Detection of enrofloxacin and its metabolite ciprofloxacin in equine hair

Hair analysis to detect drug administration has not been studied extensively in horses. This study aimed to (a) develop an analytical method for enrofloxacin and its metabolite ciprofloxacin in mane and tail hair, (b) relate measured values to doses, routes of administration, hair colour, and (c) demonstrate long-term detectability. Samples were extracted in trifluoroacetic acid at 70 degrees C. Extracts were cleaned-up by solid-phase extraction and analysed by high-performance liquid chromatography with UV-diode array detection. Analyte recoveries were > 87%. Horses were sampled after therapeutic enrofloxacin administration either orally at 7.5 mg/kg daily for 3-13 days or twice daily for 10-14 days (Group 1, n=7) or intravenously at 5.0 mg/kg daily for 12 and 15 days (Group 2, n=2). Enrofloxacin and ciprofloxacin were detected at concentrations up to 452 and 19 ng/mg, respectively, up to 10 months post-treatment. In vitro, enrofloxacin and ciprofloxacin were extensively bound to melanin (> 96%) and in vivo, their uptake was 40-fold greater in black than white hair. Enrofloxacin and ciprofloxacin concentrations correlated to enrofloxacin dose (r2=0.777 and r2=0.769). Enrofloxacin:ciprofloxacin ratios were 21:1 and 13:1 following intravenous and oral administration, respectively. Longitudinal analyte distributions correlated to treatment-sampling interval.     

Effects of proinflammatory cytokines on canine articular chondrocytes in a three-dimensional culture

OBJECTIVE: To determine the effects of interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha on canine chondrocytes cultured in an agarose-based 3-dimensional (3-D) system. SAMPLE POPULATION: Humeral head articular cartilage chondrocytes obtained from 6 adult dogs. PROCEDURE: Chondrocytes were cultured in a 3-D system for < or = 12 days in serum-free medium with IL 1alpha, IL-1beta, or TNF-alpha at concentrations of 20, 50, or 100 ng/mL. After 1, 3, 6, and 12 days, glycosaminoglycan (GAG) concentrations in 3-D constructs; nitric oxide and prostaglandin E2 (PGE2) concentrations in media samples; and relative expressions of selected genes, including metalloproteinase (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were evaluated. Control specimens were comprised of chondrocytes cultured without proinflammatory cytokines. RESULTS: In control 3-D constructs, GAG content was significantly higher than for all other constructs. Compared with control values, relative expressions of MMP-13, TIMP-1, and TIMP-2 genes in the IL-1beta (50 ng/mL) group were significantly higher at day 1; at all evaluations, media concentrations of nitric oxide were significantly higher in all TNF-alpha-treated cultures; and concentrations of PGE2 in media samples were significantly higher in the IL-1beta (50 ng/mL) and IL-1beta (100 ng/mL) groups at days 1 and 3, in the IL-1beta (100 ng/mL) group at day 6, and in all TNF-alpha groups at days 1, 3, and 6. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that TNF-alpha more readily induces production of nitric oxide and PGE2 by canine chondrocytes, compared with IL-1beta. In vitro, IL-1alpha appeared to have a minimal effect on canine chondrocytes.     

Concentration of enrofloxacin and its metabolite ciprofloxacin in canine matrices of the locomotor system

Due to its pharmacodynamic and pharmacokinetic properties, the use of enrofloxacin may be indicated in canine osteomyelitis, but there is insufficient data on its distribution within the musculoskeletal tissues. The dogs used in this study were 31 regular veterinary orthopaedic patients. Four hours after their oral or subcutaneous treatment with 10 mg/kg enrofloxacin (Baytril; Bayer, Leverkusen, Germany) once daily for 1 or 3 days, the concentration of enrofloxacin and its main metabolite ciprofloxacin was quantified in plasma, bone, musculature and other matrices of the locomotor system by high pressure liquid chromatography with fluorescence-detection after homogenization and solid phase extraction of the samples. By oral or subcutaneous administration of enrofloxacin once daily for 3 days, higher concentrations of the active constituents in the samples were achieved than by single treatment. Nevertheless, even after single injection, minimal inhibitory enrofloxacin concentrations of up to 0.5 microg ml or microg/g sample against most pathogens of osteomyelitis were exceeded. In the musculature, on average, higher concentrations of active constituents were detected than in less perfused matrices (bones and synovial membranes) at sampling time. The enrofloxacin diffusion into inflamed bone was higher compared with mechanically damaged bone, whereas for ciprofloxacin it was lower. In conclusion, a dosage of 10 mg/kg enrofloxacin is sufficient to exceed the minimal inhibitory concentrations in osteomyelitic bone against most pathogens that are sensitive in vitro, but clinical efficacy remains to be evaluated.     

Effects of carprofen and dexamethasone on canine chondrocytes in a three-dimensional culture model of osteoarthritis

OBJECTIVE: To determine effects of carprofen and dexamethasone on chondrocytes in a culture model of osteoarthritis (OA). SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of the humeral head of 5 adult dogs. PROCEDURE: Chondrocytes were harvested, cultured and subcultured in monolayer, and then cultured in a 3-dimensional (3-D) medium. Cells from each dog were distributed into 6 groups with differing content of liquid medium for each 3-D construct (agarose [AG], AG plus interleukin [IL]-1beta, AG plus carprofen [4 microg/mL], AG plus dexamethasone [1 mg/mL], AG plus IL-1beta [20 ng/mL] plus carprofen [4 microg/mL], and AG plus IL-1beta (20 ng/mL) plus dexamethasone (1 mg/mL). On days 3, 6, 12, and 20 of culture, samples from all groups were collected. Liquid media were assayed for glycosaminoglycan, prostaglandin (PG)E2, matrix metalloprotease (MMP)-3, and MMP-13 concentrations. All 3-D constructs were evaluated for viability, cell morphology, proteoglycan staining, and collagen type-II concentration. Total glycosaminoglycan content in each 3-D construct was quantitated by spectrophotometric assay. RESULTS: Addition of IL-1beta caused a significant loss of cell viability and matrix production. Addition of carprofen or dexamethasone caused significant decreases in PGE2 in the liquid media, and each was minimally effective in protecting chondrocytes against negative effects of IL-1beta. CONCLUSIONS AND CLINICAL RELEVANCE: Human recombinant IL-1beta resulted in loss of cell viability, alterations in extracellular matrix components, and production of PG and MMP Carprofen and dexamethasone had little effect on cell and matrix variables but did decrease PGE2 concentrations and primarily affected the inflammatory pathway of osteoarthritis.     

Effects of human recombinant interleukin-1beta on canine articular chondrocytes in three-dimensional culture

OBJECTIVE: To determine the effects of interleukin (IL)-1beta on matrix synthesis and degradation by chondrocytes cultured in a 3-dimensional (3-D) gel medium. SAMPLE POPULATION: Chondrocytes from 7 dogs. PROCEDURE: Articular chondrocytes were harvested and cultured in 3-D gel medium alone or with 10 or 20 ng IL-1beta/ml that was added beginning on day 0, 3, 6, or 9. On days 3, 6, 12, and 20 of 3-D culture, samples of the liquid medium were evaluated for glycosaminoglycan (GAG), prostaglandin E2 (PGE2), and matrix metalloprotease (MMP)-3 content. The 3-D plug in each well was evaluated for histologic characteristics of viability, cell morphology, and proteoglycan staining, immunohistochemically stained for collagen type II, and spectrophotometrically analyzed for GAG content. RESULTS: Significant differences for all variables were detected between controls and each IL-1beta group, among groups with different IL-1beta concentrations, and among groups with IL-1beta added at various time points. Chondrocytes exposed to IL-1beta had loss of GAG, increased PGE2 and MMP-3 concentrations, and lack of collagen type-II synthesis. These IL-1beta effects appeared to be time and concentration dependent. CONCLUSIONS: Addition of IL-1beta to chondrocytes in 3-D gel medium results in time- and concentration-dependent effects on matrix synthesis and degradation and provides an appropriate in vitro model for many of the pathophysiologic events associated with osteoarthritis.     

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.     

Effects of enrofloxacin and magnesium deficiency on matrix metabolism in equine articular cartilage

OBJECTIVE: To investigate the effects of enrofloxacin and magnesium deficiency on explants of equine articular cartilage. SAMPLE POPULATION: Articular cartilage explants and cultured chondrocytes obtained from adult and neonatal horses. PROCEDURE: Full-thickness explants and cultured chondrocytes were incubated in complete or magnesium-deficient media containing enrofloxacin at concentrations of 0, 1, 5, 25, 100, and 500 microg/ml. Incorporation and release of sulfate 35S over 24 hours were used to assess glycosaminoglycan (GAG) synthesis and degradation. An assay that measured binding of dimethylmethylene blue dye was used to compare total GAG content between groups. Northern blots of RNA from cultured chondrocytes were probed with equine cDNA of aggrecan, type-II collagen, biglycan, decorin, link protein, matrix metalloproteinases 1, 3, and 13, and tissue inhibitor of metalloproteinase 1. RESULTS: A dose-dependent suppression of 35S incorporation was observed. In cartilage of neonates, 35S incorporation was substantially decreased at enrofloxacin concentrations of 25 mg/ml. In cartilage of adult horses, 35S incorporation was decreased only at enrofloxacin concentrations of > or =100 microg/ml. Magnesium deficiency caused suppression of 35S incorporation. Enrofloxacin or magnesium deficiency did not affect GAG degradation or endogenous GAG content. Specific effects of enrofloxacin on steady-state mRNA for the various genes were not observed. CONCLUSION AND CLINICAL RELEVANCE: Enrofloxacin may have a detrimental effect on cartilage metabolism in horses, especially in neonates.     

Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in chickens

The pharmacokinetics of enrofloxacin (EFX) and ciprofloxacin (CFX) was investigated in broiler chickens. Each antimicrobial was administered intravenously at a dose of 5 mg/kg body weight. Blood was taken in different preset times: prior and at 0.03, 0.06, 0.13, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h following drug administration. The concentrations of EFX and CFX in plasma were determined by high pressure liquid chromatography (HPLC). Plasma concentrations vs. time were analysed by a compartmental independent pharmacokinetic model that provided the most important kinetic parameters. Statistically significant differences between the two antimicrobials were found for most of the pharmacokinetic parameters: Area under the curve (AUC), area under first moment curve (AUMC), mean residence time (MRT), total body cleareance (ClB), volume of distribution beta (Vd beta) and volume of distribution at the steady state (Vd(ss)). Both antimicrobials were widely distributed in chickens throughout the body with a mean Vd(ss) of 1.98+/-0.18 L/kg for EFX, and 4.04+/-0.69 L/kg for CFX. The ClB for CFX was five times higher than that obtained for EFX. AUC, MRT and the diminished half time for EFX were two-four times higher than those obtained for CFX. These results indicate that CFX remains in the body for less time than the other quinolone. This characteristic of CFX suggests the advantage of a shorter withdrawal time for food producing animals treated with this antimicrobial.     

In vitro effects of glucosamine and acetylsalicylate on canine chondrocytes in three-dimensional culture

OBJECTIVE: To determine effects of glucosamine and acetylsalicylate on canine chondrocytes in 3-dimensional culture. SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of 2 adult female dogs recently euthanatized for reasons unrelated to orthopedic abnormalities. PROCEDURE: Chondrocytes were cultured in a 3-dimensional agarose-based medium alone (control), with glucosamine (100 microg/ml; GL), or with acetylsalicylate (18 microg/ml; AS). Supernatant and agarose plugs from 4 wells/group/d were collected on days 3, 6, and 12 of culture. Agarose plugs were evaluated for percentage of viable cells, percentage of cells producing pericellular or territorial matrix, glycosaminoglycan (GAG) concentration, and type-II collagen production. Prostaglandin E2 concentration in supernatants was determined. RESULTS: Chondrocytes in all groups had characteristics indicative of viability and differentiation; however, on day 12, a lower percentage of viable cells was detected in the AS group, compared with the other 2 groups. On day 6, GAG concentration in the AS group was significantly greater than concentrations in the other 2 groups. On day 12, GAG concentrations in the GL and AS groups were significantly less than in the control group. Within the GL and AS groups, cell viability was significantly less on day 12, compared with day 3. Significant differences in PGE2 concentration among or within groups and evidence of type II collagen production were not detected. CONCLUSIONS: 3-dimensional culture of canine chondrocytes allows for production of hyaline cartilage matrix constituents and growth of cells with morphologic characteristics similar to those of articular cartilage. Acetylsalicylate and glucosamine, at the single concentration evaluated, had detrimental effects on chondrocyte viability, GAG production, or both.     

Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs

Rung, K., Riond, J.-L. & Wanner, M. Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs. J. vet
Four dogs were given 5 mg/kg body weight enrofloxacin intravenously (i.v.) and orally (p.o.) in a cross-over study. Plasma concentrations of the active ingredient enrofloxacin and its main metabolite ciprofloxacin were determined by a reversed phase liquid chromatographic method. Pharmacokinetic parameters of both substances were calculated by use of statistical moments and were compared to those of enrofloxacin described in the veterinary literature. Mean enrofloxacin t _½λZ was 2.4 h, mean Cl_s was 27.1 ml/min-kg, and mean V_ss was 7.0 1/kg. After i.v. and p.o. administration, concentrations of ciprofloxacin exceeding minimal inhibitory concentrations of several microorganisms were reached (C_max= 0.2 ng/ml, max = 2.2 h after intravenous administration; C_max= 0.2 (ig/ml, t _max= 3.6 h after oral administration). A considerable part of the antimicrobial activity is due to ciprofloxacin, the main metabolite of enrofloxacin.     

Proliferation of canine intervertebral disk chondrocytes in three-dimensional alginate microsphere culture

Proliferation of chondrocytes from nucleus pulposus (NP) and anulus fibrosus (AF) was confirmed in three-dimensional culture using alginate microspheres. Cells isolated from NP and AF were incorporated in microspheres and cultured for 14 days. Round mononuclear cells of 20-25 microm in diameter proliferated and formed aggregates. At day 14, alcian blue positive matrix surrounded the proliferating cells. The cells had cytoplasmic vacuoles stained positively by toluidine blue. On electron microscopy, the cells contained proteoglycan vacuoles and lipid droplets in the cytoplasm and synthesized collagen fibrils and electron dense granules surrounding the cell. These features of the cells were characteristic for chondrocytes. This culture system should be useful to further investigate metabolic activities of intervertebral disk chondrocytes.     

Allometric analysis of ciprofloxacin and enrofloxacin pharmacokinetics across species

The purpose of this study was to examine the allometric analysis of ciprofloxacin and enrofloxacin using pharmacokinetic data from the literature. The pharmacokinetic parameters used were half-life, clearance and volume of distribution. Relationships between body weight and the pharmacokinetic parameter were based on the empirical formula Y = aW(b), where Y is half-life, clearance or volume of distribution, W the body weight and a is an allometric coefficient (intercept) that is constant for a given drug. The exponential term b is a proportionality constant that describes the relationship between the pharmacokinetic parameter of interest and body weight. A total of 21 different species of animals were studied. Results of the allometric analyses indicated similarity between clearance and volume of distribution as they related to body weight for both drugs. Results of the current analyses indicate it is possible to use allometry to predict pharmacokinetic variables of enrofloxacin or ciprofloxacin based on body size of species. This could provide information on appropriate doses of ciprofloxacin and enrofloxacin for all species.     

RP-HPLC法测定阿莫西林-盐酸环丙沙星可溶性粉的含量

应用RP-HPLC法测定阿莫西林-盐酸环丙沙星可溶性粉的含量.采用Shim-pack CLC-ODS C18柱(150 mm×6 mm, 5 μm),以1.5×10-2 mol/L三乙胺溶液(用磷酸调pH至3.0)-乙腈 (体积比85∶ 15)为流动相,流速为1.0 mL/min,检测波长为229 nm,柱温为40 ℃.在此色谱条件下两者能完全分离,阿莫西林、盐酸环丙沙星分别在22～108 μg/mL和29～146 μg/mL范围内峰面积与浓度呈良好的线性关系,相关系数均为0.999 9.阿莫西林、盐酸环丙沙星的回收率分别为99.50%～100.20%和99.30%～99.86%(n=5);RSD分别为0.60%～1.00%和0.86%～1.02%(n=5).本法可用于阿莫西林-盐酸环丙沙星可溶性粉的质量控制.     

Effects of methylprednisolone acetate and glucosamine on proteoglycan production by equine chondrocytes in vitro

OBJECTIVE: To evaluate the effects of methylprednisolone acetate (MPA) on proteoglycan production by equine chondrocytes and to investigate whether glucosamine hydrochloride modulates these effects at clinically relevant concentrations. SAMPLE POPULATION: Articular cartilage with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age). PROCEDURES: In vitro chondrocyte pellets were pretreated with glucosamine (0, 1, 10, and 100 microg/mL) for 48 hours and exposed to MPA (0, 0.05, and 0.5 mg/mL) for 24 hours. Pellets and media were assayed for proteoglycan production (Alcian blue precipitation) and proteoglycan content (dimethylmethylene blue assay), and pellets were assayed for DNA content. RESULTS: Methylprednisolone decreased production of proteoglycan by equine chondrocytes at both concentrations studied. Glucosamine protected proteoglycan production at all 3 concentrations studied. CONCLUSIONS AND CLINICAL RELEVANCE: Methylprednisolone, under noninflammatory conditions present in this study, decreased production of proteoglycan by equine chondrocytes. Glucosamine had a protective effect against inhibition of proteoglycan production at all 3 concentrations studied. This suggested that glucosamine may be useful as an adjunct treatment when an intra-articular injection of a corticosteroid is indicated and that it may be efficacious at concentrations relevant to clinical use.     

Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in lactating dairy cows and beef steers following intravenous administration of enrofloxacin

The comparative pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin were investigated in lactating cows and beef steers. The plasma elimination half-life of either enrofloxacin or ciprofloxacin was shorter in cows than in steers. The overall production of ciprofloxacin was slightly higher in steers than in cows (metabolite ratio: 64% and 59%, respectively). There was no significant difference in plasma protein binding of enrofloxacin between cows (percent bound: 59.4%) and steers (percent bound: 60.8%). Ciprofloxacin was more extensively bound to plasma proteins in steers (percent bound: 49.6%) than in cows (percent bound: 33.8%). The steady state volume of distribution of enrofloxacin is comparable in cows (1.55 L/kg) and steers (1.59 L/kg). Within either bovine class, plasma elimination half-life of enrofloxacin and ciprofloxacin are comparable, while plasma protein binding was higher for enrofloxacin than for ciprofloxacin. Ciprofloxacin was more concentrated in milk than enrofloxacin.     

恩诺沙星及其代谢物环丙沙星在猪粪便及尿液中的排泄研究

给猪连续3d饲喂含无味恩诺沙星的饲料后，对其排泄的粪便和尿液进行了药物含量测定结果表明，粪便和尿液中恩诺沙星及其代谢产物环丙沙星的排泄量都比较高，粪便中排泄的药物浓度显著高于尿液中药物排泄的浓度，停止给药后第15天，在猪粪中可检测到恩诺沙星，停止给药后第10天，猪尿液中可检测到环丙沙星。     

Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in goats given enrofloxacin alone and in combination with probenecid.

The pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin were investigated in goats given enrofloxacin alone or in combination with probenecid. Enrofloxacin was administered i.m. at a dosage of 5 mg x kg(-1) alone or in conjunction with probenecid (40 mg x kg(-1), i.v.). Blood samples were drawn from the jugular vein at predetermined time intervals after drug injection. Plasma was separated and analysed simultaneously for enrofloxacin and ciprofloxacin by reverse-phase high performance liquid chromatography. The plasma concentration-time data for both enrofloxacin and ciprofloxacin were best described by a one-compartment open pharmacokinetic model. The elimination half-life (t(1/2beta)), area under the plasma concentration-time curve (AUC), volume of distribution (V(d(area))), mean residence time (MRT) and total systemic clearance (Cl(B)) were 1.39 h, 7.82 microg x h x mL, 1.52 L x kg(-1), 2.37 h and 802.9 mL x h(-1) x kg(-1), respectively. Enrofloxacin was metabolized to ciprofloxacin in goats and the ratio between the AUCs of ciprofloxacin and enrofloxacin was 0.34. The t(1/2beta), AUC and MRT of ciprofloxacin were 1.82 h, 2.55 microg x h x mL and 3.59 h, respectively. Following combined administration of probenecid and enrofloxacin in goats, the sum of concentrations of enrofloxacin and ciprofloxacin levels > or = 0.1 microg x mL(-1) persisted in plasma up to 12 h.Co-administration of probenecid did not affect the t(1/2beta), AUC, V(d (area)) and Cl(B) of enrofloxacin, whereas the values of t(1/2beta) (3.85 h), AUC (6.29 microg x h x mL), MRT (7.34 h) and metabolite ratio (0.86) of ciprofloxacin were significantly increased. The sum of both enrofloxacin and ciprofloxacin levels was > or = 0.1 microg x mL(-1) and was maintained in plasma up to 8 h in goats after i.m. administration of enrofloxacin alone. These data indicate that a 12 h dosing regime may be appropriate for use in goats.