The threshold capsaicin concentration for subjects to report an Urge-to-Cough was 15.6 microM (+/-2.6 SEM). The capsaicin concentration threshold for eliciting a cough was significantly greater, 39.3 microM (+/-5.6 SEM). As the capsaicin concentration increased, the magnitude estimation of the Urge to-Cough increased. The slope of the log-log relationship for the Urge-to-Cough was 0.94 (+/-0.07 SEM). As the capsaicin concentration increased, the number and intensity of the coughs increased. The administration of 30 and 60 mg codeine had no significant effect on the threshold capsaicin concentration for the Urge-to-Cough. There was also no significant codeine effect on the slope of the log-log Urge-to-Cough relationship. Thirty and sixty milligram codeine had no significant effect on the relationship between the capsaicin concentration and the number and intensity of the coughs. The results of this study demonstrate that the threshold for a subject to perceive an Urge-to-Cough was less than the capsaicin concentration that elicits the cough motor response. There was a direct relationship between the sensory intensity (magnitude estimation of the Urge-to-Cough) and the cough number and intensity. Thus, as the sense of an Urge-to-Cough increased the cough motor response increased. Neither the 30 nor 60 mg codeine affected the perceptual or motor sensitivity to capsaicin-induced cough. These results showed that the initial threshold for responding to capsaicin-induced cough is the perception of an Urge-to-Cough, followed by a motor cough response if the capsaicin is increased above the perceptual threshold. As the capsaicin concentration increases, both the perceptual need to cough and the cough motor response increase. The response of subjects to inhalation of capsaicin consisted of both a sensory component leading to perception of an Urge-to-Cough and motor cough behavior.

Capsaicin exposure elicits complex airway defensive motor patterns in normal humans in a concentration-dependent manner.

The airway defensive response to tussive agents, such as capsaicin, is frequently assessed by counting the number of cough sounds, or expulsive events. This method does not identify or differentiate important respiratory events that occur in the respiratory muscles and lungs, which are critical in assessing airway defensive responses. The purpose of this study was to characterize the airway defensive behaviours (cough and expiration reflex) to capsaicin exposure in humans. We observed complex motor behaviours in response to capsaicin exposure. These behaviours were defined as cough reacceleration (CRn) and expiration reflex (ERn), where n is the number of expulsive events with and without a preceding inspiratory phase, respectively. Airway defensive responses were defined in terms of frequency (number of expulsive events), strength (activation of abdominal muscles) and behaviour type (CRn vs. ERn). Thirty-six subjects (15 females, 24+/-4 yr) were instrumented with EMG electrodes placed over the rectus abdominis (RA), external abdominal oblique (EO) and the 8th intercostal space (IC8). A custom-designed mouth pneumotachograph was used to assess the airflow acceleration, plateau velocity and phase duration of the expulsive phase. Subjects inhaled seven concentrations of capsaicin (5-200 microM) in a randomized block order. The total number of expulsive events (frequency) and the sum of integrated EMG for the IC8, RA and EO (strength) increased in a curvilinear fashion. Differentiating the airway defense responses into type demonstrated predominately CR1 and CR2 (i.e. inspiration followed by one and two expulsive events, respectively) with very few ER's at <50 microM capsaicin. At higher concentrations (>50 microM) ER's with one or more expulsive events (ER1) appeared, and the number of CR's with three or more expulsive events (CR3) increased. The decrease in EMG activation and airflow measurements with each successive expulsive event suggests a decline in power and shear force as the number of expulsive events increased. Therefore, the airway defensive response to capsaicin is a complex motor pattern that functions to coordinate ER's and CR's with differing numbers of expulsive events possibly to prevent aspirations and keep air moving to promote clearance.

Continuous monitoring of penicillin G and gentamicin in allantoic fluid of pregnant pony mares by in vivo microdialysis.

REASONS FOR PERFORMING STUDY: Most current treatments for placentitis in mares are empirical with few control studies to evaluate their effectiveness. OBJECTIVE: To monitor drug concentrations in allantoic fluid of pregnant pony mares using in vivo microdialysis and establish if this method would be useful for determining allantoic concentrations of drugs in normal mares and those with placentitis. METHODS: Five late gestational pony mares had microdialysis probes inserted into the allantoic fluid using transabdominal ultrasound-guided allantocentesis. Single injections of penicillin G (22,000 u/kg), gentamicin (6.6 mg/kg bwt) and flunixin meglumine (1 mg/kg bwt) were administered i.v. and dialysate samples collected continuously for 24 h. In a separate study, drug concentrations were monitored in allantoic fluid of 2 mares with experimental placentitis induced by intracervical inoculation with Streptococcus equi ssp. zooepidemicus. Drug concentrations were measured by high performance liquid chromatography (penicillin G, flunixin meglumine) or enzyme-linked immunosorbent assay (gentamicin). RESULTS: Penicillin G and gentamicin achieved average peak concentrations of 9.8+/-2.2 and 8.5+/-3.1 microg/ml, respectively, in allantoic fluid of noninfected mares. Pharmacokinetic comparisons indicate that penicillin G persists much longer in allantoic fluid than blood, whereas gentamicin exhibited similar profiles in the 2 compartments. Flunixin meglumine was not detected in allantoic fluid. In infected mares, penicillin G achieved a similar peak concentration in allantoic fluid (11.2 microg/ml) whereas peak gentamicin concentration (3.9 microg/ml) appeared to be reduced relative to drug concentrations in noninfected mares. CONCLUSIONS: Microdialysis is a useful technique for continuous in vivo monitoring of drugs in equine allantoic fluid. Our results indicate that penicillin G and gentamicin undergo effective placental transfer in pregnant mares and in 2 mares that transplacental drug transfer may be altered selectively if active placental infection is present. POTENTIAL RELEVANCE: Further studies are needed to evaluate the feasibility of using increased dose intervals for penicillin G and an increased dose rate of gentamicin to effectively combat placental infections in mares.

Pharmacokinetics and pharmacodynamics of pantoprazole in clinically normal neonatal foals.

REASONS FOR PERFORMING STUDY: Proton pump inhibitors (PPIs) are a mainstay of treatment for acid-related ulceration in man and horses. Currently, only an oral preparation of omeprazole is approved for use in horses in the USA. Intravenous administration of a PPI would provide a useful therapeutic alternative for those foals in which oral medication is not feasible. OBJECTIVE: To investigate the pharmacokinetics and pharmacodynamics of pantoprazole following i.v. or intragastric administration in healthy neonatal foals. METHODS: Seven healthy foals age 6-12 days at the start of the study were evaluated. Treatments included no drug administration, i.v. pantoprazole (1.5 mg/kg bwt) and intragastric pantoprazole (1.5 mg/kg bwt). Intragastric pH was recorded for 24 h after drug administration for pharmacodynamic evaluation. Plasma pantoprazole concentrations were measured using high-performance liquid chromatography. RESULTS: Plasma concentrations of pantoprazole were detectable at the 5 min sampling point following i.v. or intragastric administration. Bioavailability of intragastric-administered pantoprazole was 41%. Baseline mean hourly pH was 1.5-6.1. There was a statistically significant increase in mean hourly pH relative to untreated foals 2-24 h after i.v. or intragastric pantoprazole administration. CONCLUSIONS: Based on these data, i.v. or intragastric administration of pantoprazole results in a significant, prolonged increase in intragastric pH. POTENTIAL RELEVANCE: The i.v. formulation of pantoprazole may provide a clinically useful alternative means of acid suppression in foals unable to tolerate enteral administration of a PPI, such as those with pyloric outflow obstruction.

Continuous measurement of caffeine and two metabolites in blood and skeletal muscle of unrestrained adult horses by semi-automated in vivo microdialysis.

Concentrations of caffeine (CA) and two metabolites were measured simultaneously in venous blood and splenius muscle of adult horses using a semi-automated in vivo microdialysis sampling technique. Dialysates from muscle and jugular vein were collected continuously for 48 h and drug levels were determined by high performance liquid chromatography (HPLC). Following i.v. injection, CA (3 mg/kg) attained a peak blood level of nearly 5400 +/- 600 ng/mL and decreased with a half-life of 15.3 +/- 0.7 h. Pharmacokinetic and statistical comparisons between CA concentrations in jugular dialysates and plasma samples revealed no significant differences between these sampling techniques. However, measurements in muscle and blood revealed unexpected pharmacokinetic differences, including significantly elevated concentrations of CA in muscle for 4 h following drug administration. In contrast, the CA metabolites theophylline (TP) and theobromine (TB) exhibited delayed appearances in muscle and blood with peak concentrations of 300 +/- 60 ng/mL (TP) and 150 +/- 50 ng/mL (TB) detected in both tissues 1 day following CA administration. This study demonstrates that our novel semi-automated microdialysis procedure for continuous monitoring of drug and metabolite levels may be useful for related studies in other domesticated large animal species.

Mixing in process vessels used in biopharmaceutical manufacturing.

The use of nonbaffled vessels for mixing applications is becoming common in the biopharmaceutical industry but is not sufficiently well studied. Orientation of the impellers off-centered and/or at an angle is necessary to enhance mixing and eliminate swirling that would result without a baffle in a standard tank. This study focuses on characterizing mixing in vessels with the hydrofoil axial flow impellers mounted off-center at 10 degrees to the vertical. Geometrically similar vessels ranging from 100 to 5000 L working volume were used in this study. Mixing performance was successfully correlated to vessel geometric factors.

Chronic ethanol exposure increases 3H-GABA release in rat hippocampus by presynaptic muscarinic receptor modulation.

BACKGROUND: Chronic ethanol treatment (CET) for 28 weeks significantly increases electrically-stimulated 3H-GABA release from hippocampal slices. This increase in GABA release may be one of the mechanisms by which CET decreases the magnitude of long-term potentiation (LTP) in the hippocampus. The present study examined whether CET increases GABA release via an alteration in heterologous presynaptic cholinergic regulation. METHODS: Animals were treated with ethanol or sucrose diet for 28 weeks followed by either no withdrawal or a 48-hr withdrawal period. The electrically-stimulated 3H-GABA release from preloaded superfused hippocampal slices of naive and CET rats was measured. RESULTS: Carbachol increased 3H-GABA release in a concentration-dependent manner, and atropine modulated 3H-GABA release in a biphasic concentration-dependent manner. Atropine (10 microM) significantly blocked the effects of carbachol. Oxotremorine, a selective muscarinic receptor agonist, also increased 3H-GABA release. Mecamylamine, a selective nicotinic antagonist, did not modulate 3H-GABA release and did not block the effects of carbachol. The effects of these agents were also tested in rats 0 or 48 hrs after withdrawal from CET. The biphasic effects of atropine were decreased, whereas the facilitating effects of carbachol were significantly increased. There were no changes in the effects of these agents on 3H-acetylcholine release from hippocampal slices of CET rats compared to sucrose-treated rats. CONCLUSION: These results suggest that presynaptic muscarinic receptors facilitate GABA release, whereas nicotinic receptors do not play a significant role in modulating GABA release in hippocampus. CET selectively alters presynaptic muscarinic regulation of GABA release in hippocampus and may help us to further understand the mechanism underlying the disruption of LTP by CET.

Reversible activation of glutamate transport in rat brain glia by protein kinase C and an okadaic acid-sensitive phosphoprotein phosphatase.

High-affinity L-glutamate (GLU) transport is an important regulator of excitatory amino acid (EAA) concentrations in brain extracellular fluid and may play a key role in excitatory synaptic transmission. In view of evidence that EAA transporters (EAAT) are heterogenous and contain consensus sites for phosphorylation, this investigation was undertaken to contrast the effects of transporter phosphorylation in fractions derived from glia and neurons (synaptosomes) of the adult rat forebrain. Treatment with phorbol-12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), increased the maximal rate of GLU transport in glial plasmalemmal vesicles by greater than 50 percent (237+/-18 vs. 365+/-27 pmol/mg protein/90s, p < 0.05) but caused no change in synaptosomes. The effect by PDBu was concentration and time-dependent and was inhibited completely by the PKC inhibitor calphostin C. Inhibition of serine-threonine phosphoprotein phosphatases with okadaic acid produced similar effects which were not additive with PDBu. Together, these results demonstrate that glial EAAT can be regulated by multiple phosphorylation processes.

Rapid determination of aspartate enantiomers in tissue samples by microdialysis coupled on-line with capillary electrophoresis.

Microdialysis was coupled on-line with derivatization by o-phthalaldehyde and beta-mercaptoethanol and optically gated capillary electrophoresis to determine D- and L-aspartate in tissue samples obtained from rats. The microdialysis probe was inserted into a homogenized tissue sample which allowed generation of a continuous sample stream that was filtered and deproteinated. With 7.5 mM beta-cyclodextrin (CD) in the electrophoresis buffer, the enantiomers of interest could be resolved in 3 s with an electric field of 2500 V/cm over a separation length of 15 mm. Values of D- and L-aspartate in different tissues agreed well with those obtained by an HPLC procedure that required protein precipitation, centrifugation, and extraction. The speed and compatibility with automation of the microdialysis/CE method may make it a general approach for a variety of applications involving high-throughput analysis or sensorlike operation.

Selective loss of phorbol-12,13-dibutyrate-facilitated L-glutamate transport in forebrain neurons of aged rats.

Phorbol-12,13-dibutyrate (PDBu), an activator of protein kinase C, was used to evaluate potential age-related changes in phosphorylation-dependent facilitation of high-affinity L-glutamate uptake in the rat central nervous system (CNS). Forebrain homogenates from male Fischer-344/brown Norway F1 hybrid rats were separated into glia-enriched (glial plasmalemmal vesicles) and neuron-enriched fractions (synaptosomes) and assayed for sodium-dependent transport of L-[3H]glutamate. Glial fractions from rats aged 5, 25, 31, and 37 months exhibited similar rates of basal L-[3H]glutamate transport and demonstrated no significant age-related differences with respect to the maximal facilitatory effect of PDBu (1-100 microM). In contrast, neuronal fractions exhibited an age-related decline in both indices, with basal L-[3H]glutamate transport decreasing from 710+/-31 to 560+/-40 pmoL/mg protein/90 s for the 5- and 37-month groups, respectively (p < .03) and PDBu having a significantly attenuated effect in aged animals. Together, these results provide support for the hypothesis that aging is associated with a decrease in the number of neuronal L-glutamate transporters as well as a diminished capacity to up-regulate these transporters through a protein kinase C-dependent pathway.

Development and use of an enzyme-linked immunosorbent assay to monitor serum and urine acepromazine concentrations in thoroghbreds, and possible changes associated with exercise.

OBJECTIVES: To develop an ELISA that is sensitive and suitable for measurement of immunoreactive acepromazine (ACP) in horse serum and urine and to determine the acute effects of exercise on immunoreactive ACP values in Thoroughbreds. ANIMALS: 12 healthy Thoroughbreds (5 mares, 5 geldings, 2 stallions), aged 2 to 8 years. PROCEDURE: A commercially available antibody and a horseradish peroxidase-conjugated oxime derivative of immunoreactive ACP were used to develop a one-step ELISA. Horses were used in a crossover design study to evaluate possible effects of treadmill exercise on serum and urine ACP concentrations after a single (25 mg) IM injection of the drug. RESULTS: Immunoreactive ACP was detectable at concentrations as low as 50 pg/ml in serum and 100 pg/ml in urine, with intra- and interassay variabilities of 1.1 and 5.2%, respectively. The antibody had some cross-reactivity with a limited number of other phenothiazines. After drug administration, serum ACP immunoreactivity achieved a peak concentration (10.5 ng/ml) within 30 minutes and could be measured up to 48 hours in serum and 120 hours in urine. Although exercise had no significant effect on serum drug concentration, immunoreactive ACP disappeared more quickly (by 48 hours) from the urine of horses in the exercised group. CONCLUSIONS: This one-step ELISA provides a simple and sensitive means to measure immunoreactive ACP in equine serum and urine. The ability to detect drug several days after administration of a low dose of ACP should augment efforts to control illicit use of this drug in performance horses. Potential changes in ACP kinetics after exercise warrant further study.