Comparing the glucuronidation capacity of the feline liver with substrate‐specific glucuronidation in dogs

This study aimed to assess the overall glucuronidation capacity of cats, using prototypic substrates identified for human UDP‐glucuronosyltransferases (UGTs). To this end, Michaelis–Menten kinetics were established for the substrates using feline hepatic microsomal fractions, and results were compared with similar experiments carried out with dog liver microsomes. Cats are known for their low capacity of glucuronide formation, and UGT1A6 was found to be a pseudogene. However, functional studies with typical substrates were not performed and knowledge of the enzymology and genetics of other glucuronidation enzymes in felidae is lacking. The results of this study showed extremely low formation of naphthol‐1‐glucuronide (1.7 ± 0.4 nmol/mg protein/min), estradiol‐17‐glucuronide (<0.7 nmol/mg protein/min), and morphine‐3‐glucuronide (0.2 ± 0.03 nmol/mg protein/min), suggesting a lack of functional UGT1A6 and UGT2B7 homologues in the cat's liver. Dog liver microsomes were producing these glucuronides in much higher amounts. Glucuronide capacity was present for the substrates 17β‐estradiol (estradiol‐3‐glucuronide, 2.9 ± 0.2 nmol/mg protein/min) and 4‐methylumbelliferone (31.3 ± 3.3 nmol/mg protein/min), assuming that cats have functional homologue enzymes to at least the human UGT1A1 and probably other UGT1A isozymes. This implies that for new drugs, glucuronidation capacity has to be investigated on a substance‐to‐substance base. Knowledge of the glucuronidation rate of a drug provides the basis for pharmacokinetic modeling and as a result proper dosage regimens can be established to avoid undesirable drug toxicity in cats.     

Integrated pharmacological assessment of flubendazole potential for use in sheep: disposition kinetics, liver metabolism and parasite diffusion ability

Flubendazole (FLBZ) is a broad spectrum benzimidazole methylcarbamate anthelmintic widely used in poultry and swine. However, there is no information available on the pharmacological behaviour of FLBZ in ruminants. The work reported here was addressed to evaluate the potential of FLBZ for use in sheep. The integrated assessment included evaluation of FLBZ and metabolites plasma disposition kinetics, liver metabolism and ex vivo ability to diffuse into the cestode parasite Moniezia benedeni. In a cross-over kinetic study, six healthy Corriedale sheep were treated with FLBZ by intravenous (i.v.) (4% solution) and intraruminal (i.r.) (4% suspension) administrations at the same dosage (5 mg/kg) with a 21-day washout period between treatments. Blood samples were collected between 0 and 72 h post-treatments. Sheep liver microsomes were incubated with 40 microm FLBZ and specimens of the cestode parasite M. benedeni, collected from untreated animals, were incubated (5-120 min) with FLBZ and its reduced (R-FLBZ) metabolite (5 microm). Samples of plasma, microsomal incubations and parasite material were prepared and analyzed by high-performance liquid chromatography to measure FLBZ and its metabolites. FLBZ parent drug showed a fast disposition being detected in the bloodstream up to 36 h after its i.v. administration. Both R-FLBZ and hydrolyzed FLBZ (H-FLBZ) metabolites were recovered in plasma as early as 5 min after the i.v. treatment in sheep. The plasma AUC ratios for R-FLBZ and FLBZ (AUC(R-FLBZ)/AUC(FLBZ)) were 4.07 i.v. and 5.55 i.r., respectively. R-FLBZ achieved a significantly higher (P < 0.01) C(max) value (0.14 microg/mL at 17.3 h post-treatment) than that observed for the parent drug FLBZ (0.04 microg/mL at 14.4 h post-treatment). Low plasma concentrations of FLBZ parent drug were measured between 6 and 48 h, and only trace concentrations of H-FLBZ were detected during a short period of time after the i.r. treatment. Consistently, sheep liver microsomes metabolized FLBZ into its reduced metabolite at a rate of 9.46 +/- 2.72 nmol/mg/h. Both FLBZ and R-FLBZ demonstrated a similar ability to quickly diffuse through the tegument of the cestode parasite. The data on FLBZ pharmacological behaviour presented here contribute to evaluate its potential to be developed as an anthelmintic for broad spectrum parasite control in ruminants.     

Hepatic biotransformation pathways and ruminal metabolic stability of the novel anthelmintic monepantel in sheep and cattle

Monepantel (MNP) is a new amino‐acetonitrile derivative anthelmintic drug used for the treatment of gastrointestinal (GI) nematodes in sheep. The present work investigated the main enzymatic pathways involved in the hepatic biotransformation of MNP in sheep and cattle. The metabolic stability in ruminal fluid of both the parent drug and its main metabolite (monepantel sulphone, MNPSO₂) was characterized as well. Additionally, the relative distribution of both anthelmintic molecules between the fluid and particulate phases of the ruminal content was studied. Liver microsomal fractions from six (6) rams and five (5) steers were incubated with a 40 μm of MNP. Heat pretreatment (50 °C for 2 min) of liver microsomes was performed for inactivation of the flavin‐monooxygenase (FMO) system. Additionally, MNP was incubated in the presence of 4, 40, and 80 μm of methimazole (MTZ), a FMO inhibitor, or equimolar concentrations of piperonyl butoxide (PBx), a well‐known general cytochrome P450 (CYP) inhibitor. In both ruminant species, MNPSO₂ was the main metabolite detected after MNP incubation with liver microsomes. The conversion rate of MNP into MNPSO₂ was fivefold higher (P < 0.05) in sheep (0.15 ± 0.08 nmol/min·mg) compared to cattle. In sheep, the relative involvement of both FMO and CYP systems (FMO/CYP) was 36/64. Virtually, only the CYP system appeared to be involved in the production of MNPSO₂ in cattle liver. Methimazole significantly reduced (41 to 79%) the rate of MNPSO₂ production in sheep liver microsomes whereas it did not inhibit MNP oxidation in cattle liver microsomes. On the other hand, PBx inhibited the production of MNPSO₂ in liver microsomes of both sheep (58 to 98%, in a dose‐dependent manner) and cattle (almost 100%, independently of the PBx concentration added). The incubation of MNP and MNPSO₂ with ruminal contents of both species showed a high chemical stability without evident metabolism and/or degradation as well as an extensive degree of adsorption (83% to 90%) to the solid phase of the ruminal content. Overall, these results are a further contribution to the understanding of the metabolic fate of this anthelmintic drug in ruminants.     

Assessment of the pharmacological interactions between the nematodicidal fenbendazole and the flukicidal triclabendazole: In vitro studies with bovine liver microsomes and slices

Parasitic diseases have a significant impact on livestock production. Nematodicidal drugs, such as fenbendazole (FBZ) or its oxidized metabolite oxfendazole (OFZ), can be used along with the trematodicidal triclabendazole (TCBZ), to broaden the spectrum of anthelmintic activity. However, co‐exposure to these compounds could lead to drug–drug (D‐D) interactions and eventually alter the clinical profile of each active principle. The aim of this study was to assess the presence of such interactions by means of two in vitro models, namely bovine liver microsomal fractions and bovine precision‐cut liver slices (PCLSs). To this end, an in vitro assessment involving incubation of FBZ and TCBZ or a combination of FBZ and TCBZ was carried out. Results with microsomal fractions showed a 78.4% reduction (p = .002) in the rate of OFZ production upon co‐incubation, whereas the sulfoxide metabolite of TCBZ (TCBZSO) exhibited a decreasing tendency. With PCLS, OFZ accumulation in the incubation medium increased 1.8‐fold upon co‐incubation, whereas TCBZSO accumulation decreased by 28%. The accumulation of FBZ and OFZ in the liver tissue increased upon 2‐hr co‐incubation, from 2.1 ± 1.5 to 18.2 ± 6.1 (p = .0009) and from 0.4 ± 0.1 to 1.3 ± 0.3 nmol (p = .0005), respectively. These results confirm the presence of D‐D interactions between FBZ and TCBZ. Further studies are needed to determine the extent of involvement of drug‐metabolizing enzymes and membrane transporters in interactions between compounds largely used in livestock production systems.     

Hematological adverse effects and pharmacokinetics of ribavirin in pigs following intramuscular administration

Ribavirin (RBV) is a synthetic guanosine analog that is used as a drug against various viral diseases in humans. The in vitro antiviral effects of ribavirin against porcine viruses were demonstrated in several studies. The purposes of this study were to evaluate the adverse effects and pharmacokinetics of ribavirin following its intramuscular (IM) injection in pigs. Ribavirin was formulated as a double‐oil emulsion (RBV‐DOE) and gel (RBV‐Gel), which were injected into the pigs as single‐dose IM injections. After injection of RBV, all of the pigs were monitored. The collected serum and whole blood samples were analyzed by liquid chromatography–tandem mass spectrometry and complete blood count analysis, respectively. All of the ribavirin‐treated pigs showed significant decreases in body weight compared to the control groups. Severe clinical signs including dyspnea, anorexia, weakness, and depression were present in ribavirin‐treated pigs until 5 days postinjection (dpi). The ribavirin‐treated groups showed significant decrease in the number of red blood cells and hemoglobin concentration until 8 dpi. The mean half‐life of the RBV‐DOE and RBV‐Gel was 27.949 ± 2.783 h and 37.374 ± 3.502 h, respectively. The mean peak serum concentration (C_max) and area under the serum concentration–time curve from time zero to infinity (AUC_inf) of RBV‐DOE were 8340.000 ± 2562.577 ng/mL and 16 0095.430 ± 61 253.400 h·ng/mL, respectively. The C_max and AUC_inf of RBV‐Gel were 15 300.000 ± 3764.306 ng/mL and 207526.260 ± 63656.390 h·ng/mL, respectively. The results of this study provided the index of side effect and pharmacokinetics of ribavirin in pigs, which should be considered before clinical application.     

Comparison of mucosal drug conjugative rates along the gastrointestinal tract of female sheep

The comparative distribution of p-nitrophenol UDP-glucuronosyl-transferase, 1-chloro-2,4-dinitrobenzene glutathione-S-transferase and sulphamethazine N-acetyltransferase activities was studied along the gastrointestinal mucosa of female Lacaune sheep. Gastrointestinal mucosa was characterized by a very low and unequal N-acetyltransferase activity when activities were expressed per g of wet organ. The duodenum contained highest activities (4.1 nmol/g min). When results were expressed per mg of cytosolic protein, the duodenal activity (0.64 nmol/mg min) was sixfold higher than in liver (0.11 nmol/mg min). There was a lack in N-acetyltransferase activity accepting isoniazid as substrate. Glucuronosyltransferase activity was approximately threefold higher in microsomal fractions of the mucosal lining of gastric and colonic intestine (0.43-0.58 nmol/g min) than in small intestine or caecum (0.10-0.26 nmol/mg min). Concerning cytosolic glutathione S-transferase activity, two- to threefold higher activities were obtained in omasum, jejunum, duodenum and ileum (1021-2164 nmol/g min) than in other parts (341-799 nmol/g min) when results were expressed per g of wet organ. These data were compared with corresponding hepatic activities determined in the same six female sheep.     

Species differences in the hepatic biotransformation of zearalenone

Zearalenone (ZEA), a Fusarium toxin, is frequently found in animal feed materials. It is known to exert oestrogenic effects in all animals tested but susceptibility varies between species, possibly reflecting differences in the metabolic processing of ZEA, which predominantly involves hydroxylations, assumed to be catalysed by 3alpha- and 3beta- hydroxysteroid dehydrogenases, as well as conjugation with glucuronic acid. In this study, the biotransformation of ZEA by hepatic subcellular fractions of various domestic animals was investigated and compared to the rat. Notable inter-species differences in terms of the rate of absolute and relative metabolite production in the different subcellular fractions were identified. The highest amount of alpha-zearalenol (alpha-ZOL) was produced by pig hepatic microsomes (V(max)=795.8+/-122.7pmol/mg/min), whereas in chicken microsomes the highest amounts of beta-zearalenol (beta-ZOL) (V(max)=1524+/-29.7pmol/mg/min) could be measured. Except for sheep and cattle, the efficiency of alpha-ZOL production (expressed as the ratio of apparent V(max)/k(m)) was higher in the microsomal fraction compared to the post-mitochondrial fraction. In contrast, the apparent efficiency of beta-ZOL production was high in pigs, cattle, chickens and rats, but very low in sheep. Conjugation of ZEA with glucuronic acid was investigated, and the results indicated significant inter-species differences in the rate of glucuronidation, which was saturable at low concentrations in all species tested, except pigs. The significant differences between the percentages of glucuronidation of ZEA, alpha-ZOL, and beta-ZOL suggest not only differences in the affinity of the individual substrate, but might also indicate the presence of different isoforms of uridine diphosphate glucuronyl transferases (UDPGTs). The results are of clinical relevance, as they contribute to the understanding of the species-specific susceptibility towards exposure to ZEA.     

Pharmacokinetics of florfenicol after intravenous administration in Escherichia coli lipopolysaccharide‐induced endotoxaemic sheep

Experiments in different animal species have shown that febrile conditions, induced by Escherichia coli lipopolysaccharide (LPS), may alter the pharmacokinetic properties of drugs. The objective was to study the effects of a LPS‐induced acute‐phase response (APR) model on plasma pharmacokinetics of florfenicol (FFC) after its intravenous administration in sheep. Six adult clinically healthy Suffolk Down sheep, 8 months old and 35.5 ± 2.2 kg in body weight (bw), were distributed through a crossover factorial 2 × 2 design, with 4 weeks of washout. Pairs of sheep similar in body weight were assigned to experimental groups: Group 1 (LPS) was treated with three intravenous doses of 1 μg/kg bw of E. coli LPS before FFC treatment. Group 2 (control) was treated with an equivalent volume of saline solution (SS) at similar intervals as LPS. At 24 h after the first injection of LPS or SS, an intravenous bolus of 20 mg/kg bw of FFC was administered. Blood samples (5 mL) were collected before drug administration and at different times between 0.05 and 48.0 h after treatment. FFC plasma concentrations were determined by liquid chromatography. A noncompartmental pharmacokinetic model was used for data analysis, and data were compared using a Mann–Whitney U‐test. The mean values of AUC_0–∞ in the endotoxaemic sheep (105.9 ± 14.3 μg·h/mL) were significantly higher (P < 0.05) than values observed in healthy sheep (78.4 ± 5.2 μg·h/mL). The total mean plasma clearance (CL_T) decreased from 257.7 ± 16.9 mL·h/kg in the control group to 198.2 ± 24.1 mL·h/kg in LPS‐treated sheep. A significant increase (P < 0.05) in the terminal half‐life was observed in the endotoxaemic sheep (16.9 ± 3.8 h) compared to the values observed in healthy sheep (10.4 ± 3.2 h). In conclusion, the APR induced by the intravenous administration of E. coli LPS in sheep produces higher plasma concentrations of FFC due to a decrease in the total body clearance of the drug.     

Pharmacokinetics,metabolism and excretion of celecoxib,a selective cyclooxygenase‐2 inhibitor,in horses

Celecoxib, a nonsteroidal anti‐inflammatory drug, is frequently used to treat arthritis in humans with minimal gastrointestinal side effect compared to traditional NSAIDs. The primary aim of this study was to determine the pharmacokinetic profile of celecoxib—a selective cyclooxygenase‐2 (COX‐2) inhibitor in horses. Six horses were administered a single oral dose of celecoxib at 2 mg/kg (body weight). After oral dosing, the drug reached a maximum concentration (mean ± SD) in blood of 1,088 ± 324 ng/ml in 4.58 hr. The elimination half‐life was 13.60 ± 3.18 hr, and the area under the curve was 24,142 ± 1,096 ng hr ml^?1. The metabolism of celecoxib in horses was via a single oxidative pathway in which the methyl group of celecoxib is oxidized to a hydroxymethyl metabolite and is further oxidized to form a carboxylic acid metabolite. Celecoxib is eliminated mainly through faeces as unchanged drug and as metabolites in urine. Therefore, instructions for a detection time following therapeutic dosing of celecoxib can be set by the racing practitioner and veterinarians to control illegal use in horse racing based on the results of this study.     

Purification of swine carbonic anhydrase isoenzyme III and measurement of its levels in tissues and plasma

The changes in the levels of carbonic anhydrase isozyme III (CA‐III) in swine plasma and urine have not been previously determined or reported. CA‐III is relatively specific to skeletal muscles, and should therefore be a useful diagnostic marker for muscle diseases. We isolated CA‐III from swine muscle tissues and determined CA‐III levels in the plasma and urine from both healthy and diseased pigs. The levels of CA‐III in the tissues of female swine (age, 3 months) and plasma of young swine (age, 1–5 months) and adult female pigs (age, 2–3 years) were determined using the ELISA system for swine CA‐III. The mean (± SD) levels of CA‐III in the skeletal muscles were 3.8 ± 3.2 mg/g (wet tissue), and in the plasma, 230 ± 193 ng/ml at 1 month, 189 ± 208 ng/ml at 2 months, 141 ± 148 ng/ml at 3 months, 78 ± 142 ng/ml at 4 months and 53 ± 99 ng/ml at 5 months. The mean level of CA‐III in the plasma samples from 2‐ to 3‐year‐old pigs was 18 ± 60 ng/ml. CA‐III in the plasma samples was found to decrease from 1 month until 3 years of age (p < 0.01). We performed far‐western blotting to clarify the cause of the observed decrease in CA‐III in plasma. Our results demonstrated that CA‐III is bound to the transferrin and albumin. In addition, we determined that the levels of CA‐III in plasma and urine samples were higher in diseased swine compared with the healthy pigs.     

Pharmacokinetics and dynamics of mycophenolate mofetil after single‐dose oral administration in juvenile dachshunds

Mycophenolate mofetil (MMF) is recommended as an alternative/complementary immunosuppressant. Pharmacokinetic and dynamic effects of MMF are unknown in young‐aged dogs. We investigated the pharmacokinetics and pharmacodynamics of single oral dose MMF metabolite, mycophenolic acid (MPA), in healthy juvenile dogs purpose‐bred for the tripeptidyl peptidase 1 gene (TPP1) mutation. The dogs were heterozygous for the mutation (nonaffected carriers). Six dogs received 13 mg/kg oral MMF and two placebo. Pharmacokinetic parameters derived from plasma MPA were evaluated. Whole‐blood mitogen‐stimulated T‐cell proliferation was determined using a flow cytometric assay. Plasma MPA C_max (mean ± SD, 9.33 ± 7.04 μg/ml) occurred at <1 hr. The AUC_0–∞ (mean ± SD, 12.84±6.62 hr*μg/ml), MRT_inf (mean ± SD, 11.09 ± 9.63 min), T1/2 (harmonic mean ± PseudoSD 5.50 ± 3.80 min), and k/d (mean ± SD, 0.002 ± 0.001 1/min). Significant differences could not be detected between % inhibition of proliferating CD5+ T lymphocytes at any time point (p = .380). No relationship was observed between MPA concentration and % inhibition of proliferating CD5+ T lymphocytes (R = .148, p = .324). Pharmacodynamics do not support the use of MMF in juvenile dogs at the administered dose based on existing therapeutic targets.     

The exogenous fluorophore,fluorescein, enables in vivo assessment of the gastrointestinal mucosa via confocal endomicroscopy: optimization of intravenous dosing in the dog model

This study described the pharmacokinetics of the intravenous fluorophore, fluorescein, and aimed to evaluate its utility for use in upper gastrointestinal confocal endomicroscopy (CEM). Six healthy, mature, mixed‐breed dogs were anesthetized and then dosed intravenously with fluorescein at 15 mg/kg. Blood samples were collected at predetermined time‐points. Dogs were examined by upper gastrointestinal confocal endomicroscopy and monitored for adverse effects. Plasma fluorescein concentrations were measured using high‐performance liquid chromatography (HPLC) with UV/Vis detection. Mean plasma concentration at 5 min was 57.6 ± 18.2 mg/L, and plasma concentrations decreased bi‐exponentially thereafter with a mean concentration of 2.5 mg/L ± 1.26 at 120 min. Mean terminal plasma elimination half‐life (t_½β) was 34.8 ± 8.94 min, and clearance was 9.1 ± 3.0 mL/kg/min. Apparent volume of distribution at steady‐state was 0.3 ± 0.06 L/kg. Fluorescein provided optimal fluorescent contrast to enable in vivo histologically equivalent evaluation of topologic mucosal morphology within the first 30 min following intravenous administration. Adverse effects were not observed. Based upon the calculated clearance, a constant rate infusion at a rate of 0.18 mg/kg/min is predicted to be adequate, following an initial loading dose (2 mg/kg), to maintain plasma concentration at 20 mg/L for optimal CEM imaging during the study period.     

Integration of pharmacokinetic–pharmacodynamic for dose optimization of doxycycline against Haemophilus parasuis in pigs

The aims of this study were to establish optimal doses of doxycycline (dox) against Haemophilus parasuis on the basis of pharmacokinetic–pharmacodynamic (PK‐PD) integration modeling. The infected model was established by intranasal inoculation of organism in pigs and confirmed by clinical signs, blood biochemistry, and microscopic examinations. The recommended dose (20 mg/kg b.w.) was administered in pigs through intramuscular routes for PK studies. The area under the concentration 0‐ to 24‐hr curve (AUC_0–24), elimination half‐life (T_½ke), and mean residence time (MRT) of dox in healthy and H. parasuis‐infected pigs were 55.51 ± 5.72 versus 57.10 ± 4.89 μg·hr/ml, 8.28 ± 0.91 versus 9.80 ± 2.38 hr, and 8.43 ± 0.27 versus 8.79 ± 0.18 hr, respectively. The minimal inhibitory concentration (MIC) of dox against 40 H. parasuis isolates was conducted through broth microdilution method, the corresponding MIC₅₀ and MIC₉₀ were 0.25 and 1 μg/ml, respectively. The Ex vivo growth inhibition data suggested that dox exhibited a concentration‐dependent killing mechanism. Based on the observed AUC_24 hr/MIC values by modeling PK‐PD data in H. parasuis‐infected pigs, the doses predicted to obtain bacteriostatic, bactericidal, and elimination effects for H. parasuis over 24 hr were 5.25, 8.55, and 10.37 mg/kg for the 50% target attainment rate (TAR), and 7.26, 13.82, and 18.17 mg/kg for 90% TAR, respectively. This study provided a more optimized alternative for clinical use and demonstrated that the dosage 20 mg/kg of dox by intramuscular administration could have an effective bactericidal activity against H. parasuis.     

The influence of Actinobacillus pleuropneumoniae infection on tulathromycin pharmacokinetics and lung tissue disposition in pigs

A tulathromycin concentration and pharmacokinetic parameters in plasma and lung tissue from healthy pigs and Actinobacillus pleuropneumoniae (App)‐infected pigs were compared. Tulathromycin was administered intramuscularly (i.m.) to all pigs at a single dose of 2.5 mg/kg. Blood and lung tissue samples were collected during 33 days postdrug application. Tulathromycin concentration in plasma and lung was determined by high‐performance liquid chromatography with tandem mass spectrometry (LC‐MS/MS) method. The mean maximum plasma concentration (C_max) in healthy pigs was 586 ± 71 ng/mL, reached by 0.5 h, while the mean value for C_max of tulathromycin in infected pigs was 386 ± 97 ng/mL after 0.5 h. The mean maximum tulathromycin concentration in lung of healthy group was calculated as 3412 ± 748 ng/g, detected at 12 h, while in pigs with App, the highest concentration in lung was 3337 ± 937 ng/g, determined at 48 h postdosing. The higher plasma and lung concentrations in pigs with no pulmonary inflammation were observed at the first time points sampling after tulathromycin administration, but slower elimination with elimination half‐life t_1/2el = 126 h in plasma and t_1/2el = 165 h in lung, as well as longer drug persistent in infected pigs, was found.     

Chemical Coding of Sensory Neurons Supplying the Hip Joint Capsule in the Sheep

Immunohistochemical properties of nerve fibres supplying the joint capsule were previously described in many mammalian species, but the localization of sensory neurons supplying this structure was studied only in laboratory animals, the rat and rabbit. However, there is no comprehensive data on the chemical coding of sensory neurons projecting to the hip joint capsule (HJC). The aim of this study was to establish immunohistochemical properties of sensory neurons supplying HJC in the sheep. The study was carried out on 10 sheep, weighing about 30–40 kg. The animals were injected with a retrograde neural tracer Fast Blue (FB) into HJC. Sections of the spinal ganglia (SpG) with FB‐positive (FB+) neurons were stained using antibodies against calcitonin gene‐related peptide (CGRP) substance P (SP), pituitary adenylate cyclase‐activating peptide (PACAP), nitric oxide synthase (n‐NOS), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), Leu‐5‐enkephalin (Leu‐Enk), galanin (GAL) and vesicular acetylcholine transporter (VACHT). The vast majority of FB+ neurons supplying HJC was found in the ganglia from the 5th lumbar to the 2nd sacral. Immunohistochemistry revealed that most of these neurons were immunoreactive to CGRP or SP (80.7 ± 8.0% or 56.4 ± 4.8%, respectively) and many of them stained for PACAP or GAL (52.9 ± 2.9% or 50.6 ± 19.7%, respectively). Other populations of FB+ neurons were those immunoreactive to n‐NOS (37.8 ± 9.7%), NPY (34.6 ± 6.7%), VIP (28.7 ± 4.8%), Leu‐Enk (27.1 ± 14.6) and VACHT (16.7 ± 9.6).     

Neuromuscular block monitoring after the administration of 1 mg/kg intravenous cis‐atracurium in the anaesthetized pig

Neuromuscular blocking agents should be included as part of a balanced anaesthetic protocol to improve anaesthetic management, although doses are not always established for each species. Cis‐atracurium is a benzylisoquinolinium neuromuscular blocking agent with an intermediate duration of action devoid of significant adverse effects previously used in pigs with a wide dosage range. Cis‐atracurium was administered at 1 mg/kg bolus to sixteen pigs to establish its time profile and effects. The pigs were premedicated intramuscularly with 4 mg/kg azaperone, 8 mg/kg ketamine and 0.2 mg/kg morphine IM and maintained with isoflurane in oxygen. After cis‐atracurium administration, neuromuscular monitoring via acceleromyography was started until the recovery of the 90% of the train of four ratio. Complete decrease in the train of four ratio was accomplished in eleven pigs. Onset of action was 70 s, with a recovery of the fourth twitch at 26 min and a recovery of a train of four ratio greater than 90% in 60 min. In conclusion, 1 mg/kg intravenous cis‐atracurium in the pig allowed for a rapid onset of action and a complete recovery after 60 min although high variability in the time profile is seen.     

Pharmacokinetic profiles of the active metamizole metabolites after four different routes of administration in healthy dogs

Metamizole (MT), an analgesic and antipyretic drug, is rapidly hydrolyzed to the active primary metabolite 4‐methylaminoantipyrine (MAA) and relatively active secondary metabolite 4‐aminoantipyrine (AA). The aim of this study was to assess the pharmacokinetic profiles of MAA and AA after dose of 25 mg/kg MT by intravenous (i.v.), intramuscular (i.m.), oral (p.o.), and rectal (RC) routes in dogs. Six dogs were randomly allocated to an open, single‐dose, four‐treatment, four‐phase, unpaired, crossover study design. Blood was collected at predetermined times within 24 hr, and plasma was analyzed by a validated HPLC‐UV method. Plasma concentrations of MAA and AA after i.v., i.m., p.o., and RC administrations of MT were detectable from 5 (i.v. and i.m.) or 30 (p.o. and RC) min to 24 hr in all dogs. The highest concentrations of MAA were found in the i.v., then i.m., p.o., and RC groups. Plasma concentrations of AA were similar for i.v., i.m., and RC, and the concentrations were approximately double those in the PO groups. The AUC_EV/IV ratio for MAA was 0.75 ± 0.11, 0.59 ± 0.08, and 0.32 ± 0.05, for i.m., p.o., and RC, respectively. The AUC_EV/IV ratio for AA was 1.21 ± 0.33, 2.17 ± 0.62, and 1.08 ± 0.19, for i.m., p.o., and RC, respectively. Although further studies are needed, rectal administration seems to be the least suitable route of administration for MT in the dog.     

Cryopreservation of immature oocytes of the indigeneous Vietnamese Ban Pig

We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus‐oocyte complexes were collected from antral follicles of 7–8 mo old female cyclic Ban pigs and vitrified in micro‐drops. Oocyte morphology, lipid content, post‐warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post‐warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo‐tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.     

Intravenous infusion of electrolyte solution changes pharmacokinetics of drugs: pharmacokinetics of ampicillin

The pharmacokinetics of ampicillin in dogs was determined after intravenous (i.v.) bolus and constant rate infusion. Ampicillin was administered to six beagle dogs as an i.v. bolus at 20 mg/kg and as a constant rate i.v. infusion (CRI) at 20 mg/kg during 8 h (0.042 mL/min/kg) in Ringer's lactate (Hartmann's) solution. The concentrations were determined by an LC/MS/MS method. After i.v. bolus, ampicillin total body clearance, apparent volume of distribution at steady‐state, mean residence time (MRT), and half‐life were 4.53 ± 0.70 mL/min/kg, 0.275 ± 0.044 L/kg, 61 ± 13 min, and 111 (85–169) min, respectively. The corresponding parameters calculated after CRI were 13.5 ± 1.06 mL/min/kg, 0.993 ± 0.415 L/kg, 73 ± 27 min, and 49 (31–69) min. Ampicillin concentration decreased by 30% in the Ringer's lactate infusion solution mostly during the first hour after preparation of the solution. Constant rate infusion of Ringer's lactate solution during 8 h caused significant changes in ampicillin pharmacokinetics. The results suggested that special attention should be given to drug pharmacokinetics when co‐administered intravenously with electrolyte solutions.     

Comparative metabolism of R(-)-fenoprofen in rats and sheep

The chiral inversion of 2-arylpropionic acids occurs in many species. It is a unique reaction specific to this group of drugs. In this study R-(-)-fenoprofen (R-(-)-FPF) was used as a model compound to investigate metabolic chiral inversion in sheep in vivo and in vitro and to compare the data with the results obtained in rats. Metabolic inversion in sheep was 80%. The apparent mean values of Km and V _max of thioester formation were: 392 μm and 2.08 nmol/min/mg in sheep and 500 μm and 22 nmol/min/mg in rats. For hydroxylation, the apparent mean values were V_max: 0.02 nmol/min/mg in rats and 0.01 nmol/min/mg in sheep. There was no correlation between in vitro thioesterification and in vivo chiral inversion in sheep as compared to rats. In sheep most of the thioester formed underwent inversion (80%) while in rats, where in vitro thioesterification was greater, in vivo inversion was less (42%). In consequence, in rats other metabolic pathways for R(-)-FPF-CoA, such as incorporation into triacylglycerols and conjugation with amino acids, may be quantitatively more important