Copper(II) complexes with non-steroidal anti-inflammatory drugs: Structural characterization, in vitro and in silico biological profile.

Six novel copper(II) complexes with the non-steroidal anti-inflammatory drugs ibuprofen, loxoprofen, fenoprofen and clonixin as ligands were synthesized and characterized by diverse techniques including single-crystal X-ray crystallography. The in vitro scavenging activity of the complexes against 1,1-diphenyl-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) free radicals and the ability to reduce H2O2 were studied in the context of the antioxidant activity studies. The complexes may interact with calf-thymus DNA via intercalation as revealed by the techniques employed. The affinity of the complexes for bovine and human serum albumins was evaluated by fluorescence emission spectroscopy and the corresponding binding constants were determined. Molecular docking simulations on the crystal structure of calf-thymus DNA, human and bovine serum albumins were also employed in order to study in silico the ability of the studied compounds to bind to these target biomacromolecules, in terms of impairment of DNA and transportation through serum albumins, to explain the observed in vitro activity and to establish a possible mechanism of action.

Identification and characterization of the enzymes responsible for the metabolism of the non-steroidal anti-inflammatory drugs, flunixin meglumine and phenylbutazone, in horses.

The in vivo metabolism and pharmacokinetics of flunixin meglumine and phenylbutazone have been extensively characterized; however, there are no published reports describing the in vitro metabolism, specifically the enzymes responsible for the biotransformation of these compounds in horses. Due to their widespread use and, therefore, increased potential for drug-drug interactions and widespread differences in drug disposition, this study aims to build on the limited current knowledge regarding P450-mediated metabolism in horses. Drugs were incubated with equine liver microsomes and a panel of recombinant equine P450s. Incubation of phenylbutazone in microsomes generated oxyphenbutazone and gamma-hydroxy phenylbutazone. Microsomal incubations with flunixin meglumine generated 5-OH flunixin, with a kinetic profile suggestive of substrate inhibition. In recombinant P450 assays, equine CYP3A97 was the only enzyme capable of generating oxyphenbutazone while several members of the equine CYP3A family and CYP1A1 were capable of catalyzing the biotransformation of flunixin to 5-OH flunixin. Flunixin meglumine metabolism by CYP1A1 and CYP3A93 showed a profile characteristic of biphasic kinetics, suggesting two substrate binding sites. The current study identifies specific enzymes responsible for the metabolism of two NSAIDs in horses and provides the basis for future study of drug-drug interactions and identification of reasons for varying pharmacokinetics between horses.

Plasma and urine pharmacokinetics of intravenously administered flunixin in greyhound dogs.

Medication control in greyhound racing requires information from administration studies that measure drug levels in the urine as well as plasma, with time points that extend into the terminal phase of excretion. To characterize the plasma and the urinary pharmacokinetics of flunixin and enable regulatory advice for greyhound racing in respect of both medication and residue control limits, flunixin meglumine was administered intravenously on one occasion to six different greyhounds at the label dose of 1 mg/kg and the levels of flunixin were measured in plasma for up to 96 hr and in urine for up to 120 hr. Using the standard methodology for medication control, the irrelevant plasma concentration was determined as 1 ng/ml and the irrelevant urine concentration was determined as 30 ng/ml. This information can be used by regulators to determine a screening limit, detection time and a residue limit. The greyhounds with the highest average urine pH had far greater flunixin exposure compared with the greyhounds that had the lowest. This is entirely consistent with the extent of ionization predicted by the Henderson-Hasselbalch equation. This variability in the urine pharmacokinetics reduces with time, and at 72 hr postadministration, in the terminal phase, the variability in urine and plasma flunixin concentrations are similar and should not affect medication control.

Analgesic and anti-inflammatory controlled-released injectable microemulsion: Pseudo-ternary phase diagrams, in vitro, ex vivo and in vivo evaluation.

PURPOSE: Development of analgesic and anti-inflammatory controlled-released injectable microemulsions utilising lysine clonixinate (LC) as model drug and generally regarded as safe (GRAS) excipients. METHODS: Different microemulsions were optimised through pseudo-ternary phase diagrams and characterised measuring droplet size, viscosity, ex vivo haemolytic activity and in vitro drug release. The anti-inflammatory and analgesic activity was tested in mice (Hot plate test) and rats (Carrageenan-induced paw edema test) respectively and their activity was compared to an aqueous solution of LC salt. RESULTS: The aqueous solution showed a faster and shorter response whereas the optimised microemulsion increased significantly (p<0.01) the potency and duration of the analgesic and anti-inflammatory activity after deep intramuscular injection. The droplet size and the viscosity were key factors to control the drug release from the systems and enhance the effect of the formulations. CONCLUSIONS: The microemulsion consisting of Labrafil®/Lauroglycol®/Polysorbate 80/water with LC (56.25/18.75/15/10, w/w) could be a promising formulation after buccal surgery due to its ability to control the drug release and significantly achieve greater analgesic and anti-inflammatory effect over 24h.

Pharmacokinetics and tissue elimination of flunixin in veal calves.

OBJECTIVE To describe plasma pharmacokinetic parameters and tissue elimination of flunixin in veal calves. ANIMALS 20 unweaned Holstein calves between 3 and 6 weeks old. PROCEDURES Each calf received flunixin (2.2 mg/kg, IV, q 24 h) for 3 days. Blood samples were collected from all calves before the first dose and at predetermined times after the first and last doses. Beginning 24 hours after injection of the last dose, 4 calves were euthanized each day for 5 days. Plasma and tissue samples were analyzed by ultraperformance liquid chromatography. Pharmacokinetic parameters were calculated by compartmental and noncompartmental methods. RESULTS Mean ± SD plasma flunixin elimination half-life, residence time, and clearance were 1.32 ± 0.94 hours, 12.54 ± 10.96 hours, and 64.6 ± 40.7 mL/h/kg, respectively. Mean hepatic and muscle flunixin concentrations decreased to below FDA-established tolerance limits (0.125 and 0.025 µg/mL, respectively) for adult cattle by 3 and 2 days, respectively, after injection of the last dose of flunixin. Detectable flunixin concentrations were present in both the liver and muscle for at least 5 days after injection of the last dose. CONCLUSIONS AND CLINICAL RELEVANCE The labeled slaughter withdrawal interval for flunixin in adult cattle is 4 days. Because administration of flunixin to veal calves represents extralabel drug use, any detectable flunixin concentrations in edible tissues are considered a violation. Results indicated that a slaughter withdrawal interval of several weeks may be necessary to ensure that violative tissue residues of flunixin are not detected in veal calves treated with that drug.

Identification of flunixin glucuronide and depletion of flunixin and its marker residue in bovine milk.

Residues of flunixin [and its marker residue 5-hydroxyflunixin (5OHFLU)] were determined in milk from cows that intravenously received therapeutic doses of the drug. The samples were collected during each milking (every 12 h) for six consecutive days, and concentrations of flunixin and its metabolites were determined by the method with and without enzymatic hydrolysis (beta-glucuronidase). The highest flunixin concentration in milk was observed 12 h after dosing (2.4 ± 1.42 µg/kg, mean ± SD). Flunixin concentrations in the samples determined with enzymatic hydrolysis were significantly higher (P < 0.05), which suggests the transfer of flunixin glucuronide to the milk. Additionally, unambiguous identification of flunixin glucuronide in the bovine milk was performed with linear ion-trap mass spectrometry. The 5OHFLU concentrations analyzed without enzymatic hydrolysis (22.3 ± 16.04 µg/kg) were similar to this obtained with enzymatic hydrolysis. Flunixin and 5OHFLU concentrations dropped below the limits of detection at 48 h after last dosing.

Examining the occurrence of residues of flunixin meglumine in cull dairy cows by use of the flunixin cull cow survey.

OBJECTIVE: To determine whether cull dairy cows with signs of certain clinical conditions, termed suspect, are more likely than healthy-appearing cull dairy cows to have violative concentrations of flunixin meglumine in their tissues at slaughter. DESIGN: Cross-sectional study. ANIMALS: 961 cull dairy cows. PROCEDURES: Suspect cull dairy cows were selected from 21 beef slaughter establishments with a high production volume of dairy cows, and kidney and liver tissues were collected for screening. Kidney tissues were screened for antibiotics and sulfonamides with the fast antimicrobial screening test (FAST). Liver tissues were screened for flunixin meglumine with an ELISA, and quantitative analysis of ELISA-positive samples was performed with high-performance liquid chromatography. During the same time period, liver tissues from 251 healthy-appearing cull dairy cows were collected for the Food Safety and Inspection Service National Residue Program Scheduled Sampling Plan, but were screened only for flunixin meglumine. RESULTS: Of 710 suspect cull dairy cows, 50 (7.04%) had liver tissue flunixin concentrations higher than the flunixin tolerance concentration (0.125 ppm). Thirty-one of 168 (18.45%) FAST-positive and 19 of 542 (3.51%) FAST-negative suspect cull dairy cows had violative tissue flunixin concentrations. Two of the 251 (0.80%) healthy-appearing cull dairy cows had violative tissue flunixin concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: Suspect cull dairy cows, especially those that were also FAST positive, had a significantly higher incidence of violative tissue flunixin concentrations than healthy-appearing cull dairy cows at slaughter. Targeted sampling plans for flunixin meglumine in suspect dairy cows can help to support more efficient use of resources and further safeguard the nation's food supply.

[Determination of residual organic solvents in flunixin meglumine raw material by headspace gas chromatography].

A method for the determination of five kinds of residual organic solvents in flunixin meglumine raw material was developed by headspace gas chromatography. An HP-FFAP capillary column (30 m x 0.32 mm x 1.0 microm), a flame ionization detector and the external standard method were used for the separation and quantitative analysis. The effects of equilibrium temperature and equilibrium time on the determination of residual organic solvents were investigated. The good results were obtained in the equilibrium temperature of 90 degrees C and equilibrium time of 30 min. The standard curves were linear in the range of 0.40-7.93 mg/L (r = 0.999 8) for ethyl acetate, 7.32-146.48 mg/L (r = 0.999 6) for methanol, 4.53-90.61 mg/L (r = 0.999 9) for isopropanol, 3.62-72.32 mg/L (r = 0.999 8) for ethanol and 2.31-46.24 mg/L (r = 0.999 6) for acetonitrile. The recoveries for the five residual organic solvents were between 95.96% and 100.31% with relative standard deviations (RSDs) (n = 6) of 1.97%-3.28%. The detection limits of ethyl acetate, methanol, isopropanol, ethanol and acetonitrile were 0.08, 0.9, 0.2, 0.4 and 0.3 mg/L, respectively. The proposed method was successfully applied to analyze the residual organic solvents in the real sample of flunixin meglumine raw material. The results showed that only isopropanol and ethanol were found in the sample with the contents of 177.44 microg/g and 69.32 microg/g, respectively. The method is rapid, sensitive and accurate for the content determination of residual solvents in flunixin meglumine raw material.

Interaction of clonixin with EPC liposomes used as membrane models.

In this work, an overall analysis of clonixin interaction with liposomes was achieved using different techniques, which allowed the evaluation of the change in different membrane's characteristics as well as the possible location of the drug in the membrane. Clonixin acidity constants were obtained and the values are 5.5 +/- 0.08 and 2.2 +/- 0.04. Clonixin partition coefficient (K(p)) between liposomes and water was also determined using derivative spectrophotometry, fluorescence quenching, and zeta-potential (zeta-potential). These three techniques yielded similar results. zeta-potential measurements were performed and an increase of the membrane negative charge with an increase of drug concentration was observed. Drug location within the bilayer was performed by fluorescence quenching using a set of n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, and 12). The fluorescence intensity of all probes was quenched by the drug. This effect is more noticeable for the outer located probe, indicating that the drug is positioning in the external part of the membrane. These same probes were used for steady-state anisotropy measurements to determine the perturbation in membrane structure induced by clonixin. Clonixin increased membrane fluidity in a concentration dependent manner, with the highest perturbation occurring nearby the 2-AS probe, closely located to the bilayer surface.

Extraction and LC determination of lysine clonixinate salt in water/oil microemulsions.

A new reversed-phase high performance liquid chromatography method has been developed and validated for the quantitative determination of lysine clonixinate salt in water/oil microemulsions. The mobile phase was acetonitrile-buffer phosphate pH 3.3. Detection was UV absorbance at 252 nm. The precision and accurately of the method were excellent. The established linearity range was 5-60 microg ml(-1) (r(2)=0.999). Microemulsions samples were dispersed with chloroform and extracted lysine clonixinate salt with water. This easy method employing chloroformic extraction has been done three times. The recovery of lysine clonixinate salt from spiked placebo and microemulsion were >90% over the linear range.

Detection and identification of flunixin after multiple intravenous and intramuscular doses to horses.

The objectives of the study were to compare various methods to determine flunixin in test samples collected periodically from horses after intramuscular (IM) and intravenous (IV) dosing at the maximum recommended dosage and to document detection times for this drug in test samples. Flunixin, a nonsteroidal anti-inflammatory drug approved for use in horses, was administered to eight mares in five consecutive daily doses of 1.1 mg per kilogram of body weight by the IM or IV route. Flunixin was detected in urine samples collected at various times after drug administration by flunixin enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatographic-mass spectrometric (GC-MS) methods. Detection time was defined as the time period over which flunixin was detected and was dependent on the method used. The shortest detection times were 24 to 48 h and were observed when the TLC method was used. On the other hand, detection times were as long as 15 days when HPLC, GC-MS, and flunixin ELISA methods were used. The use of these more sensitive tests to monitor official samples collected from racehorses could result in positive tests for flunixin when it is exerting no detectable clinical effects because it produces clinical effects lasting only 24-36 h in horses.