Short communication: Determination of the milk pharmacokinetics and depletion of milk residues of flunixin following transdermal administration to lactating Holstein cows.

Flunixin is a nonsteroidal anti-inflammatory drug and the most commonly prescribed analgesic in cattle in the United States. Recently, the US Food and Drug Administration (FDA) approved a transdermal formulation of flunixin for control of pyrexia associated with bovine respiratory disease and the control of pain associated with foot rot. The transdermal formulation is not currently approved for use in lactating dairy cattle in the United States, but extra-label use in dairy cattle is permissible under US regulations. The objectives of this study were to determine the pharmacokinetics in milk of dairy cows treated with transdermal flunixin and determine an appropriate withdrawal time for milk. Ten lactating Holstein cows were enrolled into the study in mid lactation. Following treatment, cows were milked 3 times per day through 144 h. Milk samples were collected for drug analysis using ultra-high-pressure liquid chromatography coupled with a triple quadrupole mass spectrometer. The geometric mean maximum concentration for flunixin in milk was 0.010 µg/mL and was 0.061 µg/mL for the active metabolite, 5-hydroxyflunixin. The geometric mean terminal half-life was 20.71 h for flunixin and 22.62 h for 5-hydroxyflunixin. Calculations to approximate a withdrawal time in milk following transdermal flunixin administration were accomplished using a statistical tolerance limit procedure. This analysis indicated that it would be prudent to observe a withdrawal period of 96 h following the last treatment. This is more than twice as long as the labeled withdrawal period of 36 h following use of the injectable formulation. The withdrawal period suggested by this work should be applied carefully, as this study was not conducted under the full quality control practices required by the US FDA for a full drug approval study. Caution should be taken when applying this withdrawal time to diseased animals, animals that are milked with different milking frequencies, and those in different stages of production as these have all been shown to affect drug depletion from milk.

Pharmacokinetics of multiple doses of transdermal flunixin meglumine in adult Holstein dairy cows.

A transdermal formulation of the nonsteroidal anti-inflammatory drug, flunixin meglumine, has been approved in the United States and Canada for single-dose administration. Transdermal flunixin meglumine was administered to 10 adult Holstein cows in their second or third lactation at the label dose of 3.33 mg/kg every 24 hr for three total treatments. Plasma flunixin concentrations were determined using high-pressure liquid chromatography with mass spectroscopy (HPLC-MS). Pharmacokinetic analysis was completed on each individual animal with noncompartmental methods using computer software. The time to maximum drug concentration (Tmax) was 2.81 hr, and the maximum drug concentration was 1.08 µg/ml. The mean terminal half-life (T½) was determined to be 5.20 hr. Clearance per fraction absorbed (Cl/F) was calculated to be 0.294 L/hr kg-1 , and volume of distribution of fraction (Vz/F) absorbed was 2.20 L/kg. The mean accumulation factor was 1.10 after three doses. This indicates changes in dosing may not be required when giving multiple doses of flunixin transdermal. Further work is required to investigate the clinical efficacy of transdermal flunixin after multiple daily doses.

Pharmacokinetic and pharmacodynamic characterization of ceftiofur crystalline-free acid following subcutaneous administration in domestic goats.

Pharmacokinetic (PK)-pharmacodynamic (PD) integration of crystalline ceftiofur-free acid (CCFA) was established in six healthy female goats administered subcutaneously (s.c.) on the left side of the neck at a dosage of 6.6 mg/kg body weight. Serum concentrations of ceftiofur and desfuroylceftiofur (DFC) were determined using high-performance liquid chromatography. Mutant prevention concentration (MPC), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ceftiofur were determined for Pasteurella (P.) multocida. Mean terminal half-life and mean residence time of ceftiofur + DFC were 48.6 h and 104 h, respectively. In vitro plasma protein binding of ceftiofur was 46.6% in goats. The MIC and MBC values of ceftiofur were similar in serum and MHB and a very small difference between these values confirmed bactericidal activity of drug against P. multocida. In vitro and ex vivo time-kill curves for P. multocida demonstrated a time-dependent killing action of drug. Considering target serum concentration of 0.20 µg/mL, PK-PD values for AUC24 h /MIC90 and T > MIC90 , respectively, were 302 h and 192 h against P. multocida. A MPC/MIC ratio of 10-14 indicated that selective pressure for proliferation of resistant mutants of P. multocida is minimal after CCFA single-dose administration. Based on MPC = 1.40 µg/mL for P. multocida, the PK-PD indices, viz. T > MPC and AUC24 /MPC, were 48 h and 43 h, respectively. The data suggested the use of single dose (6.6 mg/kg, s.c.) of CCFA in goats to obtain clinical and bacteriological cure of pneumonia due to P. multocida.

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 (Cmax ), terminal half-life (t1/2 K10) , 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 Emax equation provided AUC24 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 AUC24 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.

Pharmacokinetic and pharmacodynamic modelling of marbofloxacin administered alone and in combination with tolfenamic acid in calves.

In a four-period, cross-over study, the fluoroquinolone antibacterial drug marbofloxacin (MB) was administered to calves, alone and in combination with the nonsteroidal anti-inflammatory drug tolfenamic acid (TA). Both drugs were administered intramuscularly (IM) at doses of 2 mg/kg. A tissue cage model of inflammation, based on the actions of the mild irritant carrageenan, was used to evaluate the pharmacokinetics (PK) of MB and MB in combination with TA. MB mean values of area under concentration-time curve (AUC) were 15.1 µg·h/mL for serum, 12.1 µg·h/mL for inflamed tissue cage fluid (exudate) and 9.6 µg·h/mL for noninflamed tissue cage fluid (transudate). Values of C(max) were 1.84, 0.35 and 0.31 µg/mL, respectively, for serum, exudate and transudate. Mean residence time (MRT) of 23.6 h (exudate) and 22.6 h (transudate) also differed significantly from serum MRT (8.6 h). Co-administration of TA did not affect the PK profile of MB. The pharmacodynamics of MB was investigated using a bovine strain of Mannheimia haemolytica. Time-kill curves were established ex vivo on serum, exudate and transudate samples. Modelling the ex vivo serum time-kill data to the sigmoid E(max) equation provided AUC(24 h) /MIC values required for bacteriostatic (18.3 h) and bactericidal actions (92 h) of MB and for virtual eradication of the organism was 139 h. Corresponding values for MB + TA were 20.1, 69 and 106 h. These data were used to predict once daily dosage schedules for a bactericidal action, assuming a MIC(90) value of 0.24 µg/mL, a dose of 2.6 mg/kg for MB and 2.19 mg/kg for MB + TA were determined, which are similar to the currently recommended dose of 2.0 mg/kg.

Seroprevalence of Neospora caninum antibodies in cattle and water buffaloes in India.

Neospora caninum is now recognized as a major cause of abortion in cattle worldwide, but there is no report of N. caninum infection in cattle in India. Serum samples from 427 dairy cattle and 32 dairy water buffaloes from 7 organized dairy farms located in Punjab, India, were tested for N. caninum antibodies using a commercial monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Antibodies to N. caninum were found in 35 of 427 cattle from 6 of the 7 farms; 9.6% of cows, 5.1% of heifers, and 5.0% of calves were seropositive, suggesting postnatal transmission of N. caninum on the farm. Antibodies to N. caninum were found in 16 of 32 buffaloes tested from 2 dairy farms. In total, 64 cattle and 16 buffalo sera already tested by ELISA were also evaluated by an indirect fluorescent antibody test (IFAT) to verify ELISA results. Of the 64 cattle samples, 29 sera were negative by both tests and of the 35 ELISA-positive sera, 12 had IFAT titers of 1:100 or higher (1 had IFAT titer of 100, 2 had IFAT titer of 200, and 9 had IFAT titers of 400 or higher). Of the 16 buffalo sera positive by ELISA, 1 had an IFAT titer of 1:400. Thus, antibodies to N. caninum were demonstrated in cattle sera by 2 serologic methods. To our knowledge this is the first report of N. caninum infection in cattle and buffaloes in India.

Pharmacokinetic and pharmacodynamic interactions of tolfenamic acid and marbofloxacin in goats.

Pharmacokinetic and pharmacodynamic properties in goats of the non-steroidal anti-inflammatory drug tolfenamic acid (TA), administered both alone and in combination with the fluoroquinolone marbofloxacin (MB), were established in a tissue cage model of acute inflammation. Both drugs were injected intramuscularly at a dose rate of 2 mg kg(-1). After administration of TA alone and TA+MB pharmacokinetic parameters of TA (mean values) were Cmax=1.635 and 1.125 microg ml(-1), AUC=6.451 and 3.967 microgh ml(-1), t1/2K10=2.618 and 2.291 h, Vdarea/F=1.390 and 1.725Lkg(-1), and ClB/F=0.386 and 0.552 L kg(-1) h(-1), respectively. These differences were not statistically significant. Tolfenamic acid inhibited prostaglandin (PG)E2 synthesis in vivo in inflammatory exudate by 53-86% for up to 48 h after both TA treatments. Inhibition of synthesis of serum thromboxane (Tx)B2 ex vivo ranged from 16% to 66% up to 12h after both TA and TA+MB, with no significant differences between the two treatments. From the pharmacokinetic and eicosanoid inhibition data for TA, pharmacodynamic parameters after dosing with TA alone for serum TxB2 and exudate PGE2 expressing efficacy (Emax=69.4 and 89.7%), potency (IC50=0.717 and 0.073 microg ml(-1)), sensitivity (N=3.413 and 1.180) and equilibration time (t1/2Ke0=0.702 and 16.52 h), respectively, were determined by PK-PD modeling using an effect compartment model. In this model TA was a preferential inhibitor of COX-2 (COX-1:COX-2 IC50 ratio=12:1). Tolfenamic acid, both alone and co-administered with MB, did not affect leucocyte numbers in exudate, transudate or blood. Compared to placebo significant attenuation of skin temperature rise over inflamed tissue cages was obtained after administration of TA and TA+MB with no significant differences between the two treatments. Marbofloxacin alone did not significantly affect serum TxB2 and exudate PGE2 concentrations or rise in skin temperature over exudate tissue cages. These data provide a basis for the rational use of TA in combination with MB in goat medicine.

Efficacy of abamectin against ivermectin-resistant strain of Trichostrongylus colubriformis in sheep.

The efficacy of two formulations of abamectin, i.e. oral and injectable was determined against ivermectin-resistant strain of T. colubriformis in sheep. Twenty-four lambs were infected with 10,000 third stage larvae of ivermectin-resistant strain of T. colubriformis. Twenty-four days post-infection, the lambs were divided randomly into four groups of six animals each according to egg counts. The first group was left untreated and kept as a control. The second group was treated with ivermectin (oral) at 0.2mg kg(-1) body weight. The third group was treated with oral formulation of abamectin at 0.2mg kg(-1) body weight. The fourth group was treated with injectable formulation of abamectin at 0.2mg kg(-1) body weight. Fecal egg count and controlled slaughter tests were employed to determine the efficacy of abamectin (oral and injection) against ivermectin-resistant strain of T. colubriformis in sheep. Reduction in arithmetic mean fecal egg counts achieved by ivermectin (oral), abamectin (oral) and abamectin (injection) was 66, 98 and 76%, respectively 10 days after treatment. Ivermectin (oral), abamectin (oral) and abamectin (injection) reduced arithmetic mean worm burden by 63, 97 and 74%, respectively. The findings demonstrated that abamectin oral formulation was more effective than abamectin injection against ivermectin-resistant strain of T. colubriformis in sheep.

Glucose-6-phosphate dehydrogenase deficiency in blood donors: screening by micromethaemoglobin reduction test.

500 blood donors were screened for G6PD deeiciency using micromethaemoglobin reduction (microMRT) test. Most of the blood donors were young adult males (95.8%). The overall incidence of G6PD deficiency was found to be 0.8%. There, was apparently decreased frequency of G6PD deficient blood donors with increasing age, and no relation could be ascertained between G6PD) deficiency and blood groups.

Mutant prevention concentration and PK-PD relationships of enrofloxacin for Pasteurella multocida in buffalo calves.

This study validated the use of mutant prevention concentration (MPC) and pharmacokinetic and pharmacodynamic (PK-PD) modeling approach for optimization of dose regimen of enrofloxacin to contain the emergence of Pasteurella multocida resistance. The PK and PD characteristics of enrofloxacin were investigated in buffalo calves after intramuscular administration at a dose rate of 12 mg/kg. The concentration of enrofloxacin and ciprofloxacin in serum were determined by high-performance liquid chromatography. The serum peak concentration (Cmax), terminal half-life (t1/2K10), volume of distribution (Vd(area)/F) and mean residence time (MRT) of enrofloxacin were 1.89 ± 0.35 µg/ml, 5.14 ± 0.66 h, 5.59 ± 0.99 l/kg/h and 8.52 ± 1.29 h, respectively. The percent metabolite conversion ratio of ciprofloxacin to enrofloxacin was 79. The binding of enrofloxacin to plasma proteins was 11%. The MIC, MBC and MPC for enrofloxacin against P. multocida were 0.05, 0.06 µg/ml and 1.50 µg/ml.In vitro and ex-vivo bactericidal activity of enrofloxacin was concentration dependent. Modeling of ex-vivo growth inhibition data to the sigmoid Emax equation provided AUC24h/MIC values to produce bacteriostatic (19 h), bactericidal (43 h) and bacterial eradication (64 h). PK-PD data in conjunction with MPC and MIC90 data predicted dosage schedules for enrofloxacin that may achieve optimum efficacy in respect of bacteriological and clinical cure and minimize the risk of emergence of resistance.

Evaluation of factors contributing to excessive nitrate accumulation in fodder crops leading to ill-health in dairy animals.

A study was conducted to estimate nitrate content in commonly used fodder crops, viz., berseem (Trifolium alexandrinum), bajra (Pennisetum glaucum), maize (Zea mays), oats (Avena sativa), sorghum (Sorghum vulgare) and toriya (Brassica napus), collected from the fields of different villages of Punjab and farms of the university, and to evaluate the factors associated with nitrate accumulation in these crops. The nitrate level was highest in sorghum on dry matter basis, followed by oats and toriya, berseem, maize and bajra. The nitrate content was also determined in fodder samples harvested from young and mature stages and in different parts of plants. The stem part of forages had higher content than leaves; however, concentrations were low in mature crops as compared to immature ones. The environmental and soil factors associated with it are discussed and correlated with the experimental findings.

PK-PD integration and modeling of marbofloxacin in sheep.

The fluoroquinolone antimicrobial drug, marbofloxacin, was administered intravenously (IV) and intramuscularly (IM) to sheep at a dose rate of 2 mg kg(-1) in a 2-period cross-over study. Using a tissue cage model of inflammation, the pharmacokinetic properties of marbofloxacin were established for serum, inflamed tissue cage fluid (exudate) and non-inflamed tissue cage fluid (transudate). For serum, after IV dosing, mean values for pharmacokinetic parameters were: clearance 0.48 L kg(-1) h(-1); elimination half-life 3.96 h and volumes of distribution 2.77 and 1.96 L kg(-1), respectively, for V(darea) and V(ss). After IM dosing mean values for pharmacokinetic variables were: absorption half-time 0.112 h, time of maximum concentration 0.57 h, terminal half-life (T(1/2)el) 3.65 h and bioavailability 106%. For exudate, mean T(1/2)el values were 12.38 and 13.25 h, respectively, after IV and IM dosing and for transudate means were 13.39 h (IV) and 12.55 h (IM). The in vitro minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) and ex vivo time-kill curves for marbofloxacin in serum, exudate and transudate were established against a pathogenic strain of Mannheimia haemolytica. Integration of in vivo pharmacokinetic data with MIC determined in vitro provided mean values of area under curve (AUC)/MIC ratio for serum, exudate and transudate of 120.2, 156.0 and 156.6 h after IV dosing and 135.5, 165.3 and 146.2 h after IM dosing, respectively. After IM administration maximum concentration (C(max))/MIC ratios were 21.1, 6.76 and 5.91, respectively, for serum, exudate and transudate. The ex vivo growth inhibition data after IM administration were fitted to the sigmoid E(max) (Hill) equation to provide values for serum of AUC(24h)/MIC producing, bactericidal activity (22.51 h) and virtual eradication of bacteria (35.31 h). It is proposed that these findings might be used with MIC(50) or MIC(90) data to provide a rational approach to the design of dosage schedules which optimise efficacy in respect of bacteriological as well as clinical cures.

Pharmacokinetic and pharmacodynamic modelling of marbofloxacin administered alone and in combination with tolfenamic acid in goats.

In a four-period cross-over study, the fluoroquinolone antibacterial drug marbofloxacin (MB) was administered to goats intramuscularly (IM) at a dose rate of 2 mg/kg, both alone and in combination with the non-steroidal anti-inflammatory drug tolfenamic acid (TA), also administered IM at a dose rate of 2 mg/kg. Using a tissue cage model of inflammation, based on the irritant actions of carrageenan, the pharmacokinetics (PK) of MB and MB in combination with TA were determined. MB mean values of area under concentration-time curve (AUC) were similar for serum (5.60 microg h/mL), inflamed tissue cage fluid (exudate; 5.32 microg h/mL) and non-inflamed tissue cage fluid (transudate; 4.82 microg h/mL). Values of mean residence time (MRT) of MB in exudate (15.5 h) and transudate (15.8 h) differed significantly from serum MRT (4.23 h). Co-administration of TA did not affect the PK profile of MB. The pharmacodynamics of MB were investigated using a caprine strain of Mannheimia haemolytica. Integration of PK data with ex vivo bacterial time-kill curve data for serum, exudate and transudate provided AUC(24h)/minimum inhibitory concentration (MIC) ratios of 160, 133 and 121 h, respectively, for the strain of organism used. Modelling of the ex vivo time-kill data to the sigmoid E(max) equation provided AUC(24h)/MIC values required for bacteriostatic and bactericidal actions of MB and for virtual eradication of the organism of 27.6, 96.2 and 147.3 h, respectively. Corresponding values for MB+TA were 20.5, 66.5 and 103.0 h. These data were used to predict once daily dosage schedules of MB for subsequent clinical evaluation.

Influence of marbofloxacin on the pharmacokinetics and pharmacodynamics of tolfenamic acid in calves.

Pharmacokinetic and pharmacodynamic properties of tolfenamic acid (TA) in calves were determined in serum and fluids of inflamed (carrageenan administered) and non-inflamed subcutaneously implanted tissue cages after intramuscular administration both alone and in combination with marbofloxacin (MB). MB significantly altered the pharmacokinetics of TA: mean values were Cmax = 2.14 and 1.64 microg/mL, AUC = 27.38 and 16.80 microg.h/mL, Vd(area)/F = 0.87 and 1.17 L/kg, and ClB/F = 0.074 and 0.128 L/kg/h, respectively, after administration of TA alone and TA + MB. T(1/2)K10 and MRT were not significantly different for the two treatments. The pharmacodynamic properties of TA were not influenced by MB co-administration, in spite of the alterations in some TA pharmacokinetic parameters. TA inhibited prostaglandin E2 (PGE2) synthesis in vivo in inflammatory exudate by 50-88% for up to 48 h after both TA treatments. Inhibition of synthesis of serum thromboxane B2 (TxB2) ex vivo ranged from 40 to 85% up to 24 h after both TA and TA + MB. From the derived pharmacokinetic and eicosanoid inhibition data for TA, pharmacodynamic parameters for serum TxB2 and exudate PGE2 inhibition expressing efficacy (Emax = 78.1 and 97.5%), potency (IC50 = 0.256 and 0.265 microg/mL), sensitivity (N = 1.96 and 2.29) and the pharmacokinetic parameter equilibration time (t(1/2)K(e0) = 0.695 and 24.0 h), respectively, were determined. In this model TA was a nonselective inhibitor of cyclo-oxygenase (COX) (COX-1:COX-2 IC50 ratio = 1.37). TA, both alone and co-administered with MB, did not affect leucocyte numbers in exudate, transudate or blood. Partial attenuation of skin temperature rise over inflamed tissue cages and reduction of zymosan-induced skin swelling were recorded after administration of TA and TA + MB with no significant differences between the two treatments. These data provide a basis for the rational use of TA in combination with MB in calf medicine.

Serological analysis of patients treated with a new surgical hemostat containing bovine proteins and autologous plasma.

A randomized, controlled clinical study of the management of diffuse bleeding with CoStasis surgical hemostat, a new hemostat containing bovine thrombin and collagen with the patient's own plasma, included patients undergoing cardiac, hepatic, iliac, and general surgery. Sera from 92 patients treated with CoStasis and 84 control patients were collected preoperatively and at a post surgical follow-up of 8 weeks. Among the control group, 57 patients were treated with Instat collagen sponge in noncardiac indications. Results showed that antibody responses in the CoStasis clinical study were similar to the reported literature for all antigens screened and were not associated with any adverse reactions. The bovine thrombin preparations in CoStasis and other commercially available thrombins were compared with the use of SDS-PAGE and Western blot analyses. Within this clinical study, CoStasis was shown to be a safe and effective hemostatic product containing bovine thrombin and bovine collagen and no pooled human blood products.