Pradofloxacin is a third-generation fluoroquinolone approved for use in the treatment of susceptible bacterial infections in dogs and cats in Australia, Canada, and Europe. It is also approved in the United States for the treatment of skin infections in cats.
Pradofloxacin offers distinct advantages over other fluoroquinolones that warrant consideration of its use for dogs. Structurally, it contains a nitrogen ring in an S,S-pyrrolidino-piperidine at C7 and an 8-cyano substitution. Compared with earlier-generation fluoroquinolones, these structures increase the potency and broaden the antibacterial spectrum of pradofloxacin to include more gram-positive aerobes and anaerobes, including Bartonella henselae, Nocardia spp, and some Mycobacterium spp.1–4 The molecular structure of pradofloxacin, as with other later-generation fluoroquinolones, makes it so that bacteria would require more mutations in their topoisomerases than earlier-generation fluoroquinolones to become resistant to the drug, thereby potentially slowing the emergence of stepwise resistance.5–7 However, as with other fluoroquinolones, once bacteria develop resistance (which is associated with an increase in efflux pump activity), that resistance is generally high and against multiple drugs.7 Consequently, drug administration regimens should be designed to facilitate bacterial killing while minimizing the development of resistance.
In vitro efficacy of fluoroquinolones depends not only on their concentration, but, for some fluoroquinolones, also on AUC.8 Therefore, the PK-PD indices that are most predictive of fluoroquinolone efficacy and suppression of emerging resistance are the ratios of drug exposure to the MIC against the infecting microbe. The most common indices considered for this purpose are the Cmax:MIC ratio and the AUC0–24:MIC ratio.9,10 For immunocompetent patients, target values for fluoroquinolones are a Cmax:MIC ratio ≥ 8 to 10 or an AUC0–24:MIC ratio ≥ 100 to 125 hours when treating gram-negative bacterial infections,10,11 a Cmax:MIC ratio ≥ 10 or AUC0–24:MIC ratio ≥ 25 to 40 hours for gram-positive bacterial infections,10,12 and an AUC0–24:MIC ratio ≥ 7.5 hours for anaerobic bacterial infections.13,14
In addition to ratios involving MICs, PK-PD indices have also been described for MPCs. The MPC, defined as the highest MIC of the wild-type microbe population infecting the patient (that is, prior to emergent resistance), appears to be a better pharmacodynamic measure of the potential efficacy of a fluoroquinolone while concurrently minimizing emergent microbial resistance to the drug.10,15 In contrast, designing administration regimens that expose microbial isolates to drug concentrations between the MIC and MPC (the so-called mutant selection window) may promote selection of resistant strains.16 A target Cmax:MPC ratio ≥ 1 and an AUC0–24:MPC ratio ≥ 20 to 25 hours have been suggested to treat Escherichia coli infections.10,16
The pharmacokinetics of pradofloxacin in canine plasma and interstitial tissue have been described,17 and integration of plasma pharmacokinetics with pharmacodynamics of the drug has been reported.9 However, although the PK-PD indices are most commonly based on plasma analyses, pathogenic microbes are more likely to be found in tissues. Consequently, studies in which determination of indices for serum or plasma is coupled with determination of indices for tissues16–18 or inflammatory leukocytes19 may be more relevant. The purpose of the study reported here was to quantify target PK-PD indices for pradofloxacin against common canine pathogens in various fluids and leukocytes associated with infection in dogs, with enrofloxacin used as a standard for comparison.
Materials and Methods
Animals and study design
Three pharmacokinetic experiments were performed, with a common focus on drug concentrations in plasma or serum, compared with concentrations in other body fluids or tissues. In experiment 1, drug concentrations in interstitial fluid were compared with those in serum, and in experiment 2, drug concentrations in circulating leukocytes were compared with those in plasma. For both experiments, random-source hounds (n = 8; 4 males and 4 females) ranging in age from 2 to 5 years and with a mean ± SD body weight of 20 ± 5 kg were used. In experiment 3, drug concentrations in CSF, synovial fluid, aqueous humor, urine, or bile were compared with those in serum. For that experiment, 6 random-source male dogs with a mean ± SD body weight of 26 ± 4 kg were used. These dogs had been participating in a different educational exercise (associated with no drug administration) for which euthanasia (via an overdose of pentobarbitala) was the end point, and biological samples for the present study were collected after participation in the educational exercise and immediately after euthanasia.
All dogs were housed in individual runs, fed a commercially available pellet food, and provided fresh water ad libitum. Dogs were monitored to ensure regular food and water intake as well as general health. Dogs received no medication other than an orally administered heartworm preventive given once monthly and orally administered iron sulfateb to enhance hematopoiesis. Iron supplementation was discontinued 1 week before experiments began. All dogs were considered healthy on the basis of physical examination, CBC and serum biochemical analysis, and urinalysis (with urine collected by cystocentesis) findings. All study protocols were approved by the institutional laboratory animal care and use committee.
Drugs
The approved dosage of pradofloxacin for use in cats in the United States is 7.5 mg/kg and in dogs and cats in Europe is 7.5 mg/kg, PO, every 24 hours. For enrofloxacin, the dosage in dogs is 5 to 20 mg/kg, PO, every 24 hours. Doses of pradofloxacin or enrofloxacin varied with each experiment as per the directive of the investigating entity. Pradofloxacin tablets were available in 15-, 30-, and 60-mg tablets.
Experiment 1: interstitial fluid versus serum
In a parallel study design, dogs were randomly assigned (via random numbers table) to orally receive pradofloxacinc (6 mg/kg; n = 4) or enrofloxacind (10 mg/kg; 4) in the morning once daily for 5 days. Doses for both drugs were rounded to the nearest 25 mg.
One week before dose administration began, dogs were prepared for collection of interstitial fluid. Dogs were first sedated with medetomidinee (0.04 mg/kg, IV). A local anesthetic (2% lidocaine; 0.5 mL to effect) was infused SC in the lateral aspect of the thorax, and a percutaneous 14-gauge guidef was inserted into the subcutaneous space of the lateral thoracic region. An ultrafiltration probeg was inserted through the guide into the subcutaneous tissue. A 5-mL plain evacuated tubeh was connected to the end of the percutaneous catheter at the commencement of the experiment to continuously draw interstitial fluid from the sample area. After placement, the entrance site of the probe was secured so that the collection tube was easily accessible, and the site was covered with a stockinet. On the evening of the fourth day of drug administration, a 16-gauge indwelling catheteri was percutaneously placed in a jugular vein with dogs manually restrained.
Blood (5 mL) and interstitial fluid (75 μL to 2 mL) samples were collected at time 0 (immediately prior to drug administration on day 5) and then 15 (blood only), 30, and 60 minutes and 2, 3, 4, 6, 8, 12, 16, 24, 30, and 36 hours after drug administration on day 5. Interstitial fluid samples were collected by exchanging the tube attached to the probe at each sample collection time. Collected blood samples were allowed to clot at room temperature (approx 23°C) for 30 minutes, and then serum was harvested. Serum and interstitial fluid samples were frozen at −80°C within 60 minutes after collection.
Experiment 2: leukocytes versus plasma
This experiment was performed a minimum of 3 months after experiment 1. In a randomized crossover design, dogs (n = 6) orally received pradofloxacin at 3 or 12 mg/kg in the morning once daily for 5 days, with a washout period of at least 2 weeks between treatment sessions. The dose for each dog was rounded to the nearest 7.5 mg.
Before each session began, food was withheld from the dogs for 12 hours. Immediately before the final dose was given, dogs were manually restrained and a 12-inch, 16-gauge indwelling catheteri was placed aseptically into a jugular vein and secured for collection of blood samples. This catheter remained in place for the duration of each 36-hour sample collection period and was removed immediately after the final sample had been collected.
Blood samples were collected at time 0 (immediately before the final dose was given to confirm that no pradofloxacin was already present) and at 15, 30, 45, 60, and 90 minutes and 2, 4, 6, 8, 12, and 24 hours after pradofloxacin administration. At each collection point, 1 mL of blood was collected and then discarded, and then the sample was collected. After each sample collection, the volume of blood removed was replaced with physiologic saline (0.9% NaCl) solution.j For characterization of plasma drug concentrations, 5 mL of blood was collected into syringes containing 0.5 mL of 3.8% sodium citrate as anticoagulant (anticoagulant-to-blood ratio of 1:10). For characterization of leukocyte drug concentrations, an additional 20 mL of blood was collected into a syringe containing 2 mL of 3.8% sodium citrate as anticoagulant. All samples were placed immediately on ice after collection. The total volume of blood collected from each dog during each treatment session was 220 mL.
All sample manipulations, including initial centrifugation of blood samples, were performed at 5°C. Blood samples for measurement of plasma pradofloxacin concentrations were centrifuged at 1,150 × g for 15 minutes within 2 hours after collection. Plasma was harvested and frozen at −20°C until analysis. For quantification of pradofloxacin in leukocytes, blood samples were centrifugedk at 1,125 × g for 15 minutes within 2 hours after collection. The leukocyte-rich layer (buffy coat) was carefully aspirated via glass Pasteur pipettel and transferred to 2-mL conical plastic tubes.m
Residual RBCs in the leukocyte samples were lysed by adding 2 mL of a hypotonic saline (0.2% NaCl) solution for 30 seconds, and isotonicity was restored with 1 mL of saline (1.6% NaCl) solution. The resulting fluid after hypotonic lysis was centrifuged at 700 × g for 10 minutes, and the supernatant containing leukocytes was separated. The remaining supernatant was harvested and frozen at −20°C, forming a leukocyte pellet. The leukocyte pellet was thawed and resuspended in 1 mL of a modified (calcium-free) and preservative-free HBSS buffer.l A 200-μL aliquot of each reconstituted leukocyte pellet was submitted for leukocyte quantification and differential counting with an automated cell-counting system.n The remaining 800 μL of suspended leukocytes was used for quantification of pradofloxacin concentration.
Experiment 3: CSF, synovial fluid, aqueous humor, urine, and bile versus serum
In a parallel study design, dogs were randomly assigned to receive pradofloxacin (3 mg/kg; n = 4) or enrofloxacin (5 mg/kg; 2) in the morning. Doses were orally administered on days 1 to 4 and IV on day 5. Doses for both drugs were rounded to the nearest 25 mg. After the fifth dose, samples were collected approximately 3 hours after administration and immediately after euthanasia by use of a 20-gauge needle and 6-mL syringe and frozen at −80°C within 60 minutes after collection until analyzed.
Sample analysis
Leukocytes were lysed for analysis by freezing overnight at −20°C, rapidly defrosting in a hot water batho at 56°C the following morning, and immediately sonicatingp for 15 minutes. To confirm the absence of intact cells in the leukocyte samples after the lysing procedure, randomly selected samples were evaluated via low-power light microscopy. The lysed leukocyte suspensions were frozen at −20°C until analysis. All samples were analyzed within 2 weeks after collection.
All samples were analyzed with the same methods validated for each sample type. On the day of analysis, each sample was defrosted, vortexed, and centrifuged though a filtration membraneq at 1,750 × g and 4°C for 2 hours. The ultrafiltrate was directly assayed for pradofloxacin or enrofloxacin with no further sample preparation. Samples were analyzed by high-performance liquid chromatographyr involving injection through a 5-μm, 250 × 4.6-mm reversephase C18 columns and measured by fluorescence detection (290-nm excitation and 450-nm emission) modified from methods reported elsewhere.20 The injection volume was 10 μL for all fluids except interstitial fluid, for which it was 25 μL. The gradient mobile phase consisted of 1.0 g of 1-heptanesulfonic acid sodium saltl and 1.0 g of potassium dihydrogen phosphatel dissolved in 1 L of water, with pH adjusted to 2.5 by phosphoric acidl (77%) and acetonitrilel (23%). Samples containing unknown concentrations of pradofloxacin were compared with a calibration curve prepared with drug-free pooled canine interstitial fluid, serum or plasma, urine, bile, or physiologic saline solution.
Assays for pradofloxacin or enrofloxacin in interstitial fluid, CSF, synovial fluid, and aqueous humor were based on calibration curves prepared in physiologic saline solution. Control substances were prepared in samples of pooled, drug-free interstitial fluid, CSF, synovial fluid, and aqueous humor. The upper and lower limits of quantitation for pradofloxacin were 10,000 and 25 ng/mL, respectively, and for enrofloxacin were 8,000 and 100 ng/mL, respectively. Intra- and interassay variability were < 15% for all controls.
Assays for pradofloxacin in leukocyte lysate were validated on the basis of calibration curves and control substances prepared in HBSS. Controls were frozen with each set of collected samples and assayed simultaneously to confirm the stability of pradofloxacin during storage. To determine any adverse impacts of the freezing, defrosting, or sonication procedures on pradofloxacin stability, standards prepared in HBSS were also subjected to the lysing protocol. The upper and lower limits of quantification for pradofloxacin in leukocyte lysates specifically were 10,000 and 25 ng/mL, respectively. Intra- and interassay variability were < 15% for all controls.
Assays for pradofloxacin or enrofloxacin in urine and bile samples were validated on the basis of calibration curves and control substances prepared in blank urine and bile, respectively. The upper and lower limits of quantitation for pradofloxacin were 10,000 and 25 ng/mL, respectively, and for enrofloxacin were 8,000 and 100 ng/mL, respectively. Intra- and interassay variability were < 15% for all controls.
Pharmacokinetic analysis
Drug concentration-versus-time curves for pradofloxacin or enrofloxacin were subjected to noncompartmental linear regression analysist to determine the following pharmacokinetic parameters in the respective tissues21: Cmax, time to maximum concentration, AUC0–24 (by means of the log-linear trapezoidal method), mean residence time, and disappearance half-life. Ratios for serum to interstitial fluid and plasma to leukocyte lysate were determined for Cmax and AUC0–24 for pradofloxacin or enrofloxacin for each dog, as applicable.
Pradofloxacin concentrations in circulating leukocytes were determined on the basis of reported information regarding enrofloxacin accumulation in leukocytes.22 To determine the intracellular concentration of pradofloxacin in leukocytes, it was assumed that any pradofloxacin detected in the leukocyte samples originated from within the leukocytes because the HBSS added to the leukocyte pellet was free of pradofloxacin. Mean intracellular concentration of pradofloxacin in leukocytes was determined by dividing the concentration measured in the reconstituted leukocyte pellet (800 μL) by the volume of the leukocyte pellet. The volume of the leukocyte pellet was determined by multiplying the number of leukocytes in the sample, determined by cell count, by 135 μm3, which is the reported mean volume of circulating canine neutrophils.23 To account for dilution of samples with anticoagulant, all measured concentrations of pradofloxacin in leukocytes were multiplied by 1.1, derived from the ratio of anticoagulant to blood. Therefore, the concentration (y) of pradofloxacin in lysed leukocyte suspension was calculated as follows22:
where x is the mean intracellular concentration of pradofloxacin per leukocyte, as previously calculated.
The PK-PD indices for pradofloxacin and enrofloxacin, specifically those involving Cmax and AUC0–24, were calculated by use of the measured pharmacokinetic values and preestablished values for MIC90 of enrofloxacin or pradofloxacin for 3 gram-negative aerobic bacteria (E coli, Proteus mirabilis, and Pasteurella multocida), 3 gram-positive aerobic bacteria (Staphylococcus aureus, Staphylococcus pseudintermedius, and β-hemolytic Streptococcus spp), and 1 anaerobic bacterium (Bacteroides spp).1,2,24,25 The final PK-PD indices were used to assess the in vivo efficacy of pradofloxacin in all selected fluids and leukocytes, compared with that of enrofloxacin. Similarly, previously determined MPC data for uropathogenic E coli were used to calculate PK-PD indices for comparisons of pradofloxacin in serum, interstitial fluid, and leukocytes.25 For CSF, synovial fluid, aqueous humor, urine, and bile, single time point concentrations and serum ratios were reported only for pradofloxacin or enrofloxacin.
Statistical analysis
Median, minimum, and maximum values were reported for pharmacokinetic parameters (experiment 1), for 2 doses of pradofloxacin for plasma and leukocytes (experiment 2), and for pradofloxacin and enrofloxacin at 3 hours in body fluids (CSF, synovial fluid, aqueous humor, urine, and bile; experiment 3). Ratios for Cmax and AUC0–24 were determined for interstitial fluid versus serum (experiment 1), leukocytes versus plasma (experiment 2), and drug concentration at 3 hours for body fluids versus serum (experiment 3).
For inferential statistical analysis, data were first logarithmically transformed. The paired t test was used to compare Cmax and AUC0–24 between interstitial fluid versus serum (experiment 1) and leukocytes versus plasma (experiment 2) and between pradofloxacin doses for leukocytes and plasma (experiment 2). Ratios of interstitial fluid to serum concentrations and integrated PK-PD indices were also compared between pradofloxacin and enrofloxacin with the paired t test. Comparisons were made between doses and between fluids with the paired t test. Differences were considered significant with a value of P ≤ 0.05.
Results
Pharmacokinetics in all fluids
The median Cmax of pradofloxacin in serum and interstitial fluid samples when the drug was administered to dogs at 6 mg/kg was 4.7 and 4.4 μg/mL, respectively, and the median interstitial fluid-to-serum Cmax ratio was 0.86 (Table 1; Figure 1). The AUC0–∞ of pradofloxacin in serum and interstitial fluid samples was 1,999 and 2,493 μg•min/mL, respectively, and the median interstitial fluid-to-serum AUC0–∞ ratio was 1.22. The median Cmax of pradofloxacin in plasma samples at doses of 3 and 12 mg/kg was 1.6 and 6.2 μg/mL, respectively, and in leukocyte samples was 10 and 34 μg/mL, respectively (Table 2; Figure 2). Median (range) concentrations of pradofloxacin (n = 4) in CSF, synovial fluid, aqueous humor, urine, and bile samples at the 3-mg/kg dose were 0.42 μg/mL (0.4 to 0.43 μg/mL), 0.93 μg/mL (0.58 to 1.2 μg/mL), 0.33 μg/mL (0.28 to 0.36 μg/mL), 71 μg/mL (44 to 113 μg/mL), and 18 μg/mL (18 to 19 μg/mL), respectively.
Mean pradofloxacin concentrations (logarithmic values) in serum (squares) and interstitial fluid (diamonds) samples from 4 healthy dogs before receiving pradofloxacin (0 minutes) and at selected points after the final dose of pradofloxacin was given (6 mg/kg, PO in the morning, q 24 h for 5 days). Error bars represent SD.
Citation: American Journal of Veterinary Research 79, 12; 10.2460/ajvr.79.12.1268
Mean pradofloxacin concentrations (logarithmic values) in serum (squares) and interstitial fluid (diamonds) samples from 4 healthy dogs before receiving pradofloxacin (0 minutes) and at selected points after the final dose of pradofloxacin was given (6 mg/kg, PO in the morning, q 24 h for 5 days). Error bars represent SD.
Citation: American Journal of Veterinary Research 79, 12; 10.2460/ajvr.79.12.1268
Mean pradofloxacin concentrations (logarithmic values) in serum (squares) and interstitial fluid (diamonds) samples from 4 healthy dogs before receiving pradofloxacin (0 minutes) and at selected points after the final dose of pradofloxacin was given (6 mg/kg, PO in the morning, q 24 h for 5 days). Error bars represent SD.
Citation: American Journal of Veterinary Research 79, 12; 10.2460/ajvr.79.12.1268
Mean pradofloxacin concentrations (logarithmic values) in plasma (black symbols) and leukocyte (white symbols) samples from 6 healthy dogs before receiving pradofloxacin (0 minutes) and at selected points after the final dose of pradofloxacin was given (3 mg/kg [circles] or 12 mg/kg [squares], PO in the morning, q 24 h for 5 days) in a crossover study design. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 79, 12; 10.2460/ajvr.79.12.1268
Mean pradofloxacin concentrations (logarithmic values) in plasma (black symbols) and leukocyte (white symbols) samples from 6 healthy dogs before receiving pradofloxacin (0 minutes) and at selected points after the final dose of pradofloxacin was given (3 mg/kg [circles] or 12 mg/kg [squares], PO in the morning, q 24 h for 5 days) in a crossover study design. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 79, 12; 10.2460/ajvr.79.12.1268
Mean pradofloxacin concentrations (logarithmic values) in plasma (black symbols) and leukocyte (white symbols) samples from 6 healthy dogs before receiving pradofloxacin (0 minutes) and at selected points after the final dose of pradofloxacin was given (3 mg/kg [circles] or 12 mg/kg [squares], PO in the morning, q 24 h for 5 days) in a crossover study design. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 79, 12; 10.2460/ajvr.79.12.1268
Median (range) values of pharmacokinetic parameters for serum and interstitial fluid samples obtained from 4 healthy dogs that received pradofloxacin (6 mg/kg, PO) in the morning once daily for 5 days.
| Parameter | Serum | Interstitial fluid |
|---|---|---|
| Cmax (μg/mL) | 4.7 (3.0–6.7) | 4.4 (3.1–5.3) |
| Tmax (min) | 120 (60–180) | 480 (270–720)a |
| AUC0–∞ (μg•min/mL) | 1,999 (1,767–4,218) | 2,493 (1,886–3,924) |
| t1/2d (min) | 377 (193–462) | 504 (403–668) |
| kd (min−1) | 0.0018 (0.0015–0.036) | 0.0014 (0.0010–0.0017) |
| MRT (min) | 627 (616–666) | 929 (766–1,132)a |
| Cmax ratio | — | 0.86 (0.68–1.48) |
| AUC0–24 ratio | — | 1.22 (0.92–1.55) |
Reported ratios are for interstitial fluid versus serum.
Values differ significantly (P ≤ 0.02) between fluids.
— = Not applicable. kd = Disappearance rate constant. t1/2d = Disappearance half-life. Tmax = Time to maximum concentration.
Median (range) values of pharmacokinetics parameters for plasma and leukocyte samples obtained from 6 healthy dogs that received pradofloxacin (3 and 12 mg/kg, PO) in the morning once daily for 5 days in a crossover study design.
| Plasma | Leukocytes | |||
|---|---|---|---|---|
| Parameter | 3 mg/kg | 12 mg/kg | 3 mg/kg | 12 mg/kg |
| Cmax (μg/mL) | 1.6 (0.78–3.4)a,A | 6.2 (3.4–8.8)b,A | 10 (5–30)a,B | 34 (16–135)b,B |
| Tmax (min) | 111 (37–247)a,A | 73 (62–221)a,A | 120 (60–480)a,A | 60 (60–120)a,A |
| AUC0–∞ (μg•min/mL) | 839 (265–1,337)a,A | 3,782 (2,027–5,849)b,A | 6,508 (1,241–19,020)a,B | 7715 (4,760–103,325)b,B |
| t1/2d (min) | 449 (332–1,440)a,A | 347 (233–452)a,A | 551 (194–836)a,A | 343 (251–890)a,A |
| kd (min−1) | 0.0016 (0.0005–0.0021)a,A | 0.0020 (0.0015–0.0030)a,A | 0.0013 (0.0008–0.0036)a,A | 0.0020 (0.0008–0.0028)a,A |
| MRT (min) | 669 (487–2,743)a,A | 587 (476–660)a,A | 866 (399–1,528)a,A | 549 (407–1,363)a,A |
| Cmax ratio | — | — | 8.4 (2.2–39.0) | 6.2 (2.0–15.4) |
| AUC0–24 ratio | — | — | 4.0 (1.2–59.5) | 2.8 (1.0–18.0) |
Reported ratios are for leukocytes versus plasma.
Within a substance (plasma or leukocytes) for a given parameter, values with different superscript lowercase letters differ significantly (P ≤ 0.013) between doses.
Within a dose (3 or 12 mg/kg) for a given parameter, values with different superscript uppercase letters differ significantly (P ≤ 0.003) between plasma and leukocytes.
See Table 1 for remainder of key.
For enrofloxacin, the median Cmax in serum and interstitial fluid samples when the drug was administered to dogs at 10 mg/kg was 5.7 and 2.4 μg/mL, respectively. The AUC0–∞ of enrofloxacin in serum and interstitial fluid samples was 1,068 and 1,143 μg•min/mL, respectively. The interstitial fluid-to-serum ratio for enrofloxacin Cmax was 0.42 and for AUC0–∞ was 1.07. Concentrations of enrofloxacin (n = 2) in CSF, synovial fluid, aqueous humor, urine, and bile samples at a dose of 5 mg/kg were 0.79 μg/mL (0.49 to 1.1 μg/mL), 0.65 μg/mL (0.5 to 0.8 μg/mL), 0.23 μg/mL (0.15 to 0.3 μg/mL), 2.8 μg/mL (0.89 to 4.8 μg/mL), and 136 μg/mL, respectively. Ratios of CSF, synovial fluid, aqueous humor, and bile drug concentrations to serum drug concentrations were similar for both pradofloxacin and enrofloxacin. For urine, pradofloxacin-to-serum concentration ratios were numerically higher than those for enrofloxacin (Figure 3).
Mean ratios of drug concentrations in CSF, synovial fluid (SF), aqueous humor (AH), urine, and bile samples, compared with drug concentrations in serum, as measured in healthy dogs 3 hours after the final dose of pradofloxacin (3 mg/kg, PO, q 24 h for 5 days; n = 4; light gray bars) or enrofloxacin (5 mg/kg, PO, q 24 h for 5 days; 2; dark gray bars) was given. Error bars represent SD.
Citation: American Journal of Veterinary Research 79, 12; 10.2460/ajvr.79.12.1268
Mean ratios of drug concentrations in CSF, synovial fluid (SF), aqueous humor (AH), urine, and bile samples, compared with drug concentrations in serum, as measured in healthy dogs 3 hours after the final dose of pradofloxacin (3 mg/kg, PO, q 24 h for 5 days; n = 4; light gray bars) or enrofloxacin (5 mg/kg, PO, q 24 h for 5 days; 2; dark gray bars) was given. Error bars represent SD.
Citation: American Journal of Veterinary Research 79, 12; 10.2460/ajvr.79.12.1268
Mean ratios of drug concentrations in CSF, synovial fluid (SF), aqueous humor (AH), urine, and bile samples, compared with drug concentrations in serum, as measured in healthy dogs 3 hours after the final dose of pradofloxacin (3 mg/kg, PO, q 24 h for 5 days; n = 4; light gray bars) or enrofloxacin (5 mg/kg, PO, q 24 h for 5 days; 2; dark gray bars) was given. Error bars represent SD.
Citation: American Journal of Veterinary Research 79, 12; 10.2460/ajvr.79.12.1268
The pharmacokinetics of pradofloxacin was dose-dependent for Cmax and AUC0–24 in plasma samples (P ≤ 0.05) and for Cmax and AUC0–∞ in leukocyte samples (P ≤ 0.05; Table 2 and Figure 2). Leukocyte-to-plasma ratios indicated an approximate 5-fold increase for pradofloxacin Cmax and AUC0–24 at a dose of 3 mg/kg. No significant differences were identified between the pharmacokinetic values for serum and interstitial fluid samples; the interstitial fluid-to-serum ratios for pradofloxacin Cmax and AUC0–∞ approximated unity.
PK-PD integration for pradofloxacin versus enrofloxacin
Pradofloxacin administered to dogs at 3 mg/kg achieved target values for PK-PD indices in collected serum, urine, and bile samples for tested microorganisms, but not in serum samples for P mirabilis and Bacteroides spp (Supplementary Table S1, available at http://avmajournals.avma.org/doi/suppl/10.2460/ajvr.79.12.1268). Of the fluids from tissues characterized by an enhanced barrier to circulating drugs (ie, CSF, synovial fluid, and aqueous humor), only synovial fluid had an adequate Cmax:MIC90 ratio against E coli and P multocida and against S pseudintermedius. An adequate Cmax:MPC ratio (ie, ≥ 1) against E coli was achieved for all types of samples (Table 3). Concentration ratios for pradofloxacin at a dose of 6 mg/kg exceeded the Cmax:MIC90 ratio against all organisms in serum, interstitial fluid, and leukocyte samples, except against Bacteroides spp in serum and interstitial fluid samples. Values of Cmax:MPC and AUC0–∞:MPC ratios against E coli for pradofloxacin were numerically higher than those for enrofloxacin in serum, interstitial fluid, and leukocyte samples.
Values of PK-PD indices (Cmax:MIC90 and Cmax:MPC ratios) against common canine pathogens for various biological fluid samples obtained from 4 healthy dogs that received pradofloxacin (3 mg/kg, PO) or 2 healthy dogs that received enrofloxacin (5 mg/kg, PO) in the morning once daily for 5 days.
| Organism, by drug | MIC90 (μg/mL) | MPC (μg/mL) | Serum | Cerebrospinal fluid | Synovial fluid | Aqueous humor | Urine | Bile |
|---|---|---|---|---|---|---|---|---|
| Pradofloxacin | ||||||||
| Escherichia coli | 0.03 | — | 43 | 14 | 31 | 11 | 2,367 | 600 |
| Proteus mirabilis | 0.25 | — | 5.2 | 1.7 | 3.7 | 1.3 | 284 | 72 |
| Pasteurella multocida | 0.015 | — | 87 | 28 | 62 | 21 | 4,733 | 1200 |
| Staphylococcus aureus | 0.12 | — | 11 | 3.5 | 7.8 | 2.7 | 592 | 150 |
| Staphylococcus pseudintermedius | 0.06 | — | 22 | 7.0 | 16 | 5.3 | 1,183 | 300 |
| β-Hemolytic Streptococcus spp | 0.12 | — | 11 | 3.5 | 7.8 | 2.7 | 592 | 150 |
| Bacteroides spp | 1.0 | — | 1.3 | 0.42 | 0.93 | 0.32 | 71 | 18 |
| E coli | — | 0.29 | 4.5 | 1.4 | 3.2 | 1.1 | 245 | 62 |
| Enrofloxacin | ||||||||
| E coli | 1.0 | — | 1.4 | 0.79 | 0.65 | 0.23 | 2.8 | 136 |
| P mirabilis | 1.0 | — | 1.4 | 0.79 | 0.65 | 0.23 | 2.8 | 136 |
| P multocida | 0.06 | — | 23 | 13 | 11 | 3.8 | 47 | 2,267 |
| S aureus | 64 | — | 0.02 | 0.01 | 0.01 | 0.00 | 0.04 | 2.1 |
| S pseudintermedius | 0.25 | — | 5.6 | 3.2 | 2.6 | 0.92 | 11 | 544 |
| β-Hemolytic Streptococcus spp | 1.0 | — | 1.4 | 0.79 | 0.65 | 0.23 | 2.8 | 136 |
| Bacteroides spp | 8.0 | — | 0.18 | 0.10 | 0.08 | 0.03 | 0.35 | 17 |
| E coli | — | 1.55 | 0.90 | 0.51 | 0.42 | 0.15 | 1.8 | 88 |
Values for MIC901,2,24,34,46 and MPC25 were obtained from previous reports.
Enrofloxacin administered at 5 mg/kg achieved target PK-PD values (ie, ≥ 10) against only P multocida for all types of samples except aqueous humor, against S pseudintermedius in urine samples only, and against all organisms except S aureus in bile samples only. An adequate Cmax:MPC ratio (ie, ≥ 1) against E coli was achieved in urine and bile only. At a dose of 10 mg/kg, enrofloxacin achieved the target Cmax:MIC90 ratio against only P multocida in serum, interstitial fluid, and leukocyte samples and S pseudintermedius in serum and leukocyte samples (Supplementary Table S1; Table 3).
Discussion
Results of the present study suggested that pradofloxacin was superior to enrofloxacin in achieving PK-PD indices consistent with measures of treatment efficacy for other fluoroquinolones. The superiority of pradofloxacin observed in this study reflected, in part, its greater potency against E coli, P mirabilis, P multocida, S aureus, S pseudintermedius, β-hemolytic Streptococcus spp, and Bacteroides spp.9,15,24
Distribution into interstitial fluid, as inferred through Cmax, appeared superior for pradofloxacin versus enrofloxacin, given an interstitial fluid-to-serum Cmax ratio for pradofloxacin of more than twice that of enrofloxacin and an AUC0–∞ ratio that was slightly (15%) higher. Because pradofloxacin is generally more potent than enrofloxacin against the evaluated organisms, it was not surprising that pradofloxacin was generally able to achieve better PK-PD indices in the present study.
As with other fluoroquinolones, pradofloxacin accumulates in circulating leukocytes.26 Although no direct comparisons can be made, previous research involving techniques similar to those used in the present study revealed leukocyte-to-plasma concentration ratios of approximately 45 to 60 for enrofloxacin, compared with ratios of 7 to 15 for pradofloxacin.27 The role of uptake or efflux within different leukocytes was not evaluated in the present study. However, accumulation of fluoroquinolones in leukocytes appears to reflect a combination of passive diffusion into the cells followed by intracellular trapping due to binding or pH-mediated ionization.28,29 Drugs with higher partition coefficients theoretically may accumulate better in cells. Enhanced phagocytosis, ion trapping, and active transport may also contribute to differential accumulation of fluoroquinolones in leukocytes.30–32
All PK-PD indices based on MIC90 values for specific bacterial species were the same or greater for pradofloxacin in serum samples than in interstitial fluid samples in the present study. Given our findings for 7 susceptible bacterial species, only pradofloxacin administered PO at a dose of 6 mg/kg would surpass the target Cmax:MIC ratio of ≥ 10 and AUC0–24:MIC ratio of ≥ 105 hours for gram-negative organisms and respective targets of 25 to 40 hours for gram-positive organisms in both serum and interstitial fluid samples for all 6 evaluated aerobic species on the basis of respective MIC90 values. The target AUC0–24:MIC ratio of ≥ 100 hours for gram-negative organisms was used as a standard for all evaluated species to mimic the most critical scenario. Findings also suggested that care must be taken when treating tissues characterized by barriers to drug distribution. For experiment 3, the Cmax:MIC ratio was less in other fluid samples than in serum samples (Table 3). However, the dose of pradofloxacin used in this experiment (3 mg/kg) was lower than recommended,
Despite the favorable PK-PD indices achieved for pradofloxacin in the study reported here, several precautions should be considered when applying the resulting data to dogs in clinical settings. First, the clinical applicability of these targets remains to be adequately demonstrated. Second, although pradofloxacin reportedly has a wide therapeutic index (up to 5 times the recommended dose33), adverse effects of the fluoroquinolone class in general, such as chondropathy, should be considered for patients with special circumstances, such as lactating or juvenile animals. The applicability of these data to dogs previously exposed to antimicrobials in general, and fluoroquinolones in particular, should also be considered. Although previously reported MIC90 values were generally based on data for subjects with no prior antimicrobial exposure, clinical use may not be limited to such individuals. Evidence suggests that the MIC90 of enrofloxacin or pradofloxacin against clinical E coli or Staphylococcus intermedius populations exceeds 32 μg/mL1,25,34 As such, the findings of the present study were relevant only to isolates known to be susceptible to pradofloxacin or enrofloxacin. Finally, although the indices targeted in our study had the goal of both efficacy and avoiding resistance,16,35 for some infections such as Enterobacteriaceae septicemia in humans, an AUC0–24:MIC ratio ≥ 250 hours has been reported.36 Given the findings of the in vitro study reported here, pradofloxacin would also meet this more stringent target ratio in serum, interstitial fluid, and leukocytes for all aerobic bacterial species considered except P mirabilis.
Evidence is emerging to support the notion that the MPC is a more reasonable target than the MIC for maximizing treatment efficacy while avoiding emergent resistance against fluoroquinolones. When the Cmax:MPC ratio is > 1 and AUC0–24:MPC ratio is > 1,200 minutes (20 hours), prevention of resistant mutations is more likely.36 The MPC represents the highest MIC of any of the isolates comprising an infecting inoculum and ranges from several fold to 100-fold higher than the MIC, depending on the drug and isolate.37 Isolate MPC correlates poorly with, and thus cannot be predicted from, the MIC.15,38 The PK-PD values in the present study indicated that only pradofloxacin reached the target ratios of Cmax:MPC ≥ 1 and AUC0–24: MPC ≥ 20 hours in serum samples, suggesting that pradofloxacin might be preferred to enrofloxacin for avoiding resistance.
Limitations to the present study included the lack of accounting for other drug factors that may have influenced the PK-PD indices. For example, enrofloxacin is metabolized to ciprofloxacin, which can contribute to ≥ 25% of bioactivity, depending on the dose.39 As such, it is possible that enrofloxacin would come closer to achieving the magnitude of PK-PD indices achieved by pradofloxacin for either the Cmax:MIC or AUC0–24:MIC ratio. However, because the magnitude of differences in PK-PD values for pradofloxacin versus enrofloxacin was higher, such numerical superiority would presumably persist for pradofloxacin. Furthermore, the study did not take into account protein binding of either pradofloxacin or enrofloxacin. However, drug concentrations in the interstitial fluid should have been based on free, unbound drug, and the magnitude of difference between pradofloxacin and enrofloxacin was greatest for the Cmax:MIC90 ratio of interstitial fluid samples.
An additional limitation in experiment 2 was the lack of evaluation of drug accumulation on intracellular bacterial killing. As previously reported, intracellular killing of Staphylococcus spp is concentration dependent for fluoroquinolones, including ciprofloxacin (an active metabolite of enrofloxacin) and moxifloxacin, a third-generation fluoroquinolone similar in structure to pradofloxacin.40 However, the impact of drug accumulation on intracellular killing is unclear. Research involving a human monocyte cell line showed that intracellular bacterial killing by fluoroquinolones may be less than extracellular killing.41 Decreased intracellular killing may reflect less drug binding to intracellular substrates or the state of cell activity, and it appears to be pH dependent.42 Findings of previous studies43,44 indicate that intracellular uptake of antimicrobials may be impacted by the active state of the cell, the drug-structure activity relationship, and the specific pathogen. Intracellular accumulation of fluoroquinolones may also increase drug efficacy by enhancing distribution of the drug to sites of inflammation.32 Indeed, intracellular accumulation of fluoroquinolones in circulating leukocytes is associated with higher drug concentrations at sites of inflammation, as shown in a study22 involving tissue chambers implanted in enrofloxacin-treated dogs.
Despite every effort taken to provide optimal drug recovery in the present study, in vivo leukocyte drug concentrations might have been slightly higher than measured if extraction was incomplete. Although the mean volume of a neutrophil (and not other types of leukocytes) was used in calculations of mean intracellular drug concentration, we believe that neutrophils were a good representation of the whole canine leukocyte population because they are the most abundant circulating leukocyte in healthy dogs. Furthermore, neutrophil diameter (10 to 14 μm) is representative of the mean diameter of all leukocytes combined (9.6 to 14.4 μm) in humans.45
Pradofloxacin administration to healthy dogs at 6 mg/kg, PO, every day for 5 days resulted in serum and interstitial fluid concentrations that surpassed the in vitro target PK-PD index values suggested for achieving treatment efficacy and avoiding acquired antimicrobial resistance for the treatment of selected nonanaerobic gram-negative or gram-positive bacterial infections. These target values were more often achieved for pradofloxacin at 6 mg/kg, PO, than for enrofloxacin at 10 mg/kg, PO. Similarly, pradofloxacin administration at 3 mg/kg, PO, resulted in urine and bile concentrations that met target PK-PD index values for common canine bacterial pathogens. We therefore conclude that targets should be met when using the recommended dose for pradofloxacin of 7.5 mg/kg. Our findings might provide support for clinical studies of the efficacy of pradofloxacin and associated antimicrobial resistance when used to treat susceptible bacterial infections in dogs.
Acknowledgments
Supported in part by Bayer Animal Health.
Presented in abstract form at the First International Veraflox Symposium, Berlin, Germany, March 2006.
ABBREVIATIONS
| AUC | Area under the concentration-versus-time curve |
| AUC0–∞ | Area under the concentration-versus-time curve from time 0 to infinity |
| AUC0–24 | Area under the concentration-versus-time curve from time 0 to 24 hours |
| Cmax | Maximum observed serum concentration |
| HBSS | Hanks balanced salt solution |
| MIC | Minimum inhibitory concentration |
| MIC90 | Minimum concentration required to inhibit the growth of 90% of organisms |
| MPC | Mutant prevention concentration |
| PK-PD | Pharmacodynamic-pharmacokinetic |
Footnotes
Beuthanasia D, Merck Animal Health, Madison, NJ.
Pet-Tinic, SmithKline Beecham Animal Health, West Chester, Pa.
Veraflox, Bayer HealthCare LLC, Shawnee Mission, Kan.
Baytril, Bayer HealthCare LLC, Shawnee Mission, Kan.
Domitor, Zoetis Inc, Kalamazoo, Mich.
Bioanalytical Systems Inc, West Lafayette, Ind.
UF 3-2, Bioanalytical Systems Inc, West Lafayette, Ind.
Vacutainer red-top tubes, Becton, Dickinson & Co (Difco/BLL), Sparks, Md.
Venocath, Abbott Laboratories, North Chicago, Ill.
Fisher Scientific, Waltham, Mass.
Beckman GPR refrigerated centrifuge, Beckman Coulter Inc, Indianapolis, Ind.
VWR International, Radnor, Pa.
Falcon borosilicate tubes, VWR International, Radnor, Pa.
Abbott CellDyn 3500, Abbott Laboratories Philippines, Taguig City, Philippines.
Adjustable temperature water bath, Scientific Products, McGraw Park, Ill.
American Brand Sonicator water bath, Scientific Products, McGraw Park, Ill.
Centricon 10, Amicon Corp, Lexington, Mass.
Metachem Technologies Inc, Torrance, Calif.
Luna C8 column, Phenomenex Inc, Torrance, Calif.
Phoenix Winnonlin, version 6.4. Cetara, Mountain View, Calif.
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