Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p‐hydroxydiazepam [p‐OH‐DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p‐OH‐DZP was significantly less in the horse. Inhibition assays with a CYP‐specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos‐7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.     

Mutant prevention concentration,pharmacokinetic–pharmacodynamic integration,and modeling of enrofloxacin data established in diseased buffalo calves

The pharmacokinetic–pharmacodynamic (PK/PD) modeling of enrofloxacin data using mutant prevention concentration (MPC) of enrofloxacin was conducted in febrile buffalo calves to optimize dosage regimen and to prevent the emergence of antimicrobial resistance. The serum peak concentration (C_max), terminal half‐life (t_1/2K₁₀₎, apparent volume of distribution (Vd_(area)/F), and mean residence time (MRT) of enrofloxacin were 1.40 ± 0.27 μg/mL, 7.96 ± 0.86 h, 7.74 ± 1.26 L/kg, and 11.57 ± 1.01 h, respectively, following drug administration at dosage 12 mg/kg by intramuscular route. The minimum inhibitory concentration (MIC), minimum bactericidal concentration, and MPC of enrofloxacin against Pasteurella multocida were 0.055, 0.060, and 1.45 μg/mL, respectively. Modeling of ex vivo growth inhibition data to the sigmoid E_max equation provided AUC_24 h/MIC values to produce effects of bacteriostatic (33 h), bactericidal (39 h), and bacterial eradication (41 h). The estimated daily dosage of enrofloxacin in febrile buffalo calves was 3.5 and 8.4 mg/kg against P. multocida/pathogens having MIC₉₀ ≤0.125 and 0.30 μg/mL, respectively, based on the determined AUC_24 h / MIC values by modeling PK/PD data. The lipopolysaccharide‐induced fever had no direct effect on the antibacterial activity of the enrofloxacin and alterations in PK of the drug, and its metabolite will be beneficial for its use to treat infectious diseases caused by sensitive pathogens in buffalo species. In addition, in vitro MPC data in conjunction with in vivo PK data indicated that clinically it would be easier to eradicate less susceptible strains of P. multocida in diseased calves.     

Pharmacokinetics of enrofloxacin and ceftiofur in plasma,interstitial fluid,and gastrointestinal tract of calves after subcutaneous injection,and bactericidal impacts on representative enteric bacteria

This study's objectives were to determine intestinal antimicrobial concentrations in calves administered enrofloxacin or ceftiofur sodium subcutaneously, and their impact on representative enteric bacteria. Ultrafiltration devices were implanted in the ileum and colon of 12 steers, which received either enrofloxacin or ceftiofur sodium. Samples were collected over 48 h after drug administration for pharmacokinetic/pharmacodynamic analysis. Enterococcus faecalis or Salmonella enterica (5 × 10⁵ CFU/mL of each) were exposed in vitro to peak and tail (48 h postadministration) concentrations of both drugs at each location for 24 h to determine inhibition of growth and change in MIC. Enrofloxacin had tissue penetration factors of 1.6 and 2.5 in the ileum and colon, while ciprofloxacin, an active metabolite of enrofloxacin, was less able to cross into the intestine (tissue penetration factors of 0.7 and 1.7). Ceftiofur was rapidly eliminated leading to tissue penetration factors of 0.39 and 0.25. All concentrations of enrofloxacin were bactericidal for S. enterica and significantly reduced E. faecalis. Peak ceftiofur concentration was bactericidal for S. enterica, and tail concentrations significantly reduced growth. E. faecalis experienced growth at all ceftiofur concentrations. The MICs for both organisms exposed to peak and tail concentrations of antimicrobials were unchanged at the end of the study. Enrofloxacin and ceftiofur achieved intestinal concentrations capable of reducing intestinal bacteria, yet the short exposure of ceftiofur in the intestine may select for resistant organisms.     

Development of genotyping method for functionally relevant variants of cytochromes P450 in cynomolgus macaques

In cynomolgus macaques (Macaca fascicularis), widely used in drug metabolism studies, CYP2C9, CYP2C76, CYP2D6, CYP3A4, and CYP3A5, important drug‐metabolizing enzymes, are abundantly expressed in liver and metabolize cytochrome P450 substrates. CYP2C9 (c.334A>C), CYP2C76 (c.449TG>A), CYP2D6 (c.891A>G), CYP3A4 (IVS3 + 1G>del), and CYP3A5 (c.625A>T) substantially influence metabolic activity of enzymes, and thus are important variants in drug metabolism studies. In this study, a real‐time PCR method was developed for genotyping these variants. The validity of the methods was verified by genotyping two wild type, two heterozygous, and two homozygous DNAs and was used to genotype 41 cynomolgus macaques (from Cambodia, Indonesia, the Philippines, or Vietnam) for the five variants, along with another important variant CYP2C19 (c.308C>T). The CYP2C9 and CYP2C19 variants were found only in Cambodian and Vietnamese animals, while the CYP2C76 and CYP2D6 variants were found only in Indonesian and Philippine animals. The CYP3A4 and CYP3A5 variants were not found in any of the animals analyzed. Mauritian animals, genotyped using next‐generation sequencing data for comparison, possessed the CYP2C19 and CYP2D6 variants, but not the other variants. These results indicated differences in prevalence of these important variants among animal groups. Therefore, the genotyping tool developed is useful for drug metabolism studies using cynomolgus macaques.     

Pharmacokinetics of an intravenous and oral dose of enrofloxacin in white rhinoceros (Ceratotherium simum)

South Africa currently loses over 1000 white rhinoceros (Ceratotherium simum) each year to poaching incidents, and numbers of severely injured victims found alive have increased dramatically. However, little is known about the antimicrobial treatment of wounds in rhinoceros. This study explores the applicability of enrofloxacin for rhinoceros through the use of pharmacokinetic‐pharmacodynamic modelling. The pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin were evaluated in five white rhinoceros after intravenous (i.v.) and after successive i.v. and oral administration of 12.5 mg/kg enrofloxacin. After i.v. administration, the half‐life, area under the curve (AUC_tot), clearance and the volume of distribution were 12.41 ± 2.62 hr, 64.5 ± 14.44 μg ml^?1 hr^?1, 0.19 ± 0.04 L h^?1 kg^?1, and 2.09 ± 0.48 L/kg, respectively. Ciprofloxacin reached 26.42 ± 0.05% of the enrofloxacin plasma concentration. After combined i.v. and oral enrofloxacin administration oral bioavailability was 33.30 ± 38.33%. After i.v. enrofloxacin administration, the efficacy marker AUC₂₄: MIC exceeded the recommended ratio of 125 against bacteria with an MIC of 0.5 μg/mL. Subsequent intravenous and oral enrofloxacin administration resulted in a low Cmax: MIC ratio of 3.1. The results suggest that intravenous administration of injectable enrofloxacin could be a useful drug with bactericidal properties in rhinoceros. However, the maintenance of the drug plasma concentration at a bactericidal level through additional per os administration of 10% oral solution of enrofloxacin indicated for the use in chickens, turkeys and rabbits does not seem feasible.     

Midazolam oxidation in cattle liver microsomes: The role of cytochrome P450 3A

In humans, the cytochrome P450 3A (CYP3A) subfamily is involved in midazolam (MDZ) biotransformation into 1′- and 4-hydroxy metabolites, and the former serves as a probe for CYP3A catalytic activity. In veterinary species is still crucial to identify enzyme- and species-specific CYP substrates; thus, the aim of this study was to characterize MDZ oxidation in cattle liver. A HPLC-UV method was used to measure 1′- and 4-hydroxy MDZ (1′- and 4-OHMDZ, respectively) formation in cattle liver microsomes and assess the role of CYP3A by an immunoinhibition study. Moreover, MDZ hydroxylation was evaluated in 300 cattle liver samples and results were correlated with testosterone hydroxylation. Formation of both metabolites conformed to a single-enzyme Michaelis–Menten kinetics. Values of V_max and K_m were 0.67 nmol/min/mg protein and 6.16 μM for 4-OHMDZ, and 0.06 nmol/min/mg protein and 10.08 μM for 1′-OHMDZ. An anti-rat CYP3A1 polyclonal antibody inhibited up to 50% and 94% 1′- and 4-OHMDZ formation, respectively. MDZ oxidation in liver microsomes was poorly correlated with testosterone hydroxylation. In conclusion, cattle metabolized MDZ to 1′-OHMDZ and 4-OHMDZ. The immunoinhibition results indicated a major contribution of CYP3As to 4-OHMDZ formation and the involvement of other CYPs in 1′-OHMDZ production, paving the way for further investigations.     

Comparison of direct sampling and brochoalveolar lavage for determining active drug concentrations in the pulmonary epithelial lining fluid of calves injected with enrofloxacin or tilmicosin

Antibiotic distribution to interstitial fluid (ISF) and pulmonary epithelial fluid (PELF) was measured and compared to plasma drug concentrations in eight healthy calves. Enrofloxacin (Baytril^® 100) was administered at a dose of 12.5 mg/kg subcutaneously (SC), and tilmicosin (Micotil^® 300) was administered at a dose of 20 mg/kg SC. PELF, sampled by two different methods—bronchoalveolar lavage (BAL) and direct sampling (DS)—plasma, and ISF were collected from each calf and measured for tilmicosin, enrofloxacin and its metabolite ciprofloxacin by HPLC. Pharmacokinetic analysis was performed on the concentrations in each fluid, for each drug. The enrofloxacin/ciprofloxacin concentration as measured by AUC in DS samples was 137 ± 72% higher than in plasma, but in BAL samples, this value was 535 ± 403% (p < .05). The concentrations of tilmicosin in DS and BAL samples exceeded plasma drug concentrations by 567 ± 189% and 776 ± 1138%, respectively. The enrofloxacin/ciprofloxacin concentrations collected by DS were significantly different than those collected by BAL, but the tilmicosin concentrations were not significantly different between the two methods. Concentrations of enrofloxacin/ciprofloxacin exceeded the MIC values for bovine respiratory disease pathogens but tilmicosin did not reach MIC levels for these pathogens in any fluids.     

Pharmacokinetics studies of enrofloxacin injectable in situ forming gel in dogs

The objective of this study was to evaluate the pharmacokinetic characteristics of enrofloxacin (ENR) injectable in situ gel we developed in dogs following a single intramuscular (i.m.) administration. Twelve healthy dogs were randomly divided into two groups (six dogs per group), then administrated a single 20 mg/kg body weight (b.w.) ENR injectable in situ gel and a single 5 mg/kg b.w. ENR conventional injection, respectively. High‐performance liquid chromatography (HPLC) was used to determine ENR plasma concentrations. The pharmacokinetic parameters of ENR injectable in situ gel and conventional injection in dogs are as follows: MRT (mean residence time) (45.59 ± 14.05) h verse (11.40 ± 1.64) h, AUC (area under the blood concentration vs. time curve) (28.66 ± 15.41) μg·h/mL verse (11.06 ± 3.90) μg·h/mL, c_max (maximal concentration) (1.59 ± 0.35) μg/mL verse (1.46 ± 0.07) μg/mL, t_max (time needed to reach c_max) (1.25 ± 1.37) h verse (1.40 ± 0.55) h, t_1/2λz (terminal elimination half‐life) (40.27 ± 17.79) h verse (10.32 ± 0.97) h. The results demonstrated that the in situ forming gel system could increase dosing interval of ENR and thus reduced dosing frequency during long‐term treatment. Therefore, the ENR injectable in situ gel seems to be worth popularizing in veterinary clinical application.     

Impact of age on hepatic cytochrome P450 of domestic male Camborough‐29 pigs

Swine is not only an important species in veterinary medicine research but also a popular animal model for human drug discovery. It is valuable to understand the impact of pig age on abundance and activity of porcine hepatic cytochrome P450 (CYP450). Liver microsomes were prepared from Camborough‐29 intact male pigs at the age of 1 day and 2 weeks and the castrated male pigs at the age of 5, 10, and 20 weeks. Hepatic CYP450 content in the liver microsomes was measured using a UV/visible spectroscopic method. The activities of CYP450s were evaluated by metabolism of phenacetin, coumarin, tolbutamide, bufuralol, chlorzoxazone, and midazolam. The porcine hepatic CYP450 content increased with age with a plateau between age 2 and 5 weeks. Activities of all CYP450 enzymes increased with age of pigs too. The bufuralol 1’‐hydroxylase showed the highest hepatic activities compared with other CYP enzymes at all ages of pigs. The average activities at the age of 20 weeks were about five times higher than those at the age of 5 weeks for most of the CYP enzymes. With compensation of the ratio of liver to body weights, the overall CYP450 metabolism capability of the pigs may be peaked around ages of 10 to 20 weeks. Those findings suggest that metabolism can be significantly different in growing phase of pigs and that the age may be an important factor in porcine medicine evaluation and pig model development.     

The effect of breed and sex on sulfamethazine,enrofloxacin, fenbendazole and flunixin meglumine pharmacokinetic parameters in swine

Drug use in livestock has received increased attention due to welfare concerns and food safety. Characterizing heterogeneity in the way swine populations respond to drugs could allow for group‐specific dose or drug recommendations. Our objective was to determine whether drug clearance differs across genetic backgrounds and sex for sulfamethazine, enrofloxacin, fenbendazole and flunixin meglumine. Two sires from each of four breeds were mated to a common sow population. The nursery pigs generated (n = 114) were utilized in a random crossover design. Drugs were administered intravenously and blood collected a minimum of 10 times over 48 h. A non‐compartmental analysis of drug and metabolite plasma concentration vs. time profiles was performed. Within‐drug and metabolite analysis of pharmacokinetic parameters included fixed effects of drug administration date, sex and breed of sire. Breed differences existed for flunixin meglumine (P‐value<0.05; Cl, Vd_ss) and oxfendazole (P‐value<0.05, AUC_0→∞). Sex differences existed for oxfendazole (P‐value < 0.05; T_max) and sulfamethazine (P‐value < 0.05, Cl). Differences in drug clearance were seen, and future work will determine the degree of additive genetic variation utilizing a larger population.     

Antibiotic effects on cytochromes P450 content and mixed-function oxygenase (MFO) activities in the American alligator,Alligator mississippiensis

There are no Food and Drug Administration (FDA)-approved antimicrobial agents for use in cultured American alligators (Alligator mississippiensis) destined for human consumption yet some producers administer antibiotics for prophylaxis. The cytochromes P450-dependent mixed-function oxygenases (MFO) catalyze the oxidation of xenobiotic compounds such as drugs, pesticides and polycyclic aromatic hydrocarbons. Herein, we describe the effects of oxytetracycline, ceftazidime and enrofloxacin on the MFO system of the American alligator, Alligator mississippiensis. Juvenile alligators (4 animals/treatment) were administered these antibiotics intraperitoneally in an effort to induce hepatic microsomal cytochromes P450. Alligators treated with enrofloxacin exhibited emesis and convulsive spasms within 5 min of the initial injection. Total hepatic cytochromes P450 contents were significantly decreased in oxytetracycline-and enrofloxacin-pretreated alligators. In vitro hepatic microsomal benzyloxyresorufin O-dealkylase (BROD) activity was significantly decreased by enrofloxacin pretreatment. Western blots of proteins from antibiotic-pretreated alligator hepatic microsomes incubated with several mammalian and fish cytochromes P450 (CYP) antibodies exhibited little or no induction of CYP1A1, 2B, 2C and 2E1. In vitro incubation with enrofloxacin and oxytetracycline caused a concentration-dependent decrease in alkyl-substituted phenoxazone dealkylase activities catalyzed by phenobarbital- and 3-methylcholanthrene-induced alligator hepatic microsomes.     

Determination of Intracellular Concentrations of Free and Two Types of Liposome‐Encapsulated Enrofloxacin in Anatolian Shepherd Dog Monocytes

In this study, it was evaluated the accumulation of free and two types of liposome‐encapsulated enrofloxacin (LEE) at the doses of 0.25, 0.5 and 1 μg/ml, which were clinically relevant concentrations into monocytes of healthy Anatolian shepherd dogs. Enrofloxacin was encapsulated with two different types of liposome in multilamellar large vesicles (MLV). Type A MLV composed of 15 mg egg phosphatidylcholine and 35 mg cholesterol, Type B MLV composed of phosphatidylcholine (PC), cholesterol and enrofloxacin, in a molar ratio of 1 : 1 : 1. The mean sizes of Type A and Type B liposome were found to be 7.65 and 4.27 μm, respectively. However, the mean encapsulation rate determined of Type A (13 ± 2%) was found lower than Type B liposome (44 ± 3%). The amounts of intracellular enrofloxacin concentrations were determined by high performance liquid chromatography. Type B LEE accumulated significantly higher level into monocytes when compared to free drug or Type A liposome. This study showed that Type B LEE markedly concentrated within monocytes and may improve the antibacterial efficacy of the antibiotic.     

Pharmacokinetics of enrofloxacin after oral,intramuscular and bath administration in crucian carp (Carassius auratus gibelio)

The pharmacokinetics of enrofloxacin (ENR) was studied in crucian carp (Carassius auratus gibelio) after single administration by intramuscular (IM) injection and oral gavage (PO) at a dose of 10 mg/kg body weight and by 5 mg/L bath for 5 hr at 25°C. The plasma concentrations of ENR and ciprofloxacin (CIP) were determined by HPLC. Pharmacokinetic parameters were calculated based on mean ENR or CIP concentrations using WinNonlin 6.1 software. After IM, PO and bath administration, the maximum plasma concentration (C_max) of 2.29, 3.24 and 0.36 μg/ml was obtained at 4.08, 0.68 and 0 hr, respectively; the elimination half‐life (T_1/2β) was 80.95, 62.17 and 61.15 hr, respectively; the area under the concentration–time curve (AUC) values were 223.46, 162.72 and 14.91 μg hr/ml, respectively. CIP, an active metabolite of enrofloxacin, was detected and measured after all methods of drug administration except bath. It is possible and practical to obtain therapeutic blood concentrations of enrofloxacin in the crucian carp using IM, PO and bath immersion administration.     

Accumulation and elimination of enrofloxacin and its metabolite ciprofloxacin in the ridgetail white prawn Exopalaemon carinicauda following medicated feed and bath administration

Accumulation and elimination of enrofloxacin and its metabolite ciprofloxacin were evaluated in Exopalaemon carinicauda following medicated feed at dose of 10 mg/kg weight body per day for five consecutive days and 10 mg/L bath for five consecutive days at 18 °C. At different times, nine ridgetail white prawns were randomly selected from the tank and sampled after the last medicated feed or bath administration. The concentration of enrofloxacin and ciprofloxacin in the main tissues (hepatopancreas, muscle, gill, and ovary) was detected by HPLC. The results showed that the maximum concentrations of enrofloxacin were 3.408 ± 0.245, 0.554 ± 0.088, 0.789 ± 0.074, and 0.714 ± 0.123 μg/g for hepatopancreas, muscle, gill, and ovary, respectively, at 1 day after the last medicated feed treatment. The enrofloxacin concentrations were 2.389 ± 0.484, 0.656 ± 0.012, 0.951 ± 0.144, and 3.107 ± 0.721 μg/g in hepatopancreas, muscle, gill, and ovary, respectively, at 1 day after the last bath administration. Ciprofloxacin could be detected in hepatopancreas, muscle, gill, and ovary. However, the concentrations of ciprofloxacin were much lower in comparison with that of enrofloxacin in various tissues. The concentrations of enrofloxacin plus ciprofloxacin in hepatopancreas, muscle, gill, and ovary followed an eliminating pattern during the sampling time after the two routes of administration. Based on data derived from this study, to avoid the enrofloxacin and ciprofloxacin residue in E. carinicauda, it should take at least 20 and 25 days to wash out the drug from the tissues after the last medicated feed and bath administration with enrofloxacin, respectively. These results helped the Chinese fishery department to lay down the current guidelines on enrofloxacin plus ciprofloxacin withdrawal periods for farmed shrimp.     

Effects of endotoxin-induced fever and probenecid on disposition of enrofloxacin and its metabolite ciprofloxacin after intravascular administration of enrofloxacin in goats

Pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin were investigated in normal, febrile and probenecid‐treated adult goats after single intravenous (i.v.) administration of enrofloxacin (5 mg/kg). Pharmacokinetic evaluation of the plasma concentration–time data of enrofloxacin and ciprofloxacin was performed using two‐ and one‐compartment open models, respectively. Plasma enrofloxacin concentrations were significantly higher in febrile (0.75–7 h) and probenecid‐treated (5–7 h) goats than in normal goats. The sum of enrofloxacin and ciprofloxacin concentrations in plasma ≥0.1 μg/mL was maintained up to 7 and 8 h in normal and febrile or probenecid‐treated goats, respectively. The t1/2β, AUC, MRT and ClB of enrofloxacin in normal animals were determined to be 1.14 h, 6.71 μg.h/mL, 1.5 h and 807 mL/h/kg, respectively. The fraction of enrofloxacin metabolized to ciprofloxacin was 28.8%. The Cmax., t1/2β, AUC and MRT of ciprofloxacin in normal goats were 0.45 μg/mL, 1.79 h, 1.84 μg.h/mL and 3.34 h, respectively. As compared with normal goats, the values of t1/2β (1.83 h), AUC (11.68 μg ? h/mL) and MRT (2.13 h) of enrofloxacin were significantly higher, whereas its ClB (430 mL/h/kg) and metabolite conversion to ciprofloxacin (8.5%) were lower in febrile goats. The Cmax. (0.18 μg/mL) and AUC (0.99 μg.h/mL) of ciprofloxacin were significantly decreased, whereas its t1/2β (2.75 h) and MRT (4.58 h) were prolonged in febrile than in normal goats. Concomitant administration of probenecid (40 mg/kg, i.v.) with enrofloxacin did not significantly alter any of the pharmacokinetic variables of either enrofloxacin or ciprofloxacin in goats.     

Comparative Pharmacokinetics of Enrofloxacin and Tissue Concentrations of Parent Drug and Ciprofloxacin after Intramuscular Administrations of Free and Liposome‐Encapsulated Enrofloxacin in Rabbits

Pharmacokinetic properties and tissue concentrations of enrofloxacin and ciprofloxacin were compared after intramuscular (i.m.) administrations of free and liposome‐encapsulated enrofloxacin at the dose of 5 mg/kg body weight (bw). Twelve healthy adult New Zealand white rabbits were used in the experiment. Blood samples were obtained at 10, 20, 40, 60 and 90 min and 2, 4, 6, 8 and 12 h and tissue samples were collected 24 h after injection. Concentrations of drugs in serum were determined by high‐performance liquid chromatography. Pharmacokinetics were best described by a two‐compartment open model. Results indicated that absorption rate was slow, peak concentration was higher (P < 0.05), and the time to peak concentration (t_max ? 1.5 h) was significantly longer (P < 0.05) for liposome‐encapsulated enrofloxacin (LEE) when compared with free enrofloxacin. Values of elimination half‐life (t_1/2β = 12.9 h) and mean residence time (MRT = 17.6 h) of liposome‐encapsulated enrofloxacin were longer (P < 0.05) and total clearance (Cl = 0.43 l/h/kg) was lower than those of free form. Moreover, the distribution volume at steady‐state (V_d(ss) = 14.4 l/kg) of enrofloxacin administered encapsulated into liposomes was significantly higher (P < 0.05) than that of free enrofloxacin (FE). The tissue levels of enrofloxacin and ciprofloxacin after LEE injection were not different (P > 0.05) from FE. In conclusion, the result of present study suggest that LEE may be a beneficial and valuable formulation in the treatment of infectious diseases caused by sensitive pathogens in animals, providing sustained drug release from injection side and prolonged therapeutic serum concentrations after i.m. administration.     

Pharmacokinetic and pharmacodynamic evaluations of a 10 mg/kg enrofloxacin intramuscular administration in bearded dragons (Pogona vitticeps): a preliminary assessment

Enrofloxacin (E) is commonly used in veterinary medicine. It is necessary to perform pharmacokinetic/dynamic studies to minimize the selection of resistant mutants of bacteria and extend the efficacy of antimicrobial agents. Eight healthy adult Pogona vitticeps were assigned into two groups of equal size and treated with a single intramuscular injection of E at 10 mg/kg. Blood samples were withdrawn at different scheduled times for each group, and rectal swabs were collected. E and ciprofloxacin (active metabolite) blood concentrations were quantified by an HPLC validated method, while the in vitro antimicrobial susceptibility was evaluated by the Kirby–Bauer disc diffusion susceptibility test. The pharmacokinetic profiles of E gave similar pharmacokinetic parameters irrespective of the collection time schedule. Bacteria isolation showed the presence of both E. coli, Salmonella enterica subspecies enterica and subspecies 3a, Proteus spp., and Pseudomonas spp. The majority of isolated colonies were sensitive to E, but the treatment did not reduce the number of bacteria in faeces. Results suggest that E is able to reach blood concentrations high enough to kill susceptible bacteria (MIC < 0.9 μg/mL), but at the same time does not significantly affect intestinal bacteria.     

Analysis of polymorphisms of canine Cytochrome P 450-CYP2D15

Cytochrome P450 (CYP) proteins constitute a large ancient family of oxidative enzymes essential for the efficient elimination of a wide variety of clinically used drugs. Polymorphic variants of human CYP2D6 are associated with the conversion rate and efficacy of several drugs such as antidepressants. Polymorphisms of the canine orthologue CYP2D15 are of interest because these antidepressants are also used in dogs with behavioral problems and the outcome of the treatment is variable. However, the annotated CYP2D15 gene is incomplete and inaccurately assembled in CanFam3.1, hampering DNA sequence analysis of the gene in individual dogs. We elucidated the complete exon–intron structure of CYP2D15 to enable comprehensive genotyping of the gene using genomic DNA. We surveyed variations of the gene in four diverse dog breeds and identified novel polymorphisms in exon 2 in border collies. Further investigation to establish the impact of these canine CYP2D15 polymorphisms on interindividual variability in expression and function of this metabolizing enzyme is now feasible. Further knowledge of CYP pharmacogenetics will help individualize therapy and thereby increase therapeutic efficacy, especially in the use of antidepressants in veterinary behavioral medicine.     

Transient cold agglutinins associated with Mycoplasma cynos pneumonia in a dog

This report details a case of reversible cold agglutinins in a dog with Mycoplasma cynos pneumonia. An 11‐month‐old female spayed Rhodesian Ridgeback was presented for lethargy and cough. Thoracic radiographs revealed an alveolar pattern present bilaterally in the cranioventral lung lobes. Septic neutrophilic inflammation with suspected Mycoplasma sp. organisms was noted on cytologic examination of a trans‐tracheal wash, and the dog was treated empirically with IV ampicillin/sulbactam and enrofloxacin pending culture results. Red blood cell agglutination was noted unexpectedly on several blood film reviews during hospitalization; however, the dog never developed clinical or laboratory evidence of hemolysis. Cold agglutinins were demonstrated based on the results of a saline dilution and cold agglutinin test that showed agglutination at 4°C but not at room temperature (21°C) or 37°C. Based on a positive culture for M cynos, the dog was treated for 8 weeks with oral enrofloxacin. After clinical and radiographic resolution of the pneumonia, repeated saline dilution and cold agglutinin tests of peripheral blood were negative at all temperatures. Reversible, asymptomatic cold agglutinins are common in human patients with mycoplasma pneumonia, but this is the first reported case in a dog.