Tissue Cage Fluid Research Articles

Antagonistic Effect of Rifampin on the Efficacy of High-Dose Levofloxacin in Staphylococcal Experimental Foreign-Body Infection

Since levofloxacin at high doses was more active than levofloxacin at conventional doses and was the best therapy alone in a rat model of staphylococcal foreign-body infection, in this study we tested how these differences affect the activities of their respective combinations with rifampin in vitro and in vivo. In vitro studies were performed in the log and stationary phases. By using this model, rifampin at 25 mg/kg of body weight/12 h, levofloxacin at 100 mg/kg/day, levofloxacin at 100 mg/kg/day plus rifampin, levofloxacin at 50 mg/kg/day, levofloxacin at 50 mg/kg/day plus rifampin, or a control treatment was administered for 7 days; and therapy with for levofloxacin at 100 mg/kg/day alone and rifampin alone was prolonged to 14 days. We screened for the appearance of resistant strains. Killing curves in the log phase showed a clear antagonism with levofloxacin at concentrations >or=2x MIC and rifampin and tended to occur in the stationary phase. At the end of 7 days of therapy, levofloxacin at 100 mg/kg/day was the best treatment and decreased the bacterial counts from tissue cage fluid (P < 0.05 compared with the results for groups except those receiving rifampin alone). At the end of 14 days of therapy with levofloxacin at 100 mg/kg/day, levofloxacin at 100 mg/kg/day plus rifampin, and the control treatment, the bacterial counts on the coverslips were 2.24 (P < 0.05 compared with the results with the combined therapy), 3.36, and 5.4 log CFU/ml, respectively. No rifampin or levofloxacin resistance was detected in any group except that receiving rifampin alone. In conclusion, high-dose levofloxacin was the best treatment and no resistant strains appeared; the addition of rifampin showed an antagonistic effect. The efficacy of the rifampin-levofloxacin combination is not significantly improved by the dosage of levofloxacin.

Clinical efficacy of intravenous administration of marbofloxacin in a Staphylococcus aureus infection in tissue cages in ponies

Tissue cages (TC), implanted subcutaneously in the neck in eight ponies, were inoculated with Staphylococcus aureus (S. aureus) to determine the clinical efficacy of marbofloxacin in the treatment of this infection. From 21 h after inoculation, marbofloxacin (6 mg/kg) was administered intravenously (i.v.) once daily for 7 days. Samples of the tissue cage fluid (TCF) were taken to determine marbofloxacin concentrations (days 1, 3 and 7), using high-pressure liquid chromatography, and numbers of viable bacteria [colony forming units (CFU)] (days 1, 3, 7, 14 and 21). Statistical analysis was used to compare CFU before and after treatment. Clinical signs and CFU were used to evaluate the efficacy of treatment. Although, there was a slight decrease in CFU in all TC initially, the infection was not eliminated by marbofloxacin treatment in any of the ponies and abscesses formed. As the MIC (0.25 microg/mL) did not change during treatment and the concentration of marbofloxacin during treatment (mean concentration in TCF was 0.89 microg/mL on day 1, 0.80 microg/mL on day 3 and 2.77 microg/mL on day 7) was above MIC, we consider that the treatment failure might be attributable to the formation of a biofilm by S. aureus. Based on the present results, i.v. administration of marbofloxacin alone is not suitable for the elimination of S. aureus infections from secluded sites.

Efficacy of High Doses of Levofloxacin in Experimental Foreign-Body Infection by Methicillin-Susceptible Staphylococcus aureus

Antimicrobial efficacy in orthopedic device infections is diminished because of bacterial biofilms which express tolerance to antibiotics. Recently, the use of high doses of levofloxacin with rifampin has been recommended for staphylococcal infections. In the present study, we evaluated the efficacy of levofloxacin at doses of 50 mg/kg/day and 100 mg/kg/day (mimicking the usual and high human doses of 500 mg/day and 750 to 1,000 mg/day, respectively) and compared it to that of to linezolid, cloxacillin, vancomycin, and rifampin in a rat tissue cage model of experimental foreign-body infection by Staphylococcus aureus. The antimicrobial efficacy in vitro (by MIC, minimum bactericidal concentration, and kill curves) for logarithmic- and stationary-phase bacteria was compared with the in vivo efficacy. In vitro bactericidal activity at clinically relevant concentrations was reached by all drugs except rifampin and linezolid in the log-phase studies but only by levofloxacin in the stationary-phase studies. The bacterial count decreases from in vivo tissue cage fluids (means) for levofloxacin at 50 and 100 mg/kg/day, rifampin, cloxacillin, vancomycin, linezolid, and controls, respectively, were: -1.24, -2.26, -2.1, -1.56, -1.47, -1.15, and 0.33 (all groups versus controls, P < 0.05). Levofloxacin at 100 mg/kg/day (area under the concentration-time curve/MIC ratio, 234) was the most active therapy (P = 0.03 versus linezolid). Overall, in vivo efficacy was better predicted by stationary-phase studies, in which it reached a high correlation coefficient even if the rifampin group was excluded (r = 0.96; P < 0.05). Our results, including in vitro studies with nongrowing bacteria, pharmacodynamic parameters, and antimicrobial efficacy in experimental infection, provide good evidence to support the use of levofloxacin at high doses (750 to 1,000 mg/day), as recently recommended for treating patients with orthopedic prosthesis infections.

Lung IkB and NFkB with neutrophilia in vivo in a gram positive infection model

Gram-positive infections have increased over the past few years; unfortunately, our mechanistic knowledge of the related inflammatory processes remains incomplete. Transcription factor NFkB coordinates activation of many genes involved in host defense, and the IkB-NFkB cascade strongly recruits PMN in gram negative studies. To investigate the involvement of NFkB in a gram positive model, abdominal tissue cage infections were produced by a patient strain of TSST-producing S. aureus. Lungs were evaluated by histostain and laser scanning confocal microscopy to quantify IkB and NFkB. Their presence was compared to clinical infection markers. Following harvest at 1 week, lung sections were stained with anti- IkB or NFkB (Santa Cruz) followed by appropriate secondary antibodies tagged with FITC or Texas Red, and evaluated by confocal microscopy. Tissues/ fluids for clinical infection markers were collected at necropsy. Tissue TNF alpha averaged 300 pg/mL infected vs.74 in controls. Tissue cage fluid showed a single S. aureus strain, 109/mL. Lung H&E indicated granulocytic inflammatory infiltrate. Lung sections, seen by confocal microscopy double staining, showed that NFkB, normally bound to IkB in the cytoplasm, was released in bronchial epithelial cells, (34.2 stain intensity vs. 0 control). IkB was seen at high levels in the sub epithelial layer (36.1 intensity). The prominent PMN influx to lung seen with the distant gram positive infection may relate to IkB in the airway, as reported by the work of others. Our data demonstrated in vivo the processes suggested by lung cell culture, and provide a catalyst to investigations of these factors in the pathophysiology of inflammation. Funding: UMKC Med. internal

Clinical efficacy of local administration of ceftiofur in aStaphylococcus aureusinfection in tissue cages in ponies

Ceftiofur concentrations in an infected and uninfected environment were compared and the efficacy of locally administered ceftiofur was evaluated in an experimental infection with Staphylococcus aureus in tissue cages. Eight ponies had tissue cages (TCs) implanted s.c. on each side of the neck. Into one of the cages 150 mg of ceftiofur was administered and fluid samples were taken to determine ceftiofur concentrations. After 1 week the other TC was infected with S. aureus and subsequently treated with 150 mg ceftiofur administered locally into the TC once daily for 21 days. Samples of fluid were taken to determine ceftiofur concentrations and for bacterial counts. Ceftiofur concentrations did not differ significantly in the infected and uninfected environments after single dose of 150 mg of ceftiofur. Concentrations were considerably in excess of the minimum inhibitory concentration (MIC) of the S. aureus strain used. A marked decrease of viable bacteria in tissue cage fluid (TCF) occurred. In five of seven ponies; however, the infection was not eliminated and abscess formation occurred. Therefore, local application of ceftiofur alone is not advisable for infections with S. aureus in secluded sites in horses, but should be used only with adjunctive therapy.

Intensive Therapy with Ceftobiprole Medocaril of Experimental Foreign-Body Infection by Methicillin-Resistant Staphylococcus aureus

The therapeutic activity of ceftobiprole medocaril, the water-soluble prodrug of ceftobiprole, was compared to that of vancomycin in a rat tissue cage model of chronic methicillin-resistant Staphylococcus aureus (MRSA) foreign-body infection. The MICs and MBCs of ceftobiprole and vancomycin in Mueller-Hinton broth for strain MRGR3 were 1 and 4 and 1 and 2 microg/ml, respectively. In vitro elimination rates of strain MRGR3 of 4 and 8 microg/ml of ceftobiprole or vancomycin were equivalent. After 2 weeks of infection, mean +/- standard error of the mean viable counts of strain MRGR3 were 6.83 +/- 0.11 log CFU/ml of tissue cage fluid (n = 87). High-dose regimens of ceftobiprole medocaril (equivalent to 150 mg/kg of ceftobiprole) or 50 mg/kg vancomycin produced nearly identical average peak and trough levels of ceftobiprole and vancomycin in tissue cage fluid, which exceeded the MBC of either antibiotic towards strain MRGR3 for > or =75% of each dosing interval. After 7 days of therapy with ceftobiprole medocaril or vancomycin, average counts of MRGR3 decreased significantly (P < 0.02) by 0.68 +/- 0.28 (n = 29) and 0.88 +/- 0.22 (n = 28) log CFU/ml of tissue cage fluid, respectively, compared with cages of untreated animals, but were not significantly different from each other. No resistant mutants were detected on ceftobiprole-supplemented agar following therapy with this cephalosporin. The in vivo activity of ceftobiprole medocaril against chronic MRSA foreign-body infections was equivalent to that of vancomycin and did not lead to the emergence of resistant subpopulations.

Simultaneous analysis of piperacillin and tazobactam in rabbits: application to pharmacokinetic study.

A simple and rapid assay is developed for the simultaneous analysis of piperacillin and tazobactam in rabbit serum and tissue cage fluid (TCF). To eliminate endogenous interferences, a wavelength switch technique was applied, in which the programmable UV detector changed the monitoring wavelength from 218 to 254 nm at 10 min. After liquid-liquid extraction, sample analyses were performed on a C(18) column by gradient elution; the mobile phase consisted of acetonitrile and phosphate buffer (0.014 m, pH 2.4). Owing to the limited amount of rabbit TCF available, a cross-validation of a proxy matrix was evaluated. The relative standard deviation of the between- and within-batch precision of both compounds was less than 5.1%; the relative error of the between- and within-batch accuracy was less than 7.3%. The recoveries of both compounds in serum and TCF were larger than 80%. This assay was successfully applied to simultaneously analyze piperacillin and tazobactam in rabbit serum and TCF samples.

Pharmacodynamic study of beta-lactams alone and in combination with beta-lactamase inhibitors against Pseudomonas aeruginosa possessing an inducible beta-lactamase.

The antimicrobial efficacies of beta-lactams alone and in combination with beta-lactamase inhibitors were investigated by applying a rabbit tissue cage model against a strain of Pseudomonas aeruginosa with an inducible AmpC (iAmpC) beta-lactamase. Two sterilized golf Wiffle balls were surgically implanted in the rabbit dorsal cervical area. After 4 weeks, Wiffle balls had filled with tissue cage fluid (TCF), in which 2 mL of 10(6) cfu/mL of the test isolate were inoculated. To achieve the same T > MIC as in humans, 400 mg/kg of the beta-lactams alone and in combination was administered twice a day via subcutaneous injection. The dosing regimens were as follows: piperacillin alone, 4 g piperacillin/0.5 g tazobactam; ticarcillin alone, 3 g ticarcillin/0.1 g clavulanate; and 3 g ticarcillin/ 0.3 g clavulanate. The changes in bacterial counts (log cfu/mL) after the 3 day treatments were as follows: 1.03 +/- 0.97 (control), -1.31 +/- 0.61 (piperacillin), -2.81 +/- 0.53 (4 g piperacillin/0.5 g tazobactam), -1.61 +/- 0.68 (ticarcillin), -3.42 +/- 0.75 (3 g ticarcillin/0.1 g clavulanate) and -1.65 +/- 1.47 log cfu/mL (3 g ticarcillin/0.3 g clavulanate). AmpC induction by high-dose clavulanate was observed in rabbit TCF, and was confirmed by the in vitro induction study. The study indicated that tazobactam significantly enhanced the antibacterial activity of piperacillin against iAmpC P. aeruginosa; clavulanate had synergy with the antibacterial activity of ticarcillin at low concentration, but had no effect on ticarcillin at high concentration due to AmpC induction by clavulanate.

Simultaneous determination of ticarcillin and clavulanate in rabbit serum and tissue cage fluid by liquid chromatography

A gradient elution HPLC method with a wavelength switch technique was developed to simultaneously analyze the β-lactam ticarcillin and the β-lactamase inhibitor clavulanate in rabbit serum and tissue cage fluid (TCF). A C18 reversed-phase column with a programmable UV detector changing the wavelength from 218 to 254 nm at 9 min was used for chromatographic separation. The mobile phase consisted of acetonitrile, phosphate buffer and tetrabutylammonium hydrogen sulfate by following a gradient elution program at a flow-rate of 1 ml/min. Sample processing was carried out with liquid–liquid extraction. Good linearity, recoveries, precision and accuracy were obtained. The ranges of the standard curves were 1–100 μg/ml for ticarcillin, and 0.2–20 μg/ml for clavulanate. This assay has been successfully applied to analyze ticarcillin and clavulanate in rabbit serum and tissue cage fluid samples from a pharmacokinetic study.

Pharmacokinetic–pharmacodynamic integration of danofloxacin in the calf

The pharmacokinetics and pharmacodynamics of danofloxacin were studied in calves after intravenous (IV) and intramuscular (IM) administration, at a dose of 1.25 mg/kg in a two period cross-over study, using tissue cages to monitor aspects of extravascular distribution. Danofloxacin had a high volume of distribution (3.90 L/kg) and relatively rapid clearance (1.02 L/kg h) after IV dosing. Terminal half-life was 2.65 and 4.03 h, respectively, after IV and IM administration. Danofloxacin penetrated slowly into and was cleared slowly from tissue cage fluid (transudate), elimination half-life (10.2 h after IV and 8.9 h after IM dosing) being greater than for serum. The antibacterial actions of danofloxacin against the pathogen Mannheimia haemolytica 3575 were established in vitro in Mueller Hinton Broth, serum and transudate. These data were used together with in vivo pharmacokinetic parameters, C max and AUC to determine the surrogate markers of antimicrobial activity, C max/MIC, AUC/MIC and T>MIC. The antibacterial actions of danofloxacin were also determined ex vivo in serum and transudate samples harvested at pre-determined times after IM danofloxacin dosing. Ex vivo AUC/MIC data were integrated with ex vivo bacterial count to establish values producing a bacteriostatic action, inhibition of bacterial count by 50%, reduction in bacterial count by 99.9% (bactericidal action) and elimination of bacteria. Mean values were, respectively, 15.9, 16.7, 18.15 and 33.5 h for serum and 15.0, 16.34, 17.8 and 30.7 h for transudate. The AUC/MIC-effect relationships for serum may be regarded as representative of a shallow compartment of blood and well perfused tissues, whilst AUC/MIC-effect relationships for transudate may be considered to represent a deep peripheral compartment of poorly perfused tissues. A novel approach to selecting antimicrobial drug dosage for evaluation in clinical trials, using AUC/MIC values producing either bactericidal activity or elimination of bacteria together with MIC 90 values for calf pathogens, is proposed. This approach can be expected to optimise efficacy and minimise the development of resistance.

Pharmacodynamics, chiral pharmacokinetics and PK-PD modelling of ketoprofen in the goat.

There have been few studies of the pharmacodynamics of nonsteroidal antiinflammatory drugs (NSAIDs) using PK-PD modelling, yet this approach offers the advantage of defining the whole concentration-effect relationship, as well as its time course and sensitivity. In this study, ketoprofen (KTP) was administered intravenously to goats as the racemate (3.0 mg/kg total dose) and as the single enantiomers, S(+) KTP and R(-) KTP (1.5 mg/kg of each). The pharmacokinetics and pharmacodynamics of KTP were investigated using a tissue cage model of acute inflammation. The pharmacokinetics of both KTP enantiomers was characterized by rapid clearance, short mean residence time (MRT) and low volume of distribution. The penetration of R(-) KTP into inflamed (exudate) and noninflamed (transudate) tissue cage fluids was delayed but area under the curve values were only slightly less than those in plasma, whereas MRT was much longer. The S(+) enantiomer of KTP penetrated less readily into exudate and transudate. Unidirectional inversion of R(-) to S(+) KTP occurred. Both rac-KTP and the separate enantiomers produced marked inhibition of serum thromboxane B2 (TxB2) synthesis (ex vivo) and moderate inhibition of exudate prostaglandin E2 (PGE2) synthesis (in vivo); pharmacodynamic variables for S(+) KTP were Emax (%) = 94 and 100; IC50 (microg/mL) = 0.0033 and 0.0030; N = 0.45 and 0.58, respectively, where Emax is the maximal effect, IC50 the plasma drug concentration producing 50% of Emax and N the slope of log concentration/effect relationship. The IC50 ratio, serum TxB2:exudate PGE2 was 1.10. Neither rac-KTP nor the individual enantiomers suppressed skin temperature rise at, or leucocyte infiltration into, the site of acute inflammation. These data illustrate for KTP shallow concentration-response relationships, probable nonselectivity of KTP for cyclooxygenase (COX)-1 and COX-2 inhibition and lack of measurable effect on components of inflammation.

Pharmacokinetics (PK), Pharmacodynamics (PD), and PK-PD Integration of Danofloxacin in Sheep Biological Fluids

The fluoroquinolone antimicrobial drug danofloxacin was administered to sheep intravenously (i.v.) and intramuscularly (i.m.) at a dose of 1.25 mg/kg of body weight in a two-period crossover study. The pharmacokinetic properties of danofloxacin in serum, inflamed tissue cage fluid (exudate), and noninflamed tissue cage fluid (transudate) were established by using a tissue cage model. The in vitro and ex vivo activities of danofloxacin in serum, exudate, and transudate against a pathogenic strain of Mannheimia haemolytica were established. Integration of in vivo pharmacokinetic data with the in vitro MIC provided mean values for the area under the curve (AUC)/MIC for serum, exudate, and transudate of 60.5, 85.6, and 45.7 h, respectively, after i.v. dosing and 55.9, 77.9, and 49.1 h, respectively, after i.m. dosing. After i.m. dosing, the maximum concentration/MIC ratios for serum, exudate, and transudate were 10.8, 3.0, and 1.6, respectively. The ex vivo growth inhibition data after i.m. dosing were fitted to the inhibitory sigmoid E(max) equation to provide the values of AUC/MIC required to produce bacteriostasis, bactericidal activity, and elimination of bacteria. The respective values for serum were 17.8, 20.2, and 28.7 h, and slightly higher values were obtained for transudate and exudate. It is proposed that use of these data might provide a novel approach to the rational design of dosage schedules.

Pharmacokinetics and PK-PD modelling of danofloxacin in camel serum and tissue cage fluids

The pharmacokinetics and pharmacodynamics of danofloxacin were studied in the camel in a two period cross-over study. After intravenous (i.v.) administration at a dose rate of 1.25 mg/kg, the pharmacokinetics of danofloxacin indicated a high volume of distribution ( V d area =3.43 L/kg), relatively rapid clearance (0.44 L/kg/h) and half-life of 5.37 h. After intramuscular (i.m.) dosing absorption was complete ( F=114.5) and rapid ( T (1/2)abs=0.12 h) and terminal half-life was 5.71 h. Danofloxacin penetrated fairly slowly into both inflamed (exudate) and non-inflamed (transudate) tissue cage fluids and was cleared slowly from these fluids, elimination half-life being at least twice that for serum for both exudate and transudate after both i.v. and i.m. dosing. The antibacterial actions of danofloxacin against the camel pathogen Escherichia coli 0157-H7 were determined by measurement of minimum inhibitory concentration (MIC) in vitro (single measurement) and ex vivo measurements of bacterial count at nine times between one and 48 h after i.m. dosing in each of the fluids, serum, exudate, and transudate. Using in vitro MIC data and in vivo pharmacokinetic parameters, the surrogate markers of antimicrobial activity, C max/MIC, AUC/MIC and T > MIC, were determined for all three fluids. The ex vivo serum AUC 24 h /MIC data were integrated with reduction in bacterial count to provide values producing a bacteriostatic action (no change in bacterial count), inhibition of bacterial count by 50%, reduction in bacterial count by 99.9% (bactericidal action) and elimination of bacteria. Mean AUC 24 h /MIC values were 17.20, 20.07, 21.24, and 68.37 h, respectively. To describe the latter, the introduction of a new term to supplement MIC and minimum bactericidal concentration (MBC) is proposed, namely minimum elimination concentration (MEC). A novel means of designing antimicrobial drug dosage schedules for evaluation in clinical trials is proposed, using ex vivo AUC 24 h /MIC values for bactericidal activity and elimination of bacteria together with MIC 90 data for camel pathogens.

Efficacy of trimethoprim-sulfadoxine against Escherichia coli in a tissue cage model in calves.

Tissue cages implanted subcutaneously in calves were infected with Escherichia coli. Twenty-four hours later, the calves were treated either with single doses of 2.5 + 12.5 or 5 + 25 mg/kg trimethoprim (TMP) + sulfadoxine (SDX) or with five doses of 7.5 + 37.5 mg/kg TMP + SDX at 12-h intervals. In addition, one cage in each of three calves in the highest dose group was infected 3 h after initiation of treatment. Untreated calves were kept as controls. Concentrations of TMP and SDX in plasma and tissue cage fluid (TCF) and counts of viable bacteria in TCF were determined. In the highest dose group, concentrations of TMP in TCF remained above the minimum inhibitory concentration of the test strain for 94-101 h and peak to minimum inhibitory concentration (MIC) ratio was close to 10. In spite of this, an effect of treatment was noted only in cages infected after initiation of treatment. In vitro studies and analysis of thymidine content in serum and TCF from calves suggest that levels of thymidine in TCF are high enough to antagonize the antibacterial effect of TMP. The results indicate that soft tissue infections in secluded infection sites of calves are refractory to treatment with TMP + SDX.

Pharmacodynamics and enantioselective pharmacokinetics of racemic carprofen in the horse.

Carprofen is a nonsteroidal anti-inflammatory drug of the 2-arylpropionate subclass. It contains a single chiral centre and exists in two enantiomeric forms. In this study rac-carprofen, at two dosages, 0.7 and 4.0 mg/kg, and placebo were administered i.v. to six New Forest horses in a three period cross-over study. The concentration-time profiles were established for R(-) and S(+)-carprofen for plasma and both inflamed (exudate) and noninflamed (transudate) tissue cage fluids. R(-)-carprofen was the predominant enantiomer in all three fluids, as indicated by plasma area under the curve (AUC) values for R(-) and S(+)-carprofen of 117.4 and 22.6 microg h/mL (low dose carprofen) and 557.5 and 138.1 microg h/mL (high dose carprofen) respectively. Penetration of both enantiomers into exudate was slow and limited and passage into transudate was even lower. The pharmacodynamics of rac-carprofen was investigated at both the molecular level and in terms of the ability to suppress components of the tissue cage inflammatory response. Low dose carprofen produced only moderate and transient inhibition of serum thromboxane (Tx)B2 but failed to affect exudate prostaglandin (PG)E2 concentrations, whilst suppression of exudate leukotriene (LT)B4 and beta-glucuronidase was not significant. High dose carprofen produced greater and more persistent inhibition of serum TxB2 and virtually abolished exudate PGE2 synthesis. Some inhibition of LTB4 and beta-glucuronidase in exudate was also obtained. At both dosages rac-carprofen reduced the swelling produced by intradermal bradykinin injection but only high dose carprofen was anti-inflammatory as indicated by suppression of temperature rise over exudate tissue cages and neither dose affected leucocyte numbers in exudate. When considered in conjunction with previous data on carprofen, the present findings indicate that carprofen is not a selective inhibitor of cyclooxygenase (COX) isoenzymes, COX-1 and COX-2 in the horse, although it may show some preference for COX-2 inhibition. Because low dose carprofen, which is the clinically recommended dosage, produces minimal inhibition of COX, it is likely to achieve its therapeutic effects at least partially through other pathways, possibly including weak to moderate inhibition of 5-lipoxygenase and of enzyme release. The good safety margin of carprofen in clinical use might also be explained by weak COX inhibition and by other actions at the molecular level.

Pharmacokinetics and pharmacokinetic/pharmacodynamic integration of marbofloxacin in calf serum, exudate and transudate.

Marbofloxacin is a fluoroquinolone antimicrobial drug used in cattle for the treatment of respiratory infections. In this investigation the pharmacokinetics (PK) of marbofloxacin were determined after intravenous and intramuscular dosing at a dosage of 2 mg/kg. In addition the ex vivo pharmacodynamics (PD) of the drug were determined in serum and three types of tissue cage fluid (transudate, inflammatory exudate generated by carrageenan and exudate generated by lipopolysaccharide). Marbofloxacin PK was characterized by a high volume of distribution after dosing by both routes (1.28 L/kg intravenous and 1.25 L/kg intramuscular). Corresponding area under the concentration-time curve (AUC) and elimination half-life (t(1/2)el) values were 9.99 and 10.11 microg h/mL and 4.23 and 4.33 h, respectively. Values of AUC for carrageenan-induced exudate, lipopolysaccharide-induced exudate and transudate were, respectively, 8.28, 7.83 and 7.75 microg h/mL after intravenous and 8.84, 8.53 and 8.52 microg h/mL after intramuscular dosing. Maximum concentration (Cmax) values were similar for the three tissue cage fluids after intravenous and intramuscular dosing. For in vivo PK data values of AUC: minimum inhibitory concentration (MIC) (AUIC) ratio for serum were 250 and 253, respectively, after intravenous and intramuscular dosing of marbofloxacin against a pathogenic strain of Mannheimia haemolytica (MIC=0.04 microg/mL). For all tissue cage fluids AUIC values were >194 and >213 after intravenous and intramuscular dosing, and Cmax/MIC ratios were 9 or greater, indicating a likely high level of effectiveness in clinical infections caused by M. haemolytica of MIC 0.04 microg/mL or less. This was confirmed by both in vitro (serum) and ex vivo (serum, exudate and transudate) measurements, which demonstrated a concentration-dependent killing profile for marbofloxacin against M. haemolytica. Ex vivo, after 24-h incubation, virtually all bacteria were killed (<10 cfu/mL) in all samples collected up to 9 h (serum), 24 h (carrageenan-induced exudate and transudate) and 36 h (lipopolysaccharide-induced exudate). Application of the sigmoid Emax equation to the ex vivo antibacterial data provided, for serum, AUIC24 h values of 37.1 for bacteriostasis, 46.3 for bactericidal activity and 119.6 for elimination of bacteria. These data may be used as a rational basis for setting dosing schedules which optimize clinical efficacy and minimize the opportunities for emergence of resistant organisms.

In vivo emergence of subpopulations expressing teicoplanin or vancomycin resistance phenotypes in a glycopeptide-susceptible, methicillin-resistant strain of Staphylococcus aureus.

Several reports indicate the emergence of subpopulations resistant to glycopeptides in some clinical isolates of Staphylococcus aureus. While the development of glycopeptide resistance in S. aureus is easily observed in vitro, the in vivo conditions promoting emergence of glycopeptide-resistant subpopulations are unknown. Using a rat model, subcutaneous implants were chronically infected with a methicillin-resistant strain of S. aureus, MRGR3, devoid of a significant (>10(-7)) glycopeptide-resistant subpopulation at 2 mg/L of either teicoplanin or vancomycin. After 3 weeks of infection in antibiotic-untreated animals, subpopulations emerged, growing on agar containing 10 mg/L of either glycopeptide. These subpopulations were detected in all tissue cage fluids containing >7 log cfu/mL at average frequencies of 4 x 10(-5) and 2 x 10(-5) on teicoplanin- and vancomycin-containing agar, respectively. While teicoplanin MICs increased two- to 16-fold, vancomycin MICs increased by less than two-fold. Population analysis and survival kinetic studies of three teicoplanin-selected subclones indicated that transfer from solid to liquid medium conditions decreased expression of teicoplanin resistance in the bacterial population. In Mueller-Hinton broth, >90% of cells remained fully resistant to antibiotic, but did not grow in the presence of teicoplanin for an initial period of at least 6 h. All three teicoplanin-resistant subclones expressed stable teicoplanin resistance with slight cross-resistance to vancomycin after a few transfers on teicoplanin-supplemented agar. These data suggest that some in vivo conditions may lead to selection of S. aureus subpopulations exhibiting decreased glycopeptide susceptibility and growing in the presence of otherwise inhibitory concentrations of these antimicrobial agents.

Pharmacokinetics and penetration into tissue fluid of ceftizoxime in normal and hyperthermic sheep

The pharmacokinetics of ceftizoxime was studied in six sheep before and after inducing hyperthermia using Escherichia coli endotoxin. Sheep implanted subcutaneously with cages of non-reactive material for collecting tissue cage fluid (TCF) were used to conduct two trials. In Trial 1 animals with normal basal temperature (normal sheep (NS)) were given intravenous (i.v.) and intramuscular (i.m.) monodoses of ceftizoxime (20 mg/kg BW) at 1 week interval. One and 5 weeks later (Trial 2) each sheep were injected 1 μg/kg BW of endotoxin to produce hyperthermia (hyperthermic sheep (HS)) previously to i.v. administration (HSi.v.) and i.m. (HSi.m.) of ceftizoxime (20 mg/kg BW), respectively. Serum and TCF samples were collected over 6 h post-administration. Ceftizoxime concentrations in serum and TCF were determined by a microbiological assay. The concentrations in serum and TCF of ceftizoxime were analyzed through compartmental and non-compartmental models. Rectal temperature were significantly incremented in all animals during Trial 2. The half-time and constant of elimination in serum of ceftizoxime in NSi.v. ( t 1/2 = 1.1 ± 0.4 h; λ z = 0.7 ± 0.2 h −1) were statistically different those observed in HSi.v. ( t 1/2 = 1.4 ± 0.4 h; λ z = 0.5 ± 0.2 h −1). The constants of distribution in NSi.v. and HSIV were 5.1 ± 4.6 and 4.1 ± 3.4 h −1, respectively. The time to reach the maximum concentrations in TCF was latter ( p < 0.05) in NS ( t max = 2.3 ± 0.7 h) than in HS ( t max = 1.3 ± 0.6 h). After i.m. administration in NS the absorption half-life (0.12 ± 0.19 h) was latter ( p < 0.05) than in HS (0.06 ± 0.007 h) with greater areas under the curve (AUC in NS = 65.4 ± 20.8 and AUC in HS = 34.7 ± 7.5 (μg/ml) h). The maximum value of concentration in serum ( C max) and AUC in TCF were greater ( p < 0.05) in NS ( C max = 46.1 ± 10.6 μg/ml and AUC = 84.4 ± 17.4 (μg/ml) h) as compared to same HS ( C max = 27.0 ± 12.9 μg/ml and AUC = 47.9 ± 3.9 (μg/ml) h). The concentrations of ceftizoxime in TCF after i.v. and i.m. in NS and HS were elevated during a 6 h period after administration. The bioavailability of ceftizoxime in NS (101.6 ± 59.9%) and HS (87.4 ± 63.3%) was suitable for its use by the i.m. route.

Simultaneous determination of enrofloxacin and ciprofloxacin in animal biological fluids by high-performance liquid chromatography: Application in pharmacokinetic studies in pig and rabbit

A simple and rapid high-performance liquid chromatographic method for the simultaneous determination of enrofloxacin and ciprofloxacin has been developed in pig and rabbit samples. Solid-phase extraction was applied from samples on a C 18 cartridge using a mixture of methanol–hydrochloric acid (98:2, v/v). Analytical separation was performed on a C 18 column with UV detection at 277 nm under gradient conditions. The mobile phase was a mixture of orthophosphoric acid–triethylamine–acetonitrile. The method has been validated for both molecules in pig and rabbit plasma and adapted for rabbit tissue-cage fluid (TCF). The assay is specific and reproducible within the both drugs and mean recoveries for ciprofloxacin and enrofloxacin, respectively, were 92±6% and 90±5% for pig plasma over the range used. Mean recoveries for enrofloxacin were 108±9% and 102±7% for rabbit plasma and TCF, respectively, over the range used. The suitability of the assay for pharmacokinetic studies was determined by measuring enrofloxacin and ciprofloxacin concentrations either in pig plasma after administration of a single intravenous 5 mg/kg dose of enrofloxacin or in rabbit plasma and TCF during a 24 h infusion of enrofloxacin at a rate of 1.25 mg/kg per hour.

Postantibiotic effect and postantibiotic sub-mic effect of dirithromycin and erythromycin against respiratory tract pathogenic bacteria.

The postantibiotic effect (PAE) of dirithromycin and erythromycin against strains Streptococcus pyogenes group A M12, NCTC P1800, Streptococcus pneumoniae 23, Staphylococcus aureus Oxford strain 209, Moraxella catarrhalis 15616 and Haemophilus influenzae 5590 was investigated in vitro and in vivo by use of the tissue cage model in rabbits. By exposing strains to 2.5-5 x MIC levels for 6 h or 12 h, both compounds induced in vitro PAEs of 1-9 h, and in two cases >20 h. Cultures in the PAE-phase were then re-exposed to subinhibitory concentrations (0.25 x MIC and 0.5 x MIC) of antibiotic and prolonged suppression of regrowth was obtained for 2->20 h. Following i.v. antibiotic treatment of rabbits (10 mg/kg or 20 mg/kg dirithromycin and 20 mg/kg or 40 mg/kg erythromycin) and bacterial infection of the implanted tissue cages in the same rabbit, the tissue cage fluid (TCF) was sampled 6 h after infection and regrowth was monitored by sampling from new tissue cages in untreated rabbits. These i.v. single doses of both antibiotics induced in vivo PAEs of >6 h, but <20 h against S. pyogenes. Suppression of regrowth in TCF was also obtained for > or = 20 h on infection with exposed S. pyogenes in the PAE-phase in newly implanted tissue cages in rabbits that had been treated with low doses of antibiotic to produce subinhibitory concentrations in the TCE Dirithromycin was in general as active as erythromycin in inducing PAE and in prolonging suppression of bacterial regrowth in the PAE phase.