Benzydamine as a useful substrate of hepatic flavin-containing monooxygenase activity in veterinary species.

Benzydamine (BZ), a weak base and an indazole derivative with analgesic and antipyretic properties used in human and veterinary medicine, is metabolized in human, rat, cattle and rabbit to a wide range of metabolites. One of the main metabolites, BZ N-oxide (BZ-NO), is produced in the liver and brain by flavin-containing monooxygenases (FMOs), by liver and brain enzymes. To evaluate the suitability of BZ as an FMO probe in veterinary species, BZ metabolism was studied in vitro using liver microsomes from bovine, rabbit and swine. Kinetic parameters, K(m) and V(max), of BZ-NO production, were evaluated to corroborate the pivotal role of FMOs. Inhibition studies were carried out by heat inactivation and by specific FMO chemical inhibitors: trimethylamine and methimazole. The results confirmed the presence of FMO activity in the liver and the role of BZ as a suitable marker of FMO enzyme activities for the veterinary species considered.

Pharmacodynamics and pharmacokinetics of flumequine in pigs after single intravenous and intramuscular administration.

The pharmacokinetics and intramuscular (IM) bioavailability of flumequine (15 mgkg(-1)) were investigated in healthy pigs and the findings related to published minimal inhibitory concentrations (MICs) for susceptible bacteria of animal origin, and to experimentally determined MICs for susceptible strains of porcine origin. We found MICs for Escherichia coli, Salmonella spp., Pasteurella spp. and Bordetella spp. in the range 0.5 to >64 microg mL(-1) isolated from infected pigs in the Forli area of Italy; only the Pasteurella multocida strains were sensitive (MIC(90)=0.5 microg mL(-1)). After intravenous (IV) injection, flumequine was slowly distributed and eliminated (t(1/2lambda(1))1.40+/-0.16 h and t(1/2lambda(2))6.35+/-1.69 h). The distribution volume at steady state (V(dss)) was 752.59+/-84.03 mL kg(-1) and clearance (Cl(B)) was 237.19+/-17.88 mL kg(-1)h(-1). After IM administration, peak serum concentration (4.99+/-0.92 microg mL(-1)) was reached between the 2nd and the 3rd hour. The results on MIC of isolated bacteria, although only indicative, suggest that the efficacy of flumequine on Gram-negative bacteria may be impaired by the emergence of less sensitive or resistant strains.

Pharmacodynamics and pharmacokinetics of cefoperazone and cefamandole in dogs following single dose intravenous and intramuscular administration.

The pharmacokinetics and intramuscular (i.m.) bioavailability of cefoperazone and cefamandole (20mg/kg) were investigated in dogs and the findings related to minimal inhibitory concentrations (MICs) for 90 bacterial strains isolated clinically from dogs. The MICs of cefamandole for Staphylococcus intermedius (MIC(90) 0.125 microg/mL) were lower than those of cefoperazone (MIC(90) 0.5 micro/mL) although the latter was more effective against Escherichia coli strains (MIC(90) 2.0 microg/mL vs. 4.0 microg/mL). The pharmacokinetics of the drugs after intravenous administrations were similar: a rapid distribution phase was followed by a slower elimination phase (t((1/2)lambda2) 84.0+/-21.3 min for cefoperazone and 81.4+/-9.7 min for cefamandole). The apparent volume of distribution and body clearance were 0.233 L/kg and 1.96 mL/kg/min for cefoperazone, 0.190 L/kg and 1.76 mL/kg/min for cefamandole. After i.m. administration the bioavailability and peak serum concentration of cefamandole (85.1+/-13.5% and 35.9+/-5.4 microg/mL) were significantly higher than cefoperazone (41.4+/-7.1% and 24.5+/-3.0 micog/mL), but not the serum half-lives (t(1/2el) 134.3+/-12.6 min for cefoperazone and 145.4+/-12.3 min for cefamandole). The time above MIC(90) indicated that cefamandole can be administered once daily to dogs for the treatment of staphylococcal infections (T>MIC for S. intermedius 23.8+/-0.3 and for Staphylococcus aureus 21.6+/-0.6h).

Relative oral bioavailability of microgranulated amoxicillin in pigs.

A new microgranulated formulation of amoxicillin trihydrate for in-feed medication was developed using a lipogelled matrix. Its relative bioavailability was compared with powdered drug in pigs and an assessment was made to determine whether therapeutic concentrations were achieved. Microgranules containing 10% (MICR10) and 30% (MICR30) amoxicillin and free amoxicillin trihydrate powder (reference, AMX) were administered at dosages of 50 mg of amoxicillin/kg b.w. using a three-way-crossover design. Amoxicillin analysis in serum was performed by a sensitive high performance liquid chromatography (HPLC) method with fluorometric detection, using an extraction procedure already described for edible tissues of fish and adapted and validated for pig serum. The oral bioavailability of both microgranulated formulations was higher than that of the reference formulation [relative bioavailability (F): 153.9 +/- 58.2% for MICR10; 126.2 +/- 70.5% for MICR30] and the area under the concentration-time curve (AUC) values of MICR10 and AMX formulations were significantly different (P < 0.05). Differences between the mean maximum concentration (Cmax), time of Cmax (tmax) and mean residence time (MRT) of the drug formulations were not significant. Microgranulated amoxicillin is suitable for in-feed administration to pigs and, because of its higher oral bioavailability compared with the powdered compound, it may be more effective for the treatment of susceptible infections.

Biotransformation of benzydamine by microsomes and precision-cut slices prepared from cattle liver.

1. Benzydamine (BZ), a non-steroidal anti-inflammatory drug used in human and veterinary medicine, is not licensed for use in food-producing species. Biotransformation of BZ in cattle has not been reported previously and is investigated here using liver microsomes and precision-cut liver slices. 2. BZ was metabolized by cattle liver microsomes to benzydamine N-oxide (BZ-NO) and monodesmethyl-BZ (Nor-BZ). Both reactions followed Michaelis-Menten kinetics (Km = 76.4 +/- 16.0 and 58.9 +/- 0.4 microM Vmax = 6.5 +/- 0.8 and 7.4 +/- 0.5 nmolmg(-1) min(-1) respectively); sensitivity to heat and pH suggested that the N-oxidation is catalysed by the flavin-containing monooxygenases. 3. BZ-NO and Nor-BZ were the most abundant products derived from liver slice incubations, and nine other BZ metabolites were found and tentatively identified by LC-MS. Desbenzylated and hydroxylated BZ-NO analogues and a hydroxylated product of BZ were detected, which have been reported in other species. Product ion mass spectra of other metabolites, which are described here for the first time, indicated the formation of a BZ N- -glucuronide and five hydroxylated and N+-glucuronidated derivatives of BZ, BZ-NO and Nor-BZ. 4. The results indicate that BZ is extensively metabolized in cattle. Clearly, differences in metabolism compared with, for example, rat and human, will need to be considered in the event of submission for marketing authorization for use in food animals.

Absorption kinetics and bioavailability of cephalexin in the dog after oral and intramuscular administration.

The pharmacokinetics of cephalexin, a first generation cephalosporin, were investigated in dogs using two formulations marketed for humans, but also often employed by practitioners for pet therapy. Cephalexin was administered to five dogs intravenously and intramuscularly as a sodium salt and by the oral route as a monohydrate. The dosage was always 20 mg/kg of active ingredient. A microbiological assay with Sarcina lutea as the test organism was adopted to measure cephalexin concentrations in serum. The mean residence time (MRT) median values after intravenous (i.v.), intramuscular (i.m.) and oral administration (p.o.) were 86 min, 200 min, and 279 min, respectively. After i.m. and oral dosing the peak serum concentrations (24.2 +/- 1.8 micrograms/mL and 20.3 +/- 1.7 micrograms/mL, respectively) were attained at 90 min in all dogs and bioavailabilities were 63 +/- 10% and 57 +/- 5%, respectively. The time course of the cephalexin serum concentrations after oral administration was best described by a model incorporating saturable absorption kinetics of the Michaelis-Menten type: thus in the gastrointestinal tract of dogs a carrier mediated transport for cephalexin similar to that reported in humans, may exist. The predicted average serum concentrations of cephalexin after repeated i.m. and oral administration indicated that, in order to maintain the therapeutic concentrations, the 20 mg/kg b.w. dosage should be administered every 6-8 h.

Determination of tylosin residues in pig tissues using high-performance liquid chromatography.

In accordance with the maximum residue limit of 100 micrograms kg-1 established by EU legislation, a simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the measurement of tylosin residues in pig tissues (fat, kidney, liver and muscle). Tylosin, a macrolide antibiotic, is extracted with water-methanol and cleaned-up by solid-phase extraction (SPE) on cation-exchange cartridges using methanol elution. Tylosin was determined by reversed-phase HPLC with UV detection at 280 nm and the mean recovery from pig tissues fortified in the range 50-200 micrograms kg-1 was 70-85%, with intra- and inter-day RSDs in the ranges 3.4-9.1 and 3.9-10.1% respectively.

In vitro metabolism of clenbuterol and bromobuterol by pig liver microsomes.

1. Clenbuterol (CBL) and bromobuterol (BBL) were both extensively metabolized by hepatic microsomes of swine to only one polar metabolite which was separated by hplc and purified to perform mass analysis. 2. LC-MIS analysis by direct infusion into an ion trap system and after reverse-phase chromatograpy into a triple quadrupole system showed that the metabolites were the hydroxylamine-derivatives of CBL and BBL. GC-MS analysis by the CI and EI modes confirmed that the hydroxyl group was bound to the aniline nitrogen. The chemical instability of those metabolites probably as a consequence of spontaneous oxidation and reduction gave rise during the analysis to the corresponding nitroso and nitro derivatives, together with the original compound. 3. Thermal inactivation and CO complex formation were used selectively to inactivate flavin monooxygenase and cytochrome P450, respectively. Both inactivation procedures significantly reduced the formation of the hydroxyl metabolite.

Intramuscular bioavailability of ketoprofen lysine salt in horses.

Lysine salts are often used in human pharmaceuticals to increase the solubility and absorption of acidic drugs when these are administered parenterally. In this study the intramuscular bioavailability of ketoprofen administered as the lysine salt was evaluated in horses (n = 5) treated intravenously and intramuscularly (2.2 mg/kg active substance) in a cross-over study. The absorption rate of ketoprofen administered as the lysine salt was rather low: the mean residence time increased from 31.7 min after IV injection to 128.9 min (after IM injection), and the bioavailability was high (mean 92.4%). The calculated steady state plasma concentrations of ketoprofen during multiple dosage were much higher after intramuscular (0.106 g/ml) than after intravenous (0.066 microgram/ml) administration. Intramuscular injections of the ketoprofen lysine salt can therefore be given to horses, which are particularly prone to develop soft tissue reactions, since use of the lysine salt markedly reduced local irritation at the injection site.

Relative bioavailability of microgranulated sulfadimidine in veal calves.

The kinetics of free and microgranulated sulfadimidine were compared in milk-fed calves dosed orally (180 mg/kg) in a crossover study. Microgranulation results in delayed absorption of sulfadimidine and poor bioavailability, with the area under the plasma concentration-time curve (AUC(0-infinity)) reduced from 7400 to 3781 micrograms.h/mL, and maximum plasma concentration (Cmax) reduced from 188.1 +/- 39.0 to 84.41 +/- 22.6 micrograms/mL. It is concluded that sulfadimidine microgranulated with long chain fatty acids is not suitable for use in milk-fed calves; the gastrointestinal transit time is too rapid to allow full release of the drug, markedly limiting its bioavailability. In adult animals, or in the young of other animal species in which digesta transit time is slower than in calves, the bioavailability of microgranulated sulfadimidine may be much greater.

The use of cultured hepatocytes from goats and cattle to investigate xenobiotic oxidative metabolism.

The oxidative metabolism of aldicarb (ALD), a carbamate pesticide, and fenbendazole (FBZ), an anthelmintic, was studied using cultured hepatocytes obtained from 4 goats and a bullock and incubated with ALD (50 mumol/L) and FBZ (10 mumol/L). The parent compounds and the metabolites were measured by HPLC. Both compounds are metabolized at the sulphur atom via two sequential oxidations, first to the sulphoxide (aldicarb sulphoxide and oxfendazole, respectively) and then to the sulphone. Oxfendazole and fenbendazole sulphone from FBZ, and aldicarb sulphoxide from ALD were found in both species. Aldicarb sulphone was not produced by the hepatocyte preparations from the bullock. The good correlation obtained comparing the in vitro results of FBZ metabolism with published in vivo data obtained on FBZ kinetics in the same species confirmed the usefulness of in vitro models for predictive analysis of in vivo xenobiotic biotransformations.

Absorption and dosage of theophylline in the horse after single and repeated administration of a microencapsulated preparation.

The kinetics of 2 formulations of theophylline were studied in horses. In an initial cross-over study (Phase I) serum concentration-time curves were determined for granulated and microencapsulated theophylline after a single oral administration (5 mg/kg bwt). In Phase II microencapsulated theophylline was administered at 5 mg/kg bwt/12 h for 10 days at feeding time, as in normal clinical practice. Although no significant differences between the 2 preparations were found with respect to the main kinetic parameters, the microencapsulated form was more evenly and completely absorbed from the digestive tract; furthermore, after the repeated treatment, its trough-peak serum concentrations were always within the therapeutic window and no toxic effects were observed in treated animals.

Toxicokinetics and metabolism of linuron in rabbit: in vivo and in vitro studies.

1. Linuron (N'-(3,4-dichlorophenyl)-N-methoxy-N-methylurea) metabolism and kinetic behaviour were investigated after oral and i.v. administration to six New Zealand White female rabbits. 2. After i.v. dosage, linuron distributes quickly and widely to peripheral tissues and its is rapidly eliminated; rapid absorption was also observed after oral administration of the herbicide which undergoes extensive first pass metabolism in the liver. 3. The major metabolites obtained from both in vivo (serum samples) and in vitro (microsomal fractions incubated with linuron) experiments were identified by h.p.l.c.-mass spectrometry as N'-(3,4-dichlorophenyl)-N-methoxyurea, N'-(3,4-dichlorophenyl) urea, and N'-(6-hydroxy-3,4-dichlorophenyl) urea. 4. Given the common metabolites reported in rat and rabbit, and the fact that linuron is a liver enzyme inducer in rat, it may be possible that linuron also induces the P450 system in rabbit. Hence, despite the low acute toxicity of linuron in rabbit, the intake of hay and feed contaminated by the herbicide could be a health risk for these breeding animals since it could modify the effectiveness of many drugs commonly used in veterinary practice and metabolized by the same liver enzymes.

Pharmacokinetics of benzydamine in dairy cows following intravenous or intramuscular administration.

Five lactating cows were given benzydamine hydrochloride by rapid intravenous (0.45 mg/kg) and by intramuscular (0.45 and 1.2 mg/kg) injection in a crossover design. The bioavailability, pharmacokinetic parameters and excretion in milk of benzydamine were evaluated. After intravenous administration, the disposition kinetics of benzydamine was best described using a two-compartment open model. Drug disposition and elimination were fast (t1/2 alpha: 11.13 +/- 3.76 min; t1/2 beta: 71.98 +/- 24.75 min; MRT 70.69 +/- 11.97 min). Benzydamine was widely distributed in the body fluids and tissues (Vd(area): 3.549 +/- 1.301 L/kg) and characterized by a high value for body clearance (33.00 +/- 5.54 ml/kg per min). After intramuscular administration the serum concentration-time curves fitted a one-compartment open model. Following a dose of 0.45 mg/kg, the Cmax value was 38.13 +/- 4.2 ng/ml at a tmax of 67.13 +/- 4.00 min; MAT and MRT were 207.33 +/- 22.64 min and 278.01 +/- 12.22 min, respectively. Benzydamine bioavailability was very high (92.07% +/- 7.08%). An increased intramuscular dose (1.2 mg/kg) resulted in longer serum persistence (MRT 420.34 +/- 86.39 min) of the drug, which was also detectable in milk samples collected from both the first and second milking after treatment.

Pharmacokinetics of dipyridamole-beta-cyclodextrin complex in dogs.

Plasma concentrations and urinary and fecal excretion of intact dipyridamole were followed in dogs after oral administration of dipyridamole-beta-cyclodextrin complex (dip-beta-CD) (capsules containing 37.5 and 75 mg of active principle), of commercial dipyridamole and of dipyridamole. HCl (tablets and capsules of 75 mg of active principle, respectively), according to a crossover design. Dip-beta-CD afforded significantly shorter lag-times, higher Cmax, smaller interindividual variations of plasma concentrations and greater urinary excretion than the other two preparations, as a consequence of a better bioavailability of the former one. This amelioration seems to be due not only to an increased wettability and water solubility of the product, but also to a finer molecular dispersion in the gastrointestinal fluids which favors the contact of dipyridamole with a greater absorption surface.

Sensitivity to rifaximin and rifampicin of Mycobacterium tuberculosis isolated from guinea pigs treated orally with rifaximin.

The in vitro sensitivity of the H 37 RV strain of Mycobacterium tuberculosis to rifaximin and rifampicin was determined by evaluating both the antimycobacterial activity of the two rifamycin derivatives after oral administration to infected guinea pigs and the influence of rifaximin on the susceptibility to rifampicin of the isolated strain from spleen, lungs and liver. The results showed that after oral treatment with rifaximin no changes regarding the sensitivity of the strain to rifaximin and rifampicin were seen.