Horses frequently incur wounds to the distal portion of a limb (distal limb), and those wounds often involve a synovial structure and can lead to infection, which in turn can lead to permanent disability or necessitate euthanasia.1 Other causes of infection of synovial structures of the distal limb include translocation of bacteria, such as from pneumonia or an umbilical infection, and the introduction of an infective concentration of bacteria during synoviocentesis.
Administration of antimicrobials by IV-RLP can result in antimicrobial concentrations in infected synovial structures that far exceed the minimum inhibitory concentration for the infecting bacteria.2,3 Moreover, when an antimicrobial is administered by IV-RLP, the dose necessary to achieve therapeutic concentrations in targeted synovial structures of a distal limb is much less than that required to achieve therapeutic concentrations in serum, which decreases the likelihood of adverse systemic effects, such as antimicrobial-induced colitis or nephrotoxicosis.4,5
In horses, although synoviocentesis-induced infections are generally caused by gram-positive bacteria, especially Staphylococcus spp, many synovial infections, particularly those resulting from penetrating wounds, are caused by gram-negative bacteria.1,6 Additionally, synovial infections caused by gram-negative bacteria are more likely to have a poor outcome than are synovial infections caused by gram-positive bacteria.6 Therefore, identification of antimicrobials with activity against gram-negative bacteria that can achieve therapeutic concentrations in synovial structures of the distal limb without inducing adverse systemic effects is vitally important for equine practice. Polymyxin B, an antimicrobial produced by the bacterium Bacillus polymyxa, is rapidly bactericidal against most gram-negative pathogens,7,8 but its use in human medicine was nearly abandoned because the systemic dose required to achieve therapeutic tissue concentrations of the drug often results in nephrotoxicosis and neurotoxicosis.7,9,10 The veterinary literature does not contain any reports describing the clinical use of PB by either systemic administration or IV-RLP in horses. We hypothesized that administration of PB by IV-RLP to horses would result in therapeutic drug concentrations in distal limb synovial structures without inducing nephrotoxicosis or neurotoxicosis. The primary objective of the study reported here was to determine whether therapeutic concentrations of PB could be achieved in the tarsocrural joint of horses when the drug is administered by IV-RLP via a saphenous vein. A secondary objective was to document any local or systemic adverse effects associated with IV-RLP of PB in horses.
Materials and Methods
The study consisted of 2 experiments. All study procedures were reviewed and approved by the University of Tennessee Institutional Animal Use and Care Committee (protocol No. 2543-0717).
Experiment 1
Animals—Six university-owned mares that ranged in age from 13 to 24 years (mean, 19 years) and body weight from 452 to 545 kg (mean, 498 kg) were used for experiment 1. Three were American Quarter Horses, and 2 were nongaited Tennessee Walking Horse crossbreds; the breed was unknown for the remaining horse. All 6 horses were healthy and free of obvious musculoskeletal abnormalities as determined by results of complete physical and lameness examinations, a WBC count,a and serum biochemical analyses.b For the lameness examination, each horse was instrumented with wireless body-mounted inertial sensorsc and observed while trotting; data were assessed, and a baseline lameness score was assigned. Horses were individually housed in stalls for the duration of the experiment and had ad libitum access to hay and water.
IV-RLP—For each horse, 1 pelvic limb was randomly selected for IV-RLP by means of a coin toss. To minimize discomfort and movement associated with tourniquet placement during IV-RLP of PB, each horse was sedated with detomidine hydrochloride (0.008 mg/kg, IV) and the selected limb was desensitized by perineural administration of mepivacaine around the tibial nerve and superficial and deep peroneal nerves. Between 30 minutes and 2 hours after mepivacaine administration, each horse was resedated with detomidine (0.008 mg/kg, IV) and butorphanol tartrate (0.01 mg/kg, IV). The tarsocrural region and area surrounding the distal aspect of the saphenous vein of the selected limb were clipped and aseptically prepared.
To occlude venous circulation from the tarsal region, a 15-cm-wide pneumatic tourniquetd was placed around the selected limb midway between the stifle joint and tarsus proximal to the proposed injection site in the saphenous vein (proximal tourniquet). The tourniquet was inflated to a pressure of 450 mm Hg. To occlude venous circulation to the tarsal region, a 10-cm-wide rubber tourniquete was placed around the midmetatarsal region and tightly secured (distal tourniquet). All tourniquets were applied by the same investigator (JS).
Each horse was randomly assigned by means of a coin toss to receive either 25 mg (250,000 U) or 50 mg (500,000 U) of PB.f The assigned dose of PB was diluted in 60 mL of saline (0.9% NaCl) solution as described.11 The perfusate was injected into the saphenous vein between the 2 tourniquets through a winged infusion set with a 21-gauge, 1.9-cm-long needle.g The horses were resedated with detomidine (0.008 to 0.01 mg/kg, IV), as needed, to minimize movement until the tourniquets were removed 30 minutes after injection. Immediately after the needle was removed from the vein, the injection site was wrapped with a compression bandage consisting of gauze and nonelastic tape. The bandage was left in place for 60 minutes and then was removed.
Sample collection and processing—A blood sample (5 mL) and synovial fluid sample (0.5 to 2 mL) from the tarsocrural joint were obtained after the tourniquets were applied but before PB was administered (time 0; baseline) and at 30 (immediately before the tourniquets were removed) and 60 minutes and 24 hours after IV-RLP to determine PB concentration. Blood samples were obtained by jugular venipuncture and collected into additive-free blood collection tubes. Synovial fluid samples were transferred from the syringe into blood collection tubes containing EDTA as an anticoagulant (EDTA tubes). Twenty-four hours after PB administration, additional blood (3 to 6 mL) was collected into additive-free and EDTA tubes for serum biochemical analyses and determination of the WBC count.
Blood samples collected in EDTA tubes were submitted for determination of the WBC count by an automated hematology analyzer.a All other samples were centrifuged at 1,000 × g for 5 minutes. The serum or supernatant (synovial fluid samples) was harvested from each sample. Serum samples designated for biochemical analyses were evaluated within 24 hours after collection by an automated biochemical analyzer.b Serum and synovial fluid samples designated for determination of PB concentration were individually placed in cryovials and stored at −80°C until they were shipped to the laboratory. The samples were shipped to the laboratory on dry ice overnight.
Follow-up care—Following sample collection at 60 minutes after PB administration, each horse received phenylbutazone (4.4 mg/kg, IV). Additionally, the compression bandage over the injection site was removed, and a bandage composed of cast padding and elastic adhesive tape was applied to the tarsal region of the treated limb. The bandage was removed during sample collection 24 hours after PB administration. The day after IV-RLP, each horse was instrumented with wireless body-mounted inertial sensors and underwent a lameness examination. A physical examination was performed on each horse twice daily for 2 days after IV-RLP, after which the horses were turned out into a pasture where they were visually observed on a daily basis. At least 2 weeks later, the process was repeated in the contralateral pelvic limb except that the opposite dose of PB was administered.
Experiment 2
Animals—Four university-owned mares that ranged in age from 16 to 26 years (mean, 20 years) and body weight from 477 to 506 kg (mean, 496 kg) were used for experiment 2. Two were American Quarter Horses, 1 was a nongaited Tennessee Walking Horse crossbred, and 1 was an American Paint Horse. One of the 4 horses was used for experiment 1, and that horse underwent a 6-month washout period between experiments 1 and 2. All 4 horses were determined to be healthy and free of musculoskeletal abnormalities, housed, and maintained as described for the horses of experiment 1.
IV-RLP—The procedures for experiment 2 were similar to those for experiment 1, with a few minor modifications. The IV-RLP was performed on only 1 randomly selected pelvic limb, and the dose of PB administered was 300 mg (3,000,000 U). That dose was roughly equivalent to the dose of PB (0.6 mg/kg [6,000 U/kg], IV) systemically administered to horses for the treatment of endotoxemia (anti-endotoxin dose).9,12-15 Also, a 10-cm-wide rubber tourniquetc rather than a pneumatic tourniquet was used as the proximal tourniquet. This was owing to the high incidence rate of the pneumatic tourniquet failing to adequately retain the perfusate within the targeted region for the horses of experiment 1. The proximal tourniquet was applied as tightly as possible without tearing the tourniquet. As in experiment 1, the same investigator (JS) applied all tourniquets. Sample collection and processing as well as follow-up care were as described for experiment 1.
Determination of PB concentration
Serum and synovial fluid PB concentrations were determined at the K. L. Maddy Equine Analytical Chemistry Laboratory, University of California-Davis, Davis, Calif, by use of high-performance liquid chromatography-tandem mass spectometry as described.16 Polymyxin B concentrations were calculated from linear regression analyses and extrapolated from a calibration curve generated in blank equine synovial fluid samples fortified with known increasing concentrations of PB. A partial validation was performed with equine plasma and synovial fluid as the matrix. Responses were linear and yielded correlation coefficients ≥ 0.99. The precision and accuracy of the assay were determined by measuring the PB concentration in each of 6 replicates of quality control samples (PB concentrations, 0.25 and 1.0 μg/mL). Accuracy was reported as the percentage of the nominal concentration, and precision was reported as the percentage relative SD. The mean + SD accuracy was 89 + 0.02% for the 0.25-μg/mL quality control samples and 92 + 0.04% for the 1.0-μg/mL quality control samples. Mean precision was 7% and 4% for the 0.25- and 1.0-μg/mL quality controls samples, respectively. The technique was optimized to provide a limit of quantitation of 0.1 μg/mL for PB1 and PB2 and 0.6 μg/mL for PB3. The limit of detection was approximately 0.05 μg/mL for PB1 and PB2 and 0.4 μg/mL for PB3. The MIC90 of PB is ≤ 0.5 to 1.0 μg/mL for many bacterial pathogens isolated from the wounds of horses,1,7,17 and that was the definition used for the MIC90 of PB (and presumed therapeutic concentration of the drug) in the present study.
Data analysis
Mixed linear models were used to assess factors associated with serum and synovial fluid PB concentrations in both experiments 1 and 2. For experiment 1, fixed effects included in the models included the PB dose (25 or 50 mg; dose), sample acquisition time (time), and interaction between dose and time. For experiment 2, time was the only fixed effect included in the models. All models in both experiments included a random effect to account for repeated measures within horses. When the PB concentration in a sample of synovial fluid or serum was below the limit of quantification, a PB concentration of 0.05 μg/mL was assigned to that sample. Individual trials (horse-dose combination) were excluded from analyses if the serum PB concentration was greater than the synovial fluid PB concentration at any given time because that indicated the proximal tourniquet failed to retain the perfusate within the targeted area. Diagnostic analyses of residuals for normality and homoscedasticity were performed. When there was evidence that the assumptions of normality or homoscedasticity were violated, PB concentration data underwent a rank transformation for linear modeling. When warranted, pairwise comparisons between levels of a fixed effect were performed with Student t tests. Results were reported as the least squares mean + SE, and values of P < 0.05 were considered significant. All analyses were performed with statistical software.h
Results
Horses
For all horses in both experiments 1 and 2, the WBC count and results for all serum biochemical variables, including those associated with renal function, remained within the respective reference ranges before and after PB administration and did not increase significantly from baseline values after PB administration. Likewise, the lameness score the day after PB administration did not differ from that at baseline for any horse. No adverse systemic effects or signs of inflammation at the injection site were observed following PB administration.
Experiment 1
Four trials were excluded from analyses in experiment 1. For 1 horse, the trial for the 25-mg dose of PB was excluded from analyses because we were unable to inject the perfusate into the saphenous vein. For each of 3 horses, the trial for the 50-mg dose of PB was excluded from analyses because the serum PB concentration was greater than the synovial fluid PB concentration at 30 minutes after drug administration, which indicated that the proximal (pneumatic) tourniquet failed to retain the drug in the target area.
The least squares mean + SE PB concentrations over time in synovial fluid and serum samples were plotted (Figure 1). For synovial fluid samples, dose was not significantly (P = 0.07) associated with PB concentration, and the mean + SE PB concentration was 11.0 + 5.9 μg/mL for the 25-mg dose and 29.6 + 7.9 μg/mL for the 50-mg dose. However, both time (P = 0.001) and the interaction between dose and time (P = 0.02) were significantly associated with synovial fluid PB concentration. The PB concentration in synovial fluid peaked at 30 and 60 minutes after drug administration for the 50- and 25-mg doses, respectively. For the 5 horses included in the analysis for the 25-mg dose, the mean synovial fluid PB concentration was 13.4 μg/mL (range, 2.5 to 26.4 μg/mL) and 30.1 μg/mL (range, 2.4 to 72.3 μg/mL) at 30 and 60 minutes, respectively, after drug administration. For the 3 horses included in the analysis for the 50-mg dose, the mean synovial PB concentration was 87.3 μg/mL (range, 12.1 to 154 μg/mL) and 30.9 μg/mL (range, 12.2 to 48.0 μg/mL) at 30 and 60 minutes, respectively, after drug administration. For both the 25- and 50-mg doses, the mean synovial fluid PB concentration was less than the MIC90 for common gram-negative bacterial pathogens of horses (≤ 0.5 to 1.0 μg/mL; ie, below the presumed therapeutic concentration) by 24 hours after drug administration.
Least squares mean + SE PB concentration over time in tarsocrural synovial fluid (A) and serum (B) for healthy adult nonlame horses before (time 0) and at predetermined times after administration of the drug at doses of 25 mg (solid line; n = 5) and 50 mg (dashed line; 3) by means of IV-RLP into the distal portion of the saphenous vein in the tarsal region of a pelvic limb (experiment 1). Each of 6 horses received each dose of PB. Any sample with a PB concentration below the quantification limit was assigned a concentration of 0.5 μg/mL for analysis purposes. One trial (ie, horse-dose combination) was excluded from analyses for the 25-mg dose because we were unable to inject the perfusate into the saphenous vein. Three trials were excluded from the analyses for the 50-mg dose because the serum PB concentration exceeded the synovial fluid PB concentration at 30 minutes after drug administration, which indicated that the proximal (pneumatic) tourniquet failed to retain the perfusate in the target (tarsal) area. *Within the 50-mg dose, value differs significantly (P < 0.05) from other time points.
Citation: American Journal of Veterinary Research 80, 12; 10.2460/ajvr.80.12.1099
For serum samples, dose was not significantly (P = 0.27) associated with serum PB concentration, and the mean + SE PB concentration was 0.4 + 0.2 μg/mL for the 25-mg dose and 0.8 + 0.3 μg/mL for the 50-mg dose (Figure 1). Time (P = 0.003), but not the interaction between dose and time (P = 0.45), was associated with serum PB concentration. For the 5 horses included in the analysis for the 25-mg dose, the mean serum PB concentration was 0.63 μg/mL (range, 0 to 2.4 μg/mL) and 1.13 μg/mL (range, 0.34 to 2.92 μg/mL) at 30 and 60 minutes, respectively, after drug administration. For the 3 horses included in the analysis for the 50-mg dose, the mean serum PB concentration was 1.21 μg/mL (range, 1.11 to 1.935 μg/mL) and 1.62 μg/mL (range, 0.78 to 3.37 μg/mL) at 30 and 60 minutes, respectively, after drug administration. Regardless of the dose administered, PB was not detected in the serum of any horse at 24 hours after drug administration.
Experiment 2
No trials were excluded from the analyses for experiment 2. The least squares mean + SE PB concentration in synovial fluid and serum samples for the horses of experiment 2 was plotted (Figure 2). Time was significantly associated with the PB concentration in both synovial fluid (P = 0.008) and serum (P < 0.001). The mean synovial fluid PB concentration peaked at 74.6 mg/mL (range, 20.4 to 144.3 μg/mL) at 30 minutes after drug administration and decreased to 40.5 μg/mL (range, 9.6 to 77.1 μg/mL) and 1.4 μg/mL (range, 0.70 to 3.25 mg/mL) at 60 minutes and 24 hours, respectively, after drug administration. The mean serum PB concentration peaked at 6.98 μg/mL (range, 4.1 to 9.72 mg/mL) at 30 minutes after drug administration, decreased slightly to 6.5 mg/mL (range, 4.2 to 8.7 mg/mL) at 60 minutes after drug administration, and was 0.33 mg/mL (range, 0.14 to 0.5 mg/mL) at 24 hours after drug administration, which was less than the MIC90 for common gram-negative bacterial pathogens of horses.
Least squares mean + SE PB concentration over time in tarsocrural synovial fluid (dotted line) and serum (dashed line) for 4 healthy adult nonlame horses before (time 0; baseline) and at predetermined times after administration of the drug at a dose of 300 mg by means of IV-RLP into the distal portion of the saphenous vein in the tarsal region of a pelvic limb (experiment 2). Notice that the scale for synovial fluid PB concentration differs from the scale for serum PB concentration. For synovial fluid PB concentrations, mean values with different lowercase letters differ significantly (P < 0.05). For serum PB concentrations, mean values with different uppercase letters differ significantly (P < 0.05).
Citation: American Journal of Veterinary Research 80, 12; 10.2460/ajvr.80.12.1099
Discussion
Polymyxin B is a bactericidal, concentration-dependent antimicrobial that is effective against most gram-negative bacteria, which makes it an appropriate candidate for IV-RLP in horses for the treatment of wound and synovial infections of the distal aspect of limbs (distal limbs) because many of those infections are caused by gram-negative pathogens.1,7,18 Although bacterial resistance is becoming increasingly common for many antimicrobials traditionally used to treat infections in horses,19 nearly all gram-negative bacterial pathogens remain susceptible to PB, including common pathogens isolated from equine wounds such as Pseudomonas aeruginosa, Klebsiella spp, and Acinetobacter spp.1,7,11 Among Enterobacteriaceae isolates, only Proteus spp and Serratia marcescens are regularly resistant to PB during susceptibility testing.20,21 In a surveillance study7 that evaluated the activity of polymyxins against a large worldwide collection of gram-negative isolates, PB had potent in vitro activity (MIC90, ≤ 0.5 to 1 μg/mL) against most gram-negative pathogens isolated from clinical cases, including multidrug-resistant gram-negative bacilli, and a low incidence of resistance (< 0.1% to 1.5%).
Polymyxin B sulfate was discovered in the late 1940s and approved by the FDA in the late 1950s to treat human patients for infections caused by gram-negative bacteria.7,11,22-24 Despite the effectiveness of PB against most pathogenic gram-negative bacteria, its use in human medicine fell out of favor during the 1970s owing to the incidence of associated nephrotoxicosis and neurotoxicosis.8,18,24 However, the administration of PB to human patients with gram-negative bacterial infections has increased over the last 2 decades because of the emergence of multidrug-resistant gram-negative isolates.7,8,10,11,22,24-30
For human patients with multi-drug-resistant gram-negative bacterial infections and normal renal function, the recommended systemic dose of PB is 1.5 to 2.5 mg/kg, IV or IM, daily in 2 to 4 divided doses.11 Systemic dose recommendations for PB administration in horses are lacking. In an experimental study15 on the efficacy of PB for alleviation of the detrimental effects of carbohydrate overload-induced endotoxemia in horses, none of the 10 horses administered PB at a dosage of 10 mg/kg, IV, daily in 4 divided doses for 48 hours developed clinical signs of nephrotoxicosis within 72 hours after discontinuation of treatment, and none of the 4 PB-treated horses that were subsequently euthanized had histologic evidence of nephrotoxicosis. However, some of the horses did develop signs of neurotoxicosis, such as ataxia, head shaking, paresthesia, and muscular weakness, but those signs dissipated quickly after discontinuation of PB administration.15
In horses with endotoxemia, systemic administration of PB at a dose of 0.5 to 1.0 mg/kg, IV, every 12 hours, typically results in neutralization of endotoxins without the signs of nephrotoxicosis or neurotoxicosis occasionally observed when the drug is systemically administered at higher bactericidal doses.9,12-15 The 300-mg dose of PB administered by IV-RLP to the horses of experiment 2 was roughly equivalent to the systemic dose (0.6 mg/kg) administered to horses for the treatment of endotoxemia (anti-endotoxin dose). Although the anti-endotoxin dose of PB is lower than the bactericidal dose, it still has some bactericidal activity. For example, the mean serum PB concentration achieved for the horses of experiment 2 at 60 minutes after IV-RLP (ie, 30 minutes after tourniquet removal) was 6 times the MIC90 (< 0.5 to 1.0 μg/mL). Even the lower doses of PB (25 and 50 mg) administered by IV-RLP in experiment 1 resulted in mean serum concentrations of the drug that slightly exceeded the MIC90 at 60 minutes after treatment.
For the 5 horses of experiment 1 that were included in the analysis for the 25-mg dose, the tarsocrural synovial fluid PB concentration was ≥ 10 times the MIC90 (and presumed therapeutic concentration)31,32 for 3 and 4 horses at 30 and 60 minutes, respectively, after administration. We have no explanation as to why the synovial fluid PB concentration was greater at 60 minutes after administration than at 30 minutes after administration for 1 horse. All 3 horses of experiment 1 that were included in the analysis for the 50-mg dose and 4 horses that received the 300-mg dose in experiment 2 had synovial fluid PB concentrations that were ≥ 10 times the MIC90 at 30 and 60 minutes after administration. However, following administration of all 3 doses (25, 50, and 300 mg) of PB evaluated in the present study, the synovial fluid PB concentration was below the limit of quantification at 24 hours after treatment, which suggested that the drug should be administered by IV-RLP on a daily basis.
During experiment 1, the synovial fluid PB concentration failed to reach the study-defined MIC90 for 2 of the 5 horses included in the analysis for the 25-mg dose. This may have been the result of an inadequate dose, loss of PB into the systemic circulation, or a combination of both factors. The doses (25 and 50 mg) of PB administered to the horses of experiment 1 were chosen arbitrarily and were much lower than systemic doses of PB that have been safely administered to the horses of other studies.9,12-15 The goal of treatment with a concentration-dependent antimicrobial is to achieve the highest possible drug concentration in the infected tissue by administering the highest possible nontoxic dose.33 Experiment 2 was performed to determine whether administration of an anti-endotoxin dose (300 mg) of PB by IV-RLP would yield higher synovial fluid concentrations of the drug than those achieved following IV-RLP of PB at doses of 25 and 50 mg. Interestingly, the mean synovial fluid PB concentration at 30 minutes after drug administration for the 50-mg dose (87.3 μg/mL; range, 12.1 to 154 μg/mL; n = 3 horses) was slightly greater than that for the 300-mg dose (74.6 μg/mL; range, 20.4 to 144.3 μg/mL; 4).
Post hoc power analyses (α = 0.05; n = 4 horses/group) revealed that, in experiment 1, the power for detection of significant differences in synovial fluid and serum PB concentrations between the 25- and 50-mg doses was 80% and 13%, respectively. For the 300-mg dose of PB administered in experiment 2, post hoc power analyses revealed that the power for detection of significant differences in synovial fluid and serum PB concentrations between sample acquisition times was 85% and 99%, respectively. Therefore, the present study had sufficient power (> 80%) to detect differences in synovial fluid PB concentrations between the 25- and 50-mg doses and to detect differences in both synovial fluid and serum PB concentrations between 30 and 60 minutes after administration of the 300-mg dose.
Reported synovial antimicrobial concentrations within a targeted joint following IV-RLP vary widely among horses of other studies,34–38 and that variation is attributed to the inability of the tourniquets to keep the perfusate within the vasculature of the targeted region.35,37,39 It was likely that the wide variation in tarsocrural synovial fluid PB concentration observed for the horses of the present study was also caused by failure of the proximal tourniquet to retain the perfusate in the tarsal region.
The type of tourniquet used during IV-RLP has a substantial effect on vascular occlusion and retention of the perfusate within the targeted region.38,40 In experiment 1, we used a pneumatic tourniquet that was inflated to 450 mm Hg as the proximal tourniquet to standardize the vasculature occlusion pressure among horses. In 1 study,40 a pneumatic tourniquet applied proximal to the carpus of horses and inflated to 420 mm Hg was more effective in retaining amikacin sulfate in the metacarpophalangeal joint than a wide or narrow rubber tourniquet. Conversely, results of another study38 indicate that placement of a wide rubber tourniquet around the metacarpus of horses was more effective in retention of amikacin sulfate in the metacarpophalangeal joint than placement of a pneumatic tourniquet at the same site.
Movement of the horse can allow perfusate to escape from the section of a limb isolated by tourniquets.31,41 Sudden shifting of weight by a horse during IV-RLP can more than double the peripheral vascular pressure in the portion of the limb distal to the tourniquet, resulting in an intravascular pressure greater than the pressure provided by the tourniquet, which allows the perfusate to escape into the circulation.31 Two of 3 horses that were excluded from the analysis for the 50-mg dose because the serum PB concentration exceeded the corresponding synovial fluid PB concentration were noted to have moved or shifted their weight multiple times after the IV-RLP, even though the distal portion of the treated limb was desensitized and the horses were heavily sedated. In another study,42 proximal tourniquet failure (as evidenced by lower than expected antebrachiocarpal joint amikacin concentrations) was suspected in 3 of 16 (18.8%) trials in which anesthetized horses were administered amikacin by IV-RLP with a pneumatic tourniquet used as the proximal tourniquet and placed around a forelimb at the midantebrachial level. In experiment 1 of the present study, proximal tourniquet failure was suspected in 3 of 11 (27%) trials. Unlike the horses of that other study,42 the horses of the present study were sedated rather than anesthetized and therefore were more likely to move during the observation period. We used a pneumatic tourniquet for experiment 1 because we wanted to standardize the vasculature occlusion pressure provided by the proximal tourniquet for all trials. Owing to the high proximal tourniquet failure rate of experiment 1, we used a rubber tourniquet as the proximal tourniquet during experiment 2, and none of the trials had to be excluded from the analysis because of proximal tourniquet failure.
Results of the present study indicated that IV-RLP of PB into the distal portion of the saphenous vein of a pelvic limb of sedated horses at doses of 25, 50, and 300 mg resulted in tarsocrural synovial fluid concentrations of the drug that were ≥ 10 times the PB MIC90 (≤ 0.5 to 1.0 μg/mL; presumed therapeutic concentration) for most gram-negative pathogens commonly isolated from equine wounds by 30 minutes after drug administration. However, the sample size for each dose was small and the synovial fluid PB concentration varied substantially among horses, which adversely affected our ability to detect significant differences in drug concentrations among the doses. No adverse systemic or local effects were observed following IV-RLP of PB to the horses of this study. Thus, IV-RLP of PB might be a viable alternative for the treatment of distal limb wound or synovial infections caused by gram-negative bacteria in horses.
Acknowledgments
Supported by the Tennessee Equine Veterinary Research Organization at the University of Tennessee College of Veterinary Medicine, Knoxville, Tenn. The funding source was not involved in the study design, data analysis and interpretation, or writing and publication of the manuscript.
The authors declare that there were no conflicts of interest.
Presented as a poster at the American College of Veterinary Surgery Summit, Phoenix, October 2018.
ABBREVIATIONS
| IV-RLP | IV regional limb perfusion |
| MIC90 | Minimum concentration necessary to inhibit 90% of isolates |
| PB | Polymyxin B |
Footnotes
ADVIA 2120i Hematology System, Siemens Healthcare Diagnostics Inc, Tarrytown, NY.
Cobas c 501 chemistry analyzer, Roche Diagnostics, Indianapolis, Ind.
Equinosis LLC, Columbia, Mo.
Zimmer ATS 2000 Tourniquet System, Medical Products Resource, Eagan, Minn.
Esmarch bandage-tourniquet, Jorgensen Laboratories, Loveland, Colo.
XGen Pharmaceuticals Inc, Horseheads, NY.
Surflo Winged Infusion Set, Terumo Corp, Leuven, Belgium.
JMP, version 10.0.0, SAS Institute Inc, Cary, NC.
References
1. Schneider RK, Bramlage LR, Moore RM, et al. A retrospective study of 192 horses affected with septic arthritis/tenosynovitis. Equine Vet J 1992;24:436–442.
2. Oreff GL, Tatz AJ, Dahan R, et al. Pharmacokinetics of ceftazidime after regional limb perfusion in standing horses. Vet Surg 2017;46:1120–1125.
3. Rubio-Martínez LM, Elmas CR, Black B, et al. Clinical use of antimicrobial regional limb perfusion in horses: 174 cases (1999–2009). J Am Vet Med Assoc 2012;241:1650–1658.
4. Chapman AM. Acute diarrhea in hospitalized horses. Vet Clin North Am Equine Pract 2009;25:363–380.
5. van der Harst MR, Bull S, Laffont CM, et al. Gentamicin nephrotoxicity—a comparison of in vitro findings with in vivo experiments in equines. Vet Res Commun 2005;29:247–261.
6. Gilbertie JM, Schnabel LV, Stefanovski D, et al. Gram-negative multi-drug resistant bacteria influence survival to discharge for horses with septic synovial structures: 206 cases (2010–2015). Vet Microbiol 2018;226:64–73.
7. Gales AC, Jones RN, Sader HS. Contemporary activity of colistin and polymyxin B against a worldwide collection of Gram-negative pathogens: results from the SENTRY Antimicrobial Surveillance Program (2006-09). J Antimicrob Chemother 2011;66:2070–2074.
8. Zavascki AP, Goldani LZ, Li J, et al. Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother 2007;60:1206–1215.
9. Barton MH, Parviainen A, Norton N. Polymyxin B protects horses against induced endotoxaemia in vivo. Equine Vet J 2004;36:397–401.
10. Kwa AL, Lim TP, Low JG, et al. Pharmacokinetics of polymyxin B1 in patients with multidrug-resistant gram-negative bacterial infections. Diagn Microbiol Infect Dis 2008;60:163–167.
11. Landman D, Georgescu C, Martin DA, et al. Polymyxins revisited. Clin Microbiol Rev 2008;21:449–465.
12. Morresey PR, MacKay RJ. Endotoxin-neutralizing activity of polymyxin B in blood after IV administration in horses. Am J Vet Res 2006;67:642–647.
13. Morrison DC, Jacobs DM. Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides. Immunochemistry 1976;13:813–818.
14. Parviainen AK, Barton MH, Norton NN. Evaluation of polymyxin B in an ex vivo model of endotoxemia in horses. Am J Vet Res 2001;62:72–76.
15. Raisbeck MF, Garner HE, Osweiler GD. Effects of polymyxin B on selected features of equine carbohydrate overload. Vet Hum Toxicol 1989;31:422–426.
16. Cheah SE, Bulitta JB, Li J, et al. Development and validation of a liquid chromatography-mass spectrometry assay for polymyxin B in bacterial growth media. J Pharm Biomed Anal 2014;92:177–182.
17. Simar S, Sibley D, Ashcraft D, et al. Colistin and polymyxin B minimal inhibitory concentrations determined by Etest found unreliable for Gram-negative bacilli. Ochsner J 2017;17:239–242.
18. Sanders WE Jr, Sanders CC. Toxicity of antibacterial agents: mechanism of action on mammalian cells. Annu Rev Pharmacol Toxicol 1979;19:53–83.
19. Maddox TW, Clegg PD, Williams NJ, et al. Antimicrobial resistance in bacteria from horses: epidemiology of antimicrobial resistance. Equine Vet J 2015;47:756–765.
20. Lin QY, Tsai YL, Liu MC, et al. Serratia marcescens arn, a PhoP-regulated locus necessary for polymyxin B resistance. Antimicrob Agents Chemother 2014;58:5181–5190.
21. Sud IJ, Feingold DS. Mechanism of polymyxin B resistance in Proteus mirabilis. J Bacteriol 1970;104:289–294.
22. Nation RL, Li J, Cars O, et al. Framework for optimization of the clinical use of colistin and polymyxin B: the Prato polymyxin consensus. Lancet Infect Dis 2015;15:225–234.
23. Storm DR, Rosenthal KS, Swanson PE. Polymyxin and related peptide antibiotics. Annu Rev Biochem 1977;46:723–763.
24. Tran TB, Velkov T, Nation RL, et al. Pharmacokinetics/pharmacodynamics of colistin and polymyxin B: are we there yet? Int J Antimicrob Agents 2016;48:592–597.
25. Evans ME, Feola DJ, Rapp RP. Polymyxin B sulfate and colistin: old antibiotics for emerging multiresistant gram-negative bacteria. Ann Pharmacother 1999;33:960–967.
26. Falagas ME, Kasiakou SK. Toxicity of polymyxins: a systematic review of the evidence from old and recent studies. Crit Care 2006;10:R27.
27. Hermsen ED, Sullivan CJ, Rotschafer JC. Polymyxins: pharmacology, pharmacokinetics, pharmacodynamics, and clinical applications. Infect Dis Clin North Am 2003;17:545–562.
28. Humphries RM. Susceptibility testing of the polymyxins: where are we now? Pharmacotherapy 2015;35:22–27.
29. Li J, Nation RL, Turnidge JD, et al. Colistin: the re-emerging antibiotic for multidrug-resistant Gram-negative bacterial infections. Lancet Infect Dis 2006;6:589–601.
30. Phe K, Lee Y, McDaneld PM, et al. In vitro assessment and multicenter cohort study of comparative nephrotoxicity rates associated with colistimethate versus polymyxin B therapy. Antimicrob Agents Chemother 2014;58:2740–2746.
31. Errico JA, Trumble TN, Bueno AC, et al. Comparison of two indirect techniques for local delivery of a high dose of an antimicrobial in the distal portion of forelimbs of horses. Am J Vet Res 2008;69:334–342.
32. Gunderson BW, Ross GH, Ibrahim KH, et al. What do we really know about antibiotic pharmacodynamics? Pharmacotherapy 2001;21:302S–318S.
33. Moore RD, Lietman PS, Smith CR. Clinical response to aminoglycoside therapy: importance of the ratio of peak concentration to minimal inhibitory concentration. J Infect Dis 1987;155:93–99.
34. Colbath AC, Wittenburg LA, Gold JR, et al. The effects of mepivacaine hydrochloride on antimicrobial activity and mechanical nociceptive threshold during amikacin sulfate regional limb perfusion in the horse. Vet Surg 2016;45:798–803.
35. Hyde RM, Lynch TM, Clark CK, et al. The influence of perfusate volume on antimicrobial concentration in synovial fluid following intravenous regional limb perfusion in the standing horse. Can Vet J 2013;54:363–367.
36. Kelmer G, Bell GC, Martin-Jimenez T. Evaluation of regional limb perfusion with amikacin using the saphenous, cephalic, and palmar digital veins in standing horses. J Vet Pharmacol Ther 2013;36:236–240.
37. Parra-Sanchez A, Lugo J, Boothe DM, et al. Pharmacokinetics and pharmacodynamics of enrofloxicin and a low dose of amikacin administered via regional intravenous limb perfusion in standing horses. Am J Vet Res 2006;67:1687–1695.
38. Alkabes SB, Adams SB, Moore GE, et al. Comparison of two tourniquets and determination of amikacin sulfate concentrations after metacarpophalangeal joint lavage performed simultaneously with intravenous regional limb perfusion in horses. Am J Vet Res 2011;72:613–619.
39. Kilcoyne I, Dechant JE, Nieto JE. Evaluation of 10-minute versus 30-minute tourniquet time for intravenous regional limb perfusion with amikacin sulfate in standing sedated horses. Vet Rec 2016;178:585–588.
40. Levine DG, Epstein KL, Ahern BJ, et al. Efficacy of three tourniquet types for intravenous antimicrobial regional limb perfusion in standing horses. Vet Surg 2010;39:1021–1024.
41. Kelmer G. Regional limb perfusion in horses. Vet Rec 2016;178:581–584.
42. Beccar-Varela AM, Epstein KL, White CL. Effect of experimentally induced synovitis on amikacin concentrations after intravenous regional limb perfusion. Vet Surg 2011;40:891–897.
