![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 301, Issue 1, 217-222, April 2002
Food and Drug Administration, Center for Veterinary Medicine, Office of Research, Division of Animal Research, Laurel, Maryland (C.V.C., D.E.F., M.J.M.); and Division of Surveillance, Office of Surveillance and Compliance, Rockville, Maryland (L.O.P., J.D.B.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The impact of Actinobacillus pleuropneumoniae (APP) infection in swine on the pharmacokinetic parameters of enrofloxacin were determined. Twenty-four animals were used in a 2 × 2 factorial of treatment groups (six animals per group) to determine the impact of APP-induced inflammation and the anti-inflammatory drug dexamethasone on enrofloxacin pharmacokinetic parameters. All animals received enrofloxacin as a single intravenous dose (5 mg/kg). Administration of dexamethasone was associated with an increase in clearance of enrofloxacin Clearance of enrofloxacin was not affected by APP. Volume of distribution at steady state was significantly increased in the dexamethasone-treated pigs. Volume of distribution at steady state was decreased by APP infection. Dexamethasone significantly increased the terminal elimination half-life of enrofloxacin. APP infection decreased the terminal elimination half-life of enrofloxacin in the infected pigs. Infection and dexamethasone significantly decreased the urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios. This study shows that APP infection does affect plasma pharmacokinetic parameters. Dexamethasone and APP infection may reduce renal clearance of enrofloxacin with a compensatory increase in intestinal clearance. Neither infection nor dexamethasone altered the metabolism of enrofloxacin to ciprofloxacin, the principal metabolite of enrofloxacin.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Actinobacillus
pleuropneumoniae (APP) infections have an economic impact on swine
production due to mortality and medical costs incurred after an acute
outbreak. The consequences are most significant in young animals
(Nicolet, 1994
). Chronically infected animals may continue to gain
weight when the animals are asymptomatic (Nicolet, 1994). However,
these animals may serve as a focus for future infections. In contrast,
animals that are chronically affected and showing clinical signs have
decreased growth rate (Fedorka-Cray et al., 1993). Eradication of
carrier pigs is the optimum method for control, but most producers have
no alternative except for treating clinical signs of APP infection.
Most antibiotic regimens have met with limited success. Likewise, the
vaccine for preventing APP infection is not effective in preventing
field outbreaks (Nicolet, 1994).
Enrofloxacin, a fluoroquinolone antibiotic, although not approved for
swine in the United States, has been shown to be effective for treating
APP infection (Vancutsem et al., 1990; Greene and Budberg, 1993).
Enrofloxacin administered intramuscularly for 3 days at 2.5 to 5 mg/kg
beginning 8 h postinfection, or 5 mg/kg starting 12 h
postinfection, resulted in clinical improvement 12 h after
initiation of therapy. Approval for use of fluoroquinolones in
food-animals, such as swine, was sought in the early 1990s.
As part of the drug approval process for domestic animals,
pharmacokinetic studies are conducted in healthy animals, whereas efficacy studies are conducted in diseased animals. However, infected animals may have altered pharmacokinetics when compared with normal animals. Few studies have explored the possibility of altered pharmacokinetics in infected domestic animals (Monshouwer et al., 1995;
Myers and Kawalek, 1995). It is expensive to either develop accurate
disease models or conduct pharmacokinetic studies during clinical field
trials. Consequently, drug efficacy, toxicity, or tissue residue levels
may be different from those predicted. The availability of a proven
model for APP infection afforded the unique opportunity to determine
whether a bacterial disease in domestic animals would affect the
pharmacokinetics of a therapeutic drug. The purpose of this study was
to determine whether the pharmacokinetics parameter of enrofloxacin is
changed in APP-infected swine. Because infection-induced changes in
enrofloxacin pharmacokinetics would most likely be due to the
elaboration of inflammatory cytokines, a cohort of APP-infected animals
was also treated with dexamethasone to block their production. In
addition, because it is a standard veterinary practice to administer
dexamethasone to relieve suffering caused by inflammation, there exists
a possibility that these two drugs might be coadministered.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Chemicals. Acetonitrile was used for HPLC analysis (HPLC grade; Burdick and Jackson, Muskegon, MI). All other reagents were of analytical grade (Sigma, St. Louis, MO).
Animals Twenty-four Landrace-Poland China barrows were procured from a local vendor. All pigs weighed 30 to 35 kg before initiation of the study. Housing consisted of a heated building with a concrete floor, and all animals were fed 3 kg/day from a single batch of a commercial corn-based ration without antibiotics. The pigs had been fed a commercial ration medicated with Tylosin before procurement. All pigs were examined upon arrival and were clinically healthy at the start of the study. The animals were allowed 1 week to acclimate before initiation of the study. This study was approved by the Center for Veterinary Medicine Office of Research Institutional Animal Care and Use Committee.
Experimental Design.
The design consisted of a 2 × 2 factorial of treatment groups. A. pleuropneumoniae and
dexamethasone (three i.v. injections, 0.5 mg/kg) were the main effects.
The APP bacteria (serotype 1, strain L91-2; gift of Dr. Michael
Murtaugh, University of Minnesota, St. Paul, MN) were administered by
endobronchial infusion (Baarsch et al., 1995) 24 h before
administration of enrofloxacin. The virulence of this APP strain has
been attenuated by serial passage in mice, such that it possesses a
high rate of morbidity but a low rate of mortality (Baarsch et al.,
1995). Four groups of six pigs per group were intravenously
catheterized (Brocht et al., 1989) and placed in stainless steel
metabolism cages. A. pleuropneumoniae was administered as a
single endobronchial infusion to two groups, while the other two groups
received a sham saline infusion. One infected group and one sham group
received i.v. dexamethasone (Dexsone, lot 507334; Phoenix
Pharmaceuticals, St. Joseph, MO) at 
36 h, 24 h, and 12 h before
administration of enrofloxacin. A bolus dose of 5 mg/kg b.wt.
enrofloxacin (Baytril, lots 267062, 267065, and 267066; Bayer, Shawnee
Mission, KS) was administered intravenously 24 h after saline or
bacterial inoculation to all four groups (time 0). Because of the
potential for APP to be spread as an aerosol, the noninfected swine
(control and dexamethasone-only treated groups) were examined before
initiating work with the animals to be infected with APP.
Blood Sampling and Analysis.
Plasma samples for
high-performance liquid chromatography (HPLC) were drawn in heparin
tubes at 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 30, and 48 h
after i.v. administration of enrofloxacin (24 h postinfection or sham
infection). Plasma samples for IL-6 determination were taken at 0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, and 72 h after APP instillation.
The samples were placed on ice before transporting to the laboratory
and centrifuged at 1500g for 10 min. The centrifuged plasma
was harvested and aliquoted for storage at 80°C for pharmacokinetic analysis.
; Griggs and Wise, 1989; Tyczkowska et al., 1994).
This method is capable of detecting both enrofloxacin and
ciprofloxacin, the major metabolite of enrofloxacin, as well as other
minor metabolites (if present). The final internal standard concentration of the fortified samples was 250 ng pipemidic acid/ml. After vortexing for 10 to 15 s, a 200-µl aliquot was placed in a
30,000 molecular weight cut-off microfilter (Microcon 30; Millipore, Burlington, MA) and centrifuged at 14,000g in a 45°
fixed-angle rotor for 1 h. Approximately 150 µl was recovered
and placed in tapered glass sample vials. Ten microliters of the clear
ultrafiltrate were injected into the liquid chromatograph (PerkinElmer
Integral 4000; PerkinElmer Instruments, Norwalk, CT) using a reverse
phase C18 column with dimensions of 250 mm × 3.2 mm and a 5-µm particle size protected by a guard column
(Primasphere; Phenomenex, Torrance, CA). Pipemidic acid, ciprofloxacin
(main metabolite of enrofloxacin), and enrofloxacin were eluted at 5.5, 8.5, and 11.2 min, respectively.
Urine Sampling and Analysis.
Urine samples were collected
24 h after infection or sham infection and at intervals of 0 h to 24 h and 24 to 48 h after enrofloxacin administration.
The urine samples were aliquoted into cryovials and stored at 80°C.
After thawing at room temperature, sample dilution was accomplished by
adding 100 µl of the sample to 400 µl of diluent (2:1 mixture of
0.05 N sodium hydroxide and acetonitrile) in siliconized
microcentrifuge tubes and mixed by vortexing. A 50-µl aliquot was
taken from the 1:4 urine dilution and added to a sample vial containing
950 µl of the following mixture: 750 µl of diluent and 200 µl of
distilled water with 263 µg/liter of pipemidic acid. The pipemidic
acid had been added from a 5 mg/liter stock solution that was
subsequently diluted with the diluent. The pipemidic acid, water, and
diluent were mixed in a single batch before addition to the sample
vial. The final urine dilution in the sample vial was 1:99 after mixing
the 50 µl of the 1:4 diluted urine to the 950 µl. Ten-microliter
aliquots were injected onto the HPLC column.
). This approach
eliminates the problem of contamination with drinking water. Urine
creatinine was assayed using a Creatinine Test Kit (Sigma Diagnostics
Corp., St. Louis, MO). The urine samples were first diluted 10 to 30 times with water. Working standard dilutions of creatinine were
prepared by diluting 150 mg/liter stock standard with water, resulting
in a standard curve consisting of 0, 25, 50, 75, 100, and 150 mg/liter.
Sample and standards were prepared in triplicate by adding 30 to 300 µl of alkaline picrate solution into each well of a microplate
including the standard curve wells on each plate. The plates were mixed
for 30 s and let stand at room temperature for 10 min. The plates
were read at 500 nm using a microplate spectrophotometer (Spectra Max
250, Molecular Devices, Sunnyvale, CA).
Pathology. Gross necropsies were performed on all animals in the infection study. The pigs were euthanized with a sodium pentobarbital solution (Euthasol; Delmarva Laboratories, Inc., Midlothian, VA) 72 h after APP instillation, which was also 48 h after enrofloxacin administration. Brain and spinal cord were not examined. Lung weights and gross necropsy observations were recorded. The color, texture, location, and size of gross lesions were also noted. After trimming the trachea from the lung at the level of the cranial bronchi, the lung was weighed before sampling for histopathology.
The following tissues (1- to 2-mm thickness and up to 4 × 4 cm) were collected for histopathology: right bronchial lymph node, middle mediastinal lymph node, mesenteric lymph node, lung, two sections of heart from the left ventricle and septum, three sections of liver from different lobes, one section of the middle region of the spleen, and one section of each kidney. The lungs were covered with a paper towel during fixation in the specimen jar. If gross abnormalities were observed in other organs or tissues, a sample was collected for histopathology. The tissues were placed in a 4-liter specimen jar containing 10% buffered formalin, pH 7.4, in a 1:20 ratio of tissues to formalin. The tissues were allowed to fix in the formalin for 24 h, and the spent formalin was decanted and replaced with fresh formalin and allowed to fix for an additional week.Pharmacokinetic Parameters and Statistical Analyses.
Pharmacokinetic parameters, and urine enrofloxacin and ciprofloxacin
concentrations were analyzed using SigmaStat (version 2.03) and
SigmaPlot (version 4.0; SPSS, Inc., Chicago, IL), and WinNonlin
(version 2.0; Scientific Consulting Inc., Apex, NC). Pharmacokinetic
parameters were calculated using a noncompartmental model for area
under curve extrapolated from zero to infinity (AUC
0-
), area under the moment curve extrapolated from zero to infinity (AUMC 0-), terminal
half-life (t1/2 = 0.693/
), total
body clearance (CLtot = dose/AUC
0-), volume of distribution at steady state
(VdSS = MRT × CL0-), volume of distribution of the
elimination phase (Vd
= dose/( × AUC
0-), and elimination rate constant ().
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Clinical Observations and Microscopic Pathology. The APP-infected pigs became lethargic and dyspneic within 2 h after inoculation with A. pleuropneumoniae. Three infected pigs vomited, and all infected pigs were anorectic. After 24 h, the infected pigs were still depressed but ate some of their ration. Breathing was labored in all of the infected pigs until the time of necropsy. In contrast, all of the noninfected pigs were eating and drinking and did not exhibit labored breathing after sham inoculation.
Five of the noninfected pigs showed focal to extensive areas of hemorrhage, and the lesions were raised above the surface of the lung. The remaining seven noninfected pigs had normal lungs. Gross pathology observations in all of the infected pigs included fibrinous pleuropneumonia with necrosis and hemorrhage in a focal to multifocal pattern. The pulmonary lesions were raised above the surface and observed predominately on the right lung in the middle and caudal lobes. Histopathologic examination of the noninfected pigs revealed slight to moderate, chronic interstitial pneumonia that was not characteristic of A. pleuropneumoniae infection and is a typical finding in swine raised in confinement. The infected pigs had necrotizing extensive, severe, fibrinopurulent pneumonia with edema and fibrinopurulent pleuritis. One of the infected pigs died within 24 h after inoculation The gross and microscopic lesions in the infected pigs were consistent with A. pleuropneumoniae infection. Plasma IL-6 levels were elevated in APP-infected pigs (467 units/ml × h) compared with the level seen in control (32.3 units/ml × h) and dexamethasone-treated pigs (38.6 units/ml × h). Dexamethasone had no effect on IL-6 production in APP-infected pigs (1158 units/ml × h).Enrofloxacin Pharmacokinetic Parameters.
The mean
plasma concentrations over time for enrofloxacin (single intravenous
dose, 5 mg/kg) for the control (no-dexamethasone, no infection),
dexamethasone only-treated swine (dexamethasone), A. pleuropneumoniae-infected swine (APP-infected), and APP-infected swine treated with dexamethasone are shown in Fig.
1. Although ciprofloxacin was
consistently detected in a majority of the plasma samples (<0.20
mg/liter), it was below the level of quantification. The
pharmacokinetic parameters for enrofloxacin for these groups appear in
Table 1.
|
|
Urine Enrofloxacin/Creatinine and Ciprofloxacin/Creatinine
Ratios.
Enrofloxacin and ciprofloxacin were both detected in the
24- and 48-h urine samples (Figs. 2 and
3). Ciprofloxacin is the major metabolite
of enrofloxacin and was measured in the urine of the pigs. Other
possible (minor) metabolites include oxo-ciprofloxacin, desethylene-ciprofloxacin, oxo-enrofloxacin, and
desethylene-enrofloxacin; these were not detected in the urine.
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The present study demonstrated that APP infection results in a selective impact on enrofloxacin pharmacokinetic parameters, rather than a general effect as seen with dexamethasone. The main effect of APP infection was to reduce the volume of distribution of enrofloxacin. As such, the APP-induced reduction in enrofloxacin half-life (21.4 h, control versus 17.2 h, infected) was expected. In contrast, APP infection does not appear to affect enrofloxacin metabolism. This is evidenced by a lack of differences in enrofloxacin clearance and an inability to detect plasma levels of any other metabolite of enrofloxacin/ciprofloxacin.
Ciprofloxacin is the main metabolite of enrofloxacin, resulting from ethyl dealkylation at the para-nitrogen on the piperazinyl ring. It is also a human drug with antimicrobial activity of its own. Changes in metabolism (or elimination) might impact the overall efficacy of enrofloxacin. Low plasma levels of ciprofloxacin, coupled with an absence of other metabolites, are consistent with in vitro metabolism results. Using swine liver microsomes and liver S10 preparations, ciprofloxacin was not observed, whereas low levels of desethyl-enrofloxacin and desethyl-ciprofloxacin were detected (J.C. Kawalek, personal communication).
The selective effect of APP infection is further evidenced by a lack of interaction between the two main treatments used in this study, APP infection and dexamethasone. APP infection resulted in only a modest production of IL-6 without the production of other inflammatory mediators (M.J. Myers, D.E. Farrell, and L.O. Post, manuscript submitted for publication). Dexamethasone treatment did not affect the production of IL-6 during APP infection, nor did it affect the pathophysiological changes elicited after APP infection (M.J. Myers, D.E. Farrell, and L.O. Post, manuscript submitted for publication). Thus, dexamethasone does not affect the inflammatory response elicited by APP. The lack of an effect on inflammation is also consistent with the statistical analysis, which demonstrated no interaction between APP infection and dexamethasone with respect to the pharmacokinetic parameters for enrofloxacin and ciprofloxacin in plasma and urine.
Dexamethasone increased enrofloxacin total body clearance. However,
dexamethasone did not affect the metabolism of enrofloxacin and
actually decreased the total amount of drug (enrofloxacin and
ciprofloxacin) recovered in the urine. Whereas minimal plasma levels of
ciprofloxacin were observed in pigs given dexamethasone, measurable
levels were detected in urine. As with APP infection, ciprofloxacin was
detected only in the urine. The absence of other metabolites in either
plasma or urine is in agreement with the results of Nouws et al.
(1988).
The increase in enrofloxacin clearance in the dexamethasone treatment
group would lead to a prediction of an increase in urine enrofloxacin
levels via renal excretion. However, urinary enrofloxacin and
ciprofloxacin levels were significantly lower in the dexamethasone group compared with the control group (Fig. 3). Total urinary drug
recovery (enrofloxacin and ciprofloxacin together; corrected to
enrofloxacin) was approximately 35% and 24% for the control and
dexamethasone treatment groups, respectively. Extrapolation to a point
where less than 1% enrofloxacin remains in the body (7 half-lives),
results in an estimated recovery of 45% and 29% for the control and
dexamethasone treatment groups, respectively. The calculated 45%
recovery of enrofloxacin and ciprofloxacin correlates well with the
results from Nouws et al. (1988), who reported a recovery rate of
47.9% without affecting metabolism. Therefore, the lower calculated
total drug recovery in the dexamethasone treatment group, coupled with
the lack of effect on enrofloxacin metabolism, suggests that
dexamethasone is increasing fecal drug elimination.
The increased clearance of enrofloxacin in the dexamethasone-treated
pigs may be due to increased glomerular filtration rate. A 5-day
regimen of adrenocorticotropin and glucocorticoids raised blood
pressure, caused marked antinatriuresis and expansion of extracellular
fluid and plasma volume, and raised glomerular filtration rate in
humans, as demonstrated by inulin clearance (Connell et al., 1987). In
addition, filtration fraction increases during both adrenocorticotropin
and glucocorticoid treatment in humans, but renal blood flow decreases
during glucocorticoid treatment (Connell et al., 1987). Effective renal
plasma flow, measured by para-aminohippurate clearance,
remained unchanged. In the present study, total urinary creatinine
excretion was not affected by dexamethasone treatment compared with the
control group (Table 2). However,
creatinine clearance has been proven to be an unreliable index
of glomerular filtration rate during steroid administration in humans
(Connell et al., 1987). It is unknown whether steroids affect swine in
a similar manner.
|
The plasma levels of enrofloxacin and ciprofloxacin would lead to a
prediction of similar levels in the urine; that is, urinary enrofloxacin levels should be significantly greater than urinary ciprofloxacin levels. However, equivalent urinary levels of
enrofloxacin and ciprofloxacin were observed in this study. These
findings suggest that enrofloxacin is being metabolized by either the
kidney or the bladder regardless of treatment. Flavin-containing
monooxygenase may be converting enrofloxacin to ciprofloxacin in the
kidney. It would help explain why there are relatively equal amounts of enrofloxacin and ciprofloxacin in the urine but very little
ciprofloxacin in plasma (Parkinson, 1996).
Reabsorption can also influence renal tubular concentrations of
fluoroquinolones (Sorgel and Kinzig, 1993a) and may explain a part of
the compensatory clearance of enrofloxacin by the intestine. The
N4'-methylated derivatives of fluoroquinolones were the most lipophilic, and addition or removal of the methyl group can frequently affect renal tubular reabsorption (Sorgel and Kinzig, 1993b). Enrofloxacin has an ethyl group attached to the N4'-position of the
piperazinyl ring, which may impart greater lipophilicity and facilitate
reabsorption. Dexamethasone and APP infection inhibit renal tubular
secretion and/or enhance reabsorption of enrofloxacin and
ciprofloxacin. Thus, changes in renal tubular secretion or reabsorption
could account for a reduction in renal clearance and compensatory
increase in intestinal clearance of enrofloxacin and ciprofloxacin in
this study.
Alternatively, enrofloxacin and ciprofloxacin may be excreted at different rates. The urinary concentrations of these two agents are derived from the total amounts excreted into the urine over a 24-h period. Thus, differences in hourly excretion rates would be masked by the manner of sample collection.
Similar arguments can also be made for urinary drug elimination by
APP-infected animals. The concentration of drug in the urine of
infected animals is less than that in control animals, although only
for the first 24-h period. During the subsequent 24-h collection period
(24-48 h after drug administration), the amount of drug in the urine
of infected animals is identical to the amount in control animals.
A. pleuropneumoniae infection does not affect the clearance
of either indocyanine green or creatinine (Monshouwer et al., 1995),
indicating that APP infection does not impact normal hepatic blood flow
or renal excretion. This suggests that 1) the mechanism for this shift
in infected animals is transient, and 2) it is different from the
mechanism in dexamethasone-treated animals.
Although efficacy was not addressed in this study, the subtle changes in the pharmacokinetic parameters in the APP-infected swine group do not suggest that this is a cause for concern. However, the potential for more unchanged drug to be fecally excreted is of concern, because more drug is excreted than was predicted from results in normal, uninfected animals. This finding, if substantiated, might have an unexpected impact on normal gut flora not predicted based on work in normal, uninfected swine.
| |
Acknowledgments |
|---|
We thank Dr. Ron Snider (Louisiana State University, School of Veterinary Medicine, Baton Rouge, LA) for processing and reading the histopathology slides, Dr. Michael P. Murtaugh (University of Minnesota) for the generous gift of the APP bacteria and the conditions to produce an infection, and Mary Bartholomew, Center for Veterinary Medicine, Food and Drug Administration, for graciously providing assistance with the statistical analyses.
| |
Footnotes |
|---|
Accepted for publication December 13, 2001.
Received for publication October 15, 2001.
Address correspondence to: Dr. Michael J. Myers, Food and Drug Administration, Center for Veterinary Medicine, Office of Research, Division of Animal Research, 8401 Muirkirk Road, Laurel, MD 20708. E-mail: mmyers{at}cvm.fda.gov
| |
Abbreviations |
|---|
APP, Actinobacillus
pleuropneumoniae;
HPLC, high-performance liquid chromatography;
IL-6, interleukin-6;
AUC, area under curve;
AUMC, area under the moment
curve;
CLtot, total body clearance;
Vd, volume of distribution of the elimination phase;
VdSS, volume of distribution at steady state;
, elimination rate
constant.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  L. O. Post, D. E. Farrell, C. V. Cope, J. D. Baker, and M. J. Myers The Effect of Endotoxin and Dexamethasone on Enrofloxacin Pharmacokinetic Parameters in Swine J. Pharmacol. Exp. Ther., February 1, 2003; 304(2): 889 - 895. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
