Editorial Type:
Pharmacology

Endotoxin-neutralizing activity of polymyxin B in blood after IV administration in horses

Peter R. Morresey Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

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Robert J. MacKay Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

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 BVSc, PhD
DOI:
https://doi.org/10.2460/ajvr.67.4.642
Volume/Issue:
Volume 67: Issue 4
Online Publication Date:
01 Apr 2006
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Abstract

Objectives—To measure serum polymyxin B concentration after single and repeated IV infusions in horses.

Animals—5 healthy horses.

Procedures—In study 1, 1 mg (6,000 U) of polymyxin B/kg was given IV and blood samples were collected for 24 hours. In study 2, 1 mg of polymyxin B/kg was given IV every 8 hours for 5 treatments and blood samples were collected until 24 hours after the last dose. Polymyxin B concentration was measured as the ability to suppress nitrite production by murine macrophages stimulated with lipopolysaccharide and interferon-α. Urine was collected prior to the first drug infusion and 24 hours after the fifth drug infusion for determination of urinary γ-glutamyl transferase (GGT)to-creatinine ratios.

Results—In study 1, mean ± SEM maximal serum polymyxin B concentration was 2.93 ± 0.38 μg/mL. Polymyxin B was undetectable 18 hours after infusion. In study 2, maximal polymyxin B concentrations after the first and fifth doses were 2.98 ± 0.81 μg/mL and 1.91 ± 0.50 μg/mL, respectively. Mean trough concentration for all doses was 0.22 ± 0.01 μg/mL. A significant effect of repeated administration on peak and trough serum concentration was not detected. Urine GGT-to-creatinine ratios were not affected by polymyxin B administration.

Conclusions and Clinical Relevance—Polymyxin B given as multiple infusions to healthy horses by use of this protocol did not accumulate in the vascular compartment and appeared safe. Results support repeated IV use of 1 mg of polymyxin B/kg at 8-hour intervals as treatment for endotoxemia.

Abstract

Objectives—To measure serum polymyxin B concentration after single and repeated IV infusions in horses.

Animals—5 healthy horses.

Procedures—In study 1, 1 mg (6,000 U) of polymyxin B/kg was given IV and blood samples were collected for 24 hours. In study 2, 1 mg of polymyxin B/kg was given IV every 8 hours for 5 treatments and blood samples were collected until 24 hours after the last dose. Polymyxin B concentration was measured as the ability to suppress nitrite production by murine macrophages stimulated with lipopolysaccharide and interferon-α. Urine was collected prior to the first drug infusion and 24 hours after the fifth drug infusion for determination of urinary γ-glutamyl transferase (GGT)to-creatinine ratios.

Results—In study 1, mean ± SEM maximal serum polymyxin B concentration was 2.93 ± 0.38 μg/mL. Polymyxin B was undetectable 18 hours after infusion. In study 2, maximal polymyxin B concentrations after the first and fifth doses were 2.98 ± 0.81 μg/mL and 1.91 ± 0.50 μg/mL, respectively. Mean trough concentration for all doses was 0.22 ± 0.01 μg/mL. A significant effect of repeated administration on peak and trough serum concentration was not detected. Urine GGT-to-creatinine ratios were not affected by polymyxin B administration.

Conclusions and Clinical Relevance—Polymyxin B given as multiple infusions to healthy horses by use of this protocol did not accumulate in the vascular compartment and appeared safe. Results support repeated IV use of 1 mg of polymyxin B/kg at 8-hour intervals as treatment for endotoxemia.

Endotoxin (LPS) is known to be an important mediator of gram-negative bacterial sepsis.1 Endotoxemia in neonatal sepsis, peritonitis, pleuropneumonia, colic, and colitis is associated with high mortality rates in horses.2–4

Lipopolysaccharide is released from the cell wall of gram-negative bacteria via several pathways, including interaction with endogenous serum proteins and as a result of the actions of antibacterial drugs.5 This release of LPS has potentially adverse effects.5,6 Exposure of cells and plasma proteins to LPS initiates inflammatory cascades that lead to production of a multitude of proinflammatory cytokines and mediators.7,8 Widespread immunologic and physiologic changes occur.

Because many of the pathophysiologic features of gram-negative sepsis can be reproduced by experimental administration of purified LPS, it is reasonable to consider that antagonism of LPS activity may result in clinical improvement of affected animals. Substances that have been assessed for therapeutic benefit are of 3 major categories: anti-LPS antibodies, lipid A analogues, and chelating agents.

Monoclonal antilipid A antibodies have been evaluated in humans but do not decrease mortality rate significantly.9 In vitro, anti-lipid A antibodies do not have the ability to neutralize LPS, prevent LPS-induced B-cell proliferation, or inhibit LPS-induced cytokine secretion.10 By contrast, polyclonal antibodies have inconsistent clinical benefit in humans and horses with sepsis.11,12 In 1 study,13 pretreatment with specific antiserum exacerbated the effects of subsequent LPS administration in horses. Lipid A analogues are structurally similar to LPS and have the ability to interfere with LPS-initiated cellular responses.14–16 Such interference occurs by blocking of LPS receptor recognition and depletion of LPS-binding protein.

Polymyxin B sulfate is a basic cationic cyclic polypeptide antimicrobial with a broad range of activity against gram-negative bacteria.17,18 Functioning as a chelating agent, polymyxin B binds the lipid A subunit of LPS avidly in a ratio of 1:1, thereby neutralizing it.19,20 Interaction of LPS with humoral and cellular receptors is thus prevented, and initiation of a proinflammatory cascade is avoided. Recent approaches to sepsis control have concentrated on polymyxin B and synthetic peptides derived from it.21 The lipid A subunit of LPS is largely conserved across all species of gram-negative bacteria and is therefore a logical target for treatment.22 A recent study23 has also revealed the inhibitory effects of polymyxin B on activation of nuclear factor κB, a pivotal transcription factor in the production of proinflammatory cytokines.

Systemic use of polymyxin B has been limited by its toxic effects. Toxicity results from the ability of polymyxin B to bind to phospholipid membranes.24 The resulting cellular disruption can compromise a wide range of organ systems. Clinically, this occurs in horses as neurologic and renal dysfunction.18

Despite these concerns about potential toxicity, polymyxin B has been investigated at dosages of 1,000 to 6,000 U/kg in horses given endotoxin.13,25 In both studies, there was substantial amelioration of endotoxin-induced clinical signs without evidence of renal toxicosis.

Conjugation of polymyxin B to other molecules reduces its toxicity. Experimental preemptive treatment of horses with polymyxin B-dextran 70 conjugate completely blocks LPS-induced changes in heart and respiratory rates; rectal temperature; leukocyte count; and plasma concentrations of TNF-α, interleukin-6, thromboxane B2, and prostaglandin F1α.26 Transient tachypnea, sweating, and increased plasma thromboxane concentration associated with polymyxin B-dextran 70 conjugate usage (with or without experimental LPS challenge) in horses were eliminated by prior treatment with ketoprofen, a nonsteroidal anti-inflammatory drug. Renal function was not affected by polymyxin B-dextran 70 conjugate.

An ex vivo model of the effect of polymyxin B on responses of horses to LPS has been investigated.27 Polymyxin B caused a significant dose- and time-dependent decrease in endotoxin-induced TNF-α activity. Serum creatinine concentrations, monitored as an indication of toxicosis, remained within reference range.

Serum concentration studies of polymyxin B in horses are lacking. The purpose of the study reported here was to measure serum polymyxin B concentration after single and repeated IV infusion in horses.

Materials and Methods

Horses—Five mature horses purchased at regional sales (2 mares, 3 geldings; weight, 340 to 509 kg; age, 2 to 11 years) were used. One was an Arabian, and 4 were Standardbreds. Horses were verified as healthy by use of results of physical examination, CBC, and serum biochemical analyses, before commencement of each part of the study. Horses were regularly dewormed and vaccinated. Prior to beginning the experiments, horses were acclimated to 3.6 × 3.6-m stalls, and free-choice grass hay and water were provided throughout the experiments.

Procedure—Protocols for this study were approved by the University of Florida Animal Care and Use Committee. Horses were monitored continuously for development of neurologic abnormalities. Commercially available polymyxin B sulfatea was reconstituted in sterile saline (0.9% NaCl) solution and used in all experiments.

In study 1, serum concentrations of polymyxin B were investigated after IV administration of a single dose. Catheters were inserted into both jugular veins of each horse, and patency was maintained by regular flushing with heparinized saline solution. All drug infusions were via the left jugular catheter; all blood collections were from the right catheter. Polymyxin B was given IV by infusion pump at a dosage of 1 mg (6,000 U)/kg in 1 L of sterile saline solution during a 15-minute period. Blood samples were collected before infusion and at intervals for 24 hours after completion of infusion. Samples were allowed to clot at ambient temperature (approx 21°C), and serum was separated by centrifugation. Serum samples were stored at −70°C until assayed.

In study 2, serum concentrations of polymyxin B were investigated during 5 successive infusions. At least 2 weeks elapsed between study 1 and study 2 to allow washout of drug. Catheters were inserted into jugular veins and managed as described. For each infusion, polymyxin B was given IV in 1 L of sterile saline solution during a 15-minute period, every 8 hours for 5 treatments. Blood samples were collected before infusion, at intervals for 8 hours after the first infusion was begun, and for 24 hours after the fifth infusion. Samples also were collected immediately before and after doses 2 through 4 were given.

Urine was obtained by sterile catheterization immediately before administration of polymyxin B and 24 hours after completion of the fifth infusion for determination of urinary creatinine concentration and GGT activity. All samples were refrigerated and analyzed within 12 hours of collection. Urinary GGT (U/L)-to-creatinine (mg/dL) ratio was calculated.

Assays—The optimal concentration of LPS to be used in polymyxin B activity assays was determined in preliminary dose-response experiments for each horse. Nitrite concentration was measured in the supernatants of murine macrophage cultures stimulated with LPS and IFN-γ in the presence of preinfusion horse serum. In brief, cells of the J774A.1 murine monocytic line were maintained in Eagle minimal essential medium supplemented with 15mM HEPES, 2mM L-glutamine, 0.1mM nonessential amino acids, streptomycin (100 μg/mL), penicillin G (100 U/mL), and 15% FBS. For each assay, 50 μL of medium that contained 2 × 104 J774A.1 cells in recombinant murine IFN-γb (500 U/mL) and 2% FBS was added to each well of a 96-well tissue-culture plate. After incubation for 2 hours at 37°C in 5% CO2, 50 μL of horse serum was added to each well and the plate was incubated for an additional 30 minutes. Finally, LPSc dilutions were added to 100 μL of medium, and supernatants were removed after 72 hours for assay of nitrite concentration.28 For nitrite assays, nitrite standards were prepared in tissue culture medium and 50 μL of either standards or test supernatants were added to the wells of 96-well tissue culture plates. Next, 50 μL of Greiss reagent (1% sulfanilamide, 0.1% N[-1-napthyl]ethylendiamine dihydrochloride, and 2.5% H3PO4) was added to each well, and plates were incubated for 5 minutes at room temperature (22°C). The absorbance of each well was read at 540 nm on a microplate reader.d Data-capture softwaree was used to calculate supernatant nitrite concentration by interpolation into the standard curve. Data were transferred to a graphic program,f and semilogarithmic plots were produced for nitrite versus LPS concentration. Sigmoidal curves were fit to the data by use of 4-parameter logistic regression.

For each horse, the concentration of LPS to be used in polymyxin B assays was the 90% point on the LPS dose-response curve produced in preinfusion serum from that horse. To quantify polymyxin B activity (ie, LPS-neutralizing activity) in postinfusion serum samples, J774A.1 cells in IFN-γ (500 U/mL) and 2% FBS were prepared and added to wells of 96-well plates and incubated for 2 hours, as described. First, polymyxin B standards (3.16 ng/mL to 3.16 μg/mL) were prepared in preinfusion horse serum and added to wells. Postinfusion serum samples were added in the same way. All samples were assayed both undiluted and diluted 10-fold in preinfusion serum. Plates were incubated for 30 minutes, and LPS was added to 50 μL of medium. Supernatant was removed 72 hours later for nitrite assay. For each horse, a curve was fitted by use of 4-parameter logistic regression to semilogarithmic plots of nitrite concentration versus concentration of polymyxin B standards. The concentration of polymyxin B (μg/mL) in test samples was determined by interpolation into the standard curve. The assay had sensitivity for polymyxin B of approximately 0.01 μg/mL.

Analysis of time-concentration curves—Initial inspection and attempts at curve fitting revealed that the data were too variable for meaningful multiple-component modeling. Therefore, a single-component (first-order) mathematical model was fit to serum log concentration versus time data by use of a noncompartmental pharmacokinetics computer program.g The fitted line was described by the equation Ct = E Xe−g X t where Ct is the serum drug concentration at time t, e is the base of the Naperian logarithm, and E and γ are the intercept and rate constant, respectively. Elimination half-life was calculated as the natural logarithm of 2 divided by γ. Pharmacokinetic values were calculated on the basis of noncompartmental kinetics.29 The AUC and area under the first moment of the concentration-time curve were calculated by use of the trapezoidal rule for 24 hours of observed data with extrapolation to infinity. Mean residence time was calculated as (area under the first moment of the concentration-time curve)/AUC. Apparent volume of distribution based on the AUC was calculated as dose/AUC × γ, and clearance was calculated as dose/AUC. The same software package was also used to estimate steady-state parameters after multiple IV doses.

Statistical analysis—Data did not meet the homogeneity of variance and normal distribution assumptions for ANOVA, so data were analyzed by use of nonparametric tests of significance. Data for peak and trough polymyxin B concentrations after each of 5 infusions were examined by use of Friedman tests. γ-Glutamyltransferase activities before and after polymyxin B infusions were tested for significant differences by use of Wilcoxon signed rank tests. In all instances, P ≤ 0.05 was considered significant.

Results

Sigmoidal semilogarithmic plots were obtained for nitrite concentration versus LPS concentration and nitrite concentration versus concentration of polymyxin B standards in assays of serum antiendotoxic activity (Figures 1 and 2).

Figure 1—
Figure 1—

Nitrite concentration in supernatants of J774A.1 cells stimulated with increasing concentrations of LPS in the presence of serum from a horse. The resulting equation was solved to yield the LPS concentration (100.55 = 3.6 ng/mL) that induced 90% maximal nitrite concentration. This LPS concentration was used in all subsequent assays for polymyxin B concentration in that horse.

Citation: American Journal of Veterinary Research 67, 4; 10.2460/ajvr.67.4.642

Figure 2—
Figure 2—

Standard curve for supernatant nitrite concentration versus polymyxin B concentration in serum of the horse in Figure 1. J774 cells were stimulated with LPS (3.6 ng/mL) and recombinant murine IFN-γ (500 U/mL) in the presence of preinfusion serum. This standard curve was used to calculate the polymyxin B concentration in postinfusion serum samples from that horse.

Citation: American Journal of Veterinary Research 67, 4; 10.2460/ajvr.67.4.642

In study 1, mean ± SEM maximal concentration of polymyxin B was 2.93 ± 0.38 μg/mL at 4 minutes after infusion and decreased to become undetectable by 24 hours (Figure 3). Polymyxin B had a harmonic mean elimination half-life of 3.33 hours and body clearance of 0.14 L/h·kg. From preliminary experiments in vitro, it was estimated that polymyxin B concentration of 0.20 μg/mL or greater was needed to neutralize at least 75% of the nitrite-inducing activity of 1 ng of LPS/mL. On the basis of single-dose results, it was estimated that treatment every 8 hours with polymyxin B at 1 mg (6,000 U)/kg would be sufficient to keep mean serum trough polymyxin B concentration > 0.22 μg/mL.

Figure 3—
Figure 3—

Plot of mean ± SEM log10 serum polymyxin B concentration versus time for 5 horses given 1 mg of polymyxin B/kg by IV infusion during a 15-minute period. Infusion was begun at time 0. Straight line indicates the single-order best-fit line used to calculate pharmacokinetic values.

Citation: American Journal of Veterinary Research 67, 4; 10.2460/ajvr.67.4.642

In study 2, after the first polymyxin B infusion, mean maximal serum concentration of polymyxin B was 2.98 ± 0.81 μg/mL 10 minutes after infusion and decreased to 0.23 ± 0.081 μg/mL 8 hours after beginning the infusion (Figure 4). After the fifth infusion, maximal polymyxin B concentration was 1.91 ± 0.50 μg/mL and decreased to 0.22 ± 0.04 μg/mL at 8 hours. Serum polymyxin B was undetectable by 18 hours after completion of the final infusion.

Figure 4—
Figure 4—

Plot of mean ± SEM log10 serum polymyxin B concentration versus time for 5 horses given 1 mg of polymyxin B/kg by IV infusion during a 15-minute period beginning at 0, 8 16, 24, and 30 hours. Blood samples were obtained at frequent intervals after the first and fifth infusions and immediately before and after the infusions for the second to fourth infusions.

Citation: American Journal of Veterinary Research 67, 4; 10.2460/ajvr.67.4.642

The polymyxin B concentration profile after the fifth infusion did not differ significantly from that for the first infusion. Mean trough concentration was 0.22 ± 0.01 μg/mL, and trough concentrations did not differ significantly among the 5 infusions.

Pre- and postinfusion urinary GGT-to-creatinine ratios were calculated for each horse. This ratio is an earlier and a more sensitive indicator of renal compromise than is an increase in either the serum creatinine concentration or urinary protein-to-creatinine ratio.30 Repeated infusion of polymyxin B did not significantly alter urinary GGT-to-creatinine ratio. Also, urinary GGT-to-creatinine ratio did not exceed 100 U/g for any sample (a value considered to indicate renal tubular necrosis).30,h Signs of neurologic toxicosis from polymyxin B administration were not observed at any time during the experiment.

Discussion

The dose of 1 mg of polymyxin B/kg achieved biologically important antiendotoxic activity in blood for at least 8 hours after administration. Polymyxin B was administered as an infusion during a 15-minute period to approximate the manner in which it is used in clinical situations. With repeated doses at 8-hour intervals, peak and trough concentrations did not change significantly between the first and last dose. Therefore, polymyxin B did not accumulate in the vascular compartment during the 5 infusions.

The polymyxin B dose and treatment interval were chosen to achieve a trough concentration sufficient to block 75% of the nitrite-inducing activity of 1 ng of LPS/mL. A wide range of serum LPS concentrations has been detected in horses referred for colic evaluation, with some exceeding 1 ng/mL.31 Circulating LPS was associated with high heart rate, high PCV, and poor prognosis. Horses are considered to be exquisitely susceptible to the effects of LPS, and circulating concentrations as low as 0.01 ng/mL may be associated with clinical signs of endotoxemia.32 Residual LPS activity in vivo may be beneficial in stimulating an appropriate immune response to enable clearance of the inciting organism or resolution of the initiating event.27 Experimentally, although it improves clinical variables in the short-term, total suppression of inflammatory response to LPS is associated with increased overall mortality rates.33 Dosage of polymyxin B, as in this study, to maintain residual LPS activity may therefore be more rational therapeutically than completely abolishing LPS-induced immune responses.

Polymyxin B provides a readily available, effective, economic means of LPS neutralization, compared with specific antisera. In an experimental endotoxemia model, preemptive treatment of foals with specific Salmonella typhimurium antiserum and polymyxin B (6,000 U/kg) was investigated.13 Compared with LPS given without pretreatment, use of specific antiserum was associated with significantly higher respiratory rate, maximal plasma interleukin-6 activity, and total TNF response. There was no positive effect of specific antiserum on the actions of LPS in the model, and under certain conditions, actions of LPS were exacerbated. In contrast, polymyxin B significantly improved clinical variables in LPS-challenged foals. Similar results were obtained in a more recent study,25 in which polymyxin B at either 1,000 or 5,000 U/kg was given to horses with experimentally induced endotoxemia. Rectal temperature, heart and respiratory rates, and serum TNF were significantly reduced by administration of polymyxin B. At the higher dosage, antiendotoxic effects were apparent even when polymyxin B was given 30 minutes after endotoxin administration was started.

In response to experimentally induced sepsis in mice, treatment with polymyxin B significantly protected against sepsis-associated reduction in mean arterial blood pressure, blood pH, and bicarbonate concentrations, compared with controls.34 When given in conjunction with an LPS challenge, polymyxin B prevents leukopenia and thrombocytopenia.35

Results of the present study were consistent with those of an ex vivo model of polymyxin B usage in horses.27 In that study, LPS-induced TNF activity in blood from horses given polymyxin B decreased in a timeand dose-dependent manner. However, no attempt was made to quantify the pharmacokinetics of polymyxin B after single or multiple doses, as was done in the present study.

Previous investigators have measured available and tissue-bound polymyxin B with a variety of microbiologic and immunologic techniques.35,36 In the present study, active polymyxin B concentration was determined by quantifying its ability to suppress nitrite production by LPS-stimulated macrophages. It would have been useful to compare the profiles generated in this way with concentrations determined by immunoassay.

Pharmacokinetic and drug disposition studies in vivo in horses are lacking. In calves, high concentrations of polymyxin B are found in the liver, kidney, lung, brain, and heart muscle.37 The free form is not detected in the brain. Polymyxin B is not bound in the bile or urine. Analysis of the plasma profile determined that the 3-compartment open model was appropriate to describe of polymyxin B pharmacokinetics because of extensive protein and cell membrane binding. Continuous administration did not result in accumulation in the serum but was associated with high and increasing tissue concentrations. Differences in binding among organs are related to variations in membrane phospholipid content and total membrane mass.38

Systemic use of polymyxin B is limited by its toxicity. Lipid solubility of polymyxin B allows accumulation to a high concentration in the kidneys, chiefly through interaction with proximal tubular membranes. Acute tubular necrosis can result. Renal toxicosis is manifest as a decrease in creatinine clearance and the occurrence of proteinuria and cellular casts.39 In a model of equine carbohydrate overload, doses of 18,000 to 36,000 U/kg did not protect against laminitis but did cause renal compromise, as assessed by use of serum creatinine concentration.18 Neural tissue is also a site of accumulation, with ototoxicosis manifest at the Organ of Corti.40 Neuromuscular blockade and cardiovascular suppression have also been reported.41,42

Systemic administration of polymyxin B in the present study did not result in toxicosis. When it occurs, toxicosis results from cell membrane accumulation in tissues that is not reflected by increased serum concentrations. Polymyxin B is tightly bound, with toxicosis occurring after tissues are saturated. Although polymyxin B did not accumulate in the circulation in horses in the present study, it is likely that tissue binding increases over time; thus, caution must be exercised when using polymyxin B by repeated infusion.

Conjugation of polymyxin B to dextran 70 lessens toxicity and prolongs activity26; however, this product is not yet available commercially. Covalent binding of polymyxin B to human IgG results in nontoxic, dose-dependent protection against death in a murine sepsis model.43 Polymyxin B nonapeptide, a cleavage product of polymyxin B, retains endotoxin-binding activity but is much less potent.44 In clinical situations, the only commercially available form, polymyxin B sulfate (the subject of this experiment), is used.

In humans, nephrotoxicosis and neurotoxicosis associated with the systemic use of polymyxins have a reported frequency of 20.2% and 10%, respectively.45 The occurrence of both conditions is considered to be dose dependent and to resolve with cessation of administration. Inappropriate dose regimens are thought to have contributed to the frequency of toxicosis. The only available human pharmacokinetic data are derived from metabolically unstable patients that were given polymyxin B IM.46 After a 50-mg dose, elimination half-life is 6 hours in healthy humans and 48 to 72 hours in patients with renal compromise, and 60% of the dose is recovered in the urine.47

Although concern about toxicity of polymyxins persists, the rise of multidrug-resistant bacteria has necessitated reexamination of polymyxin use in human medicine.48 Extracorporeal techniques have been developed to avoid toxicosis caused by systemic polymyxin B use. Perfusion of blood from human patients with sepsis through immobilized fibers containing polymyxin B decreases inflammatory mediator concentrations.49,50 Polymyxin B immobilized on inert fibers was completely effective in removing the effects of endotoxin in a perfusion study51 conducted in horses.

Endotoxemia is a complication of many disease processes in horses. Chelation of LPS by polymyxin B may provide a cost-effective means of halting the inflammatory cascade and ameliorating clinical signs. Healthy horses were used in this experiment, in contrast to clinical situations in which polymyxin B might be used. As such, clinical variables affected by LPS (circulating volume, cardiac performance, and renal perfusion) may have a bearing on the pharmacokinetic profile of polymyxin B and should be monitored in any patient receiving this drug. When polymyxin B was given according to the schedule used in the present study, there was no evidence of renal dysfunction or abnormal neurologic signs. On the basis of this and previous research, we propose that polymyxin B can be given to horses with signs of endotoxemia at a dosage of 1 mg (6,000 U)/kg every 8 hours for up to 5 treatments.

ABBREVIATIONS

LPS

Lipopolysaccharide
TNF-α

Tumor necrosis factor-α
GGT

γ-Glutamyltransferase
IFN-γ

Interferon-γ
FBS

Fetal bovine serum
AUC

Area under the time-concentration curve
a.

Polymyxin B sulfate, Bedford Laboratories, Bedford, Ohio.

b.

Recombinant murine γ-interferon, Roche Applied Science, Indianapolis, Ind.

c.

LPS, Escherichia coli O55:B5, Sigma Chemical Co, St Louis, Mo.

d.

Bio-Tek Instruments Inc, Winooski, Vt.

e.

KC3, Bio-Tek Instruments Inc, Winooski, Vt.

f.

SigmaPlot, version 4.0, Jandel Scientific, San Rafael, Calif.

g.

PK solutions, version 2.0, Summit Research Services, Montrose, Colo.

h.

Hinchcliff KW, McGuirk SM, MacWilliams PS. Gentamicin nephrotoxicity (abstr), in Proceedings. 33rd Annu Meet Am Assoc Equine Pract 1987;67.

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    Sanders WE Jr, Sanders CC. Toxicity of antibacterial agents: mechanism of action on mammalian cells. Annu Rev Pharmacol Toxicol 1979; 19: 53–83.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25↑

    Barton MH, Parviainen A, Norton N. Polymyxin B protects horses against induced endotoxaemia in vivo. Equine Vet J 2004; 36: 397–401.

    • Search Google Scholar
    • Export Citation
  • 26↑

    MacKay RJ, Clark CK, Logdberg L, et al. Effect of a conjugate of polymyxin B-dextran 70 in horses with experimentally induced endotoxemia. Am J Vet Res 1999; 60: 68–75.

    • Search Google Scholar
    • Export Citation
  • 27↑

    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.

  • 28↑

    Green SJ. Oxidative metabolism of murine macrophages.. In: Editorial Board, eds. Current protocols in immunology. New York: Greene Publications Associates & Wiley-Interscience, 1998; 14.5.6–14.5.11.

    • Search Google Scholar
    • Export Citation
  • 29↑

    Gibaldi M, Perrier D. Noncompartmental analysis based on statistical moment theory.. In: Gibaldi M, Perrier D, eds. Pharmacokinetics. 2nd ed. New York: Marcel Dekker Inc, 1982; 409–417.

    • Search Google Scholar
    • Export Citation
  • 30↑

    Meyer DS, Harvey JW. Assessment of renal function, urinalysis and water balance.. In: Meyer DS, Harvey JW, eds. Veterinary laboratory medicine. Philadelphia: WB Saunders Co, 1998; 221–235.

    • Search Google Scholar
    • Export Citation
  • 31↑

    King JN, Gerring EL. Detection of endotoxin in cases of equine colic. Vet Rec 1988; 123: 269–271.

  • 32↑

    Mollett TA, Elmore RG, Blanchard TL, et al. Effects of intrauterine infusion of Escherichia coli endotoxin in anestrous and steroid treated pony mares. Theriogenology 1985; 23: 597–606.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33↑

    Jack RS, Fan X, Bernheiden M, et al. Lipopolysaccharide-binding protein is required to combat a murine gram-negative bacterial infection. Nature 1997; 389: 742–745.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34↑

    Flynn PM, Shenep JL, Stokes DC, et al. Polymyxin B moderates acidosis and hypotension in established, experimental gramnegative septicemia. J Infect Dis 1987; 156: 706–712.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35↑

    Kunin CM, Bugg A. Recovery of tissue bound polymyxin B and colistimethate. Proc Soc Exp Biol Med 1971; 137: 786–790.

  • 36

    Danner RL, Joiner KA, Rubin M, et al. Purification, toxicity, and antiendotoxin activity of polymyxin B nonapeptide. Antimicrob Agents Chemother 1989; 33: 1428–1434.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37↑

    Ziv G, Nouws JF, van Ginneken CA. The pharmacokinetics and tissue levels of polymyxin B, colistin and gentamicin in calves. J Vet Pharmacol Ther 1982; 5: 45–58.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38↑

    Kunin CM, Bugg A. Binding of polymyxin antibiotics to tissues: the major determinant of distribution and persistence in the body. J Infect Dis 1971; 124: 394–400.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39↑

    Kunin CM. Nephrotoxicity of antibiotics. JAMA 1967; 202: 204–208.

  • 40↑

    Komiya S, Tachibana M. Target sites of polymyxin B ototoxicity. Eur Arch Otorhinolaryngol 1990; 247: 129–130.

  • 41

    de Silva AJ, Lee C. Neuromuscular and cardiovascular depression produced by prolonged exposure to polymyxin B. Can Anaesth Soc J 1978; 25: 303–306.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42

    Bruckner J, Thomas KC Jr, Bikhazi GB, et al. Neuromuscular drug interactions of clinical importance. Anesth Analg 1980; 59: 678–682.

    • Search Google Scholar
    • Export Citation
  • 43↑

    Drabick JJ, Bhattacharjee AK, Hoover DL, et al. Covalent polymyxin B conjugate with human immunoglobulin G as an antiendotoxin reagent. Antimicrob Agents Chemother 1998; 42: 583–588.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44↑

    Larter WE, Corrigan JJ Jr, Fulginiti VA. In vivo effect of polymyxin B on pertussis vaccine. Am J Dis Child 1984; 138: 281–283.

  • 45↑

    Bosso JA, Liptak CA, Seilheimer DK, et al. Toxicity of colistin in cystic fibrosis patients. DICP 1991; 25: 1168–1170.

  • 46↑

    Hermsen ED, Sullivan CJ, Rotschafer JC. Polymyxins: pharmacology, pharmacokinetics, pharmacodynamics, and clinical applications. Infect Dis Clin North Am 2003; 17: 545–562.

    • Search Google Scholar
    • Export Citation
  • 47↑

    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.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48↑

    Beringer P. The clinical use of colistin in patients with cystic fibrosis. Curr Opin Pulm Med 2001; 7: 434–440.

  • 49

    Nakamura T, Ebihara I, Shoji H, et al. Treatment with polymyxin B-immobilized fiber reduces platelet activation in septic shock patients: decrease in plasma levels of soluble P-selectin, platelet factor 4 and beta-thromboglobulin. Inflamm Res 1999; 48: 171–175.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50

    Tani T, Hanasawa K, Kodama M, et al. Correlation between plasma endotoxin, plasma cytokines, and plasminogen activator inhibitor-1 activities in septic patients. World J Surg 2001; 25: 660–668.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51↑

    Veenman JN, Dujardint CL, Hoek A, et al. High volume continuous venovenous haemofiltration (HV-CVVH) in an equine endotoxaemic shock model. Equine Vet J 2002; 34: 516–522.

    • Search Google Scholar
    • Export Citation

Contributor Notes

Dr. Morresey's present address is Rood and Riddle Equine Hospital, Lexington, KY 40580.

Dr. MacKay.
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
Publication Date:
01 Apr 2006
  • Figure 1—
    View in gallery
    Figure 1—

    Nitrite concentration in supernatants of J774A.1 cells stimulated with increasing concentrations of LPS in the presence of serum from a horse. The resulting equation was solved to yield the LPS concentration (100.55 = 3.6 ng/mL) that induced 90% maximal nitrite concentration. This LPS concentration was used in all subsequent assays for polymyxin B concentration in that horse.

  • Figure 2—
    View in gallery
    Figure 2—

    Standard curve for supernatant nitrite concentration versus polymyxin B concentration in serum of the horse in Figure 1. J774 cells were stimulated with LPS (3.6 ng/mL) and recombinant murine IFN-γ (500 U/mL) in the presence of preinfusion serum. This standard curve was used to calculate the polymyxin B concentration in postinfusion serum samples from that horse.

  • Figure 3—
    View in gallery
    Figure 3—

    Plot of mean ± SEM log10 serum polymyxin B concentration versus time for 5 horses given 1 mg of polymyxin B/kg by IV infusion during a 15-minute period. Infusion was begun at time 0. Straight line indicates the single-order best-fit line used to calculate pharmacokinetic values.

  • Figure 4—
    View in gallery
    Figure 4—

    Plot of mean ± SEM log10 serum polymyxin B concentration versus time for 5 horses given 1 mg of polymyxin B/kg by IV infusion during a 15-minute period beginning at 0, 8 16, 24, and 30 hours. Blood samples were obtained at frequent intervals after the first and fifth infusions and immediately before and after the infusions for the second to fourth infusions.

Figure 1—

Nitrite concentration in supernatants of J774A.1 cells stimulated with increasing concentrations of LPS in the presence of serum from a horse. The resulting equation was solved to yield the LPS concentration (100.55 = 3.6 ng/mL) that induced 90% maximal nitrite concentration. This LPS concentration was used in all subsequent assays for polymyxin B concentration in that horse.
Figure 2—

Standard curve for supernatant nitrite concentration versus polymyxin B concentration in serum of the horse in Figure 1. J774 cells were stimulated with LPS (3.6 ng/mL) and recombinant murine IFN-γ (500 U/mL) in the presence of preinfusion serum. This standard curve was used to calculate the polymyxin B concentration in postinfusion serum samples from that horse.
Figure 3—

Plot of mean ± SEM log10 serum polymyxin B concentration versus time for 5 horses given 1 mg of polymyxin B/kg by IV infusion during a 15-minute period. Infusion was begun at time 0. Straight line indicates the single-order best-fit line used to calculate pharmacokinetic values.
Figure 4—

Plot of mean ± SEM log10 serum polymyxin B concentration versus time for 5 horses given 1 mg of polymyxin B/kg by IV infusion during a 15-minute period beginning at 0, 8 16, 24, and 30 hours. Blood samples were obtained at frequent intervals after the first and fifth infusions and immediately before and after the infusions for the second to fourth infusions.
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    Barton MH, Morris DD, Norton N, et al. Hemostatic and fibrinolytic indices in neonatal foals with presumed septicemia. J Vet Intern Med 1998; 12: 26–35.

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  • 3

    Barton MH, Collatos C. Tumor necrosis factor and interleukin-6 activity and endotoxin concentration in peritoneal fluid and blood of horses with acute abdominal disease. J Vet Intern Med 1999; 13: 457–464.

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  • 13

    Durando MM, MacKay RJ, Linda S, et al. Effects of polymyxin B and Salmonella typhimurium antiserum on horses given endotoxin intravenously. Am J Vet Res 1994; 55: 921–927.

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  • 14

    Aida Y, Kusumoto K, Nakatomi K, et al. An analogue of lipid A and LPS from Rhodobacter sphaeroides inhibits neutrophil responses to LPS by blocking receptor recognition of LPS and by depleting LPS-binding protein in plasma. J Leukoc Biol 1995; 58: 675–682.

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  • 15

    Chase JJ, Kubey W, Dulek MH, et al. Effect of monophosphoryl lipid A on host resistance to bacterial infection. Infect Immun 1986; 53: 711–712.

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    Danner RL, Van Dervort AL, Doerfler ME, et al. Antiendotoxin activity of lipid A analogues: requirements of the chemical structure. Pharm Res 1990; 7: 260–263.

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  • 17

    Storm DR, Rosenthal KS, Swanson PE. Polymyxin and related peptide antibiotics. Annu Rev Biochem 1977; 46: 723–763.

  • 18

    Raisbeck MF, Garner HE, Osweiler GD. Effects of polymyxin B on selected features of equine carbohydrate overload. Vet Hum Toxicol 1989; 31: 422–426.

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  • 19

    Bader J, Teuber M. Action of polymyxin B on bacterial membranes. 1. Binding to the O-antigenic lipopolysaccharide of Salmonella typhimurium. Z Naturforsch [C] 1973; 28: 422–430.

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  • 20

    Morrison DC, Jacobs DM. Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides. Immunochemistry 1976; 13: 813–818.

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  • 21

    Rustici A, Velucchi M, Faggioni R, et al. Molecular mapping and detoxification of the lipid A binding site by synthetic peptides. Science 1993; 259: 361–365.

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  • 22

    Ried C, Wahl C, Miethke T, et al. High affinity endotoxin-binding and neutralizing peptides based on the crystal structure of recombinant Limulus anti-lipopolysaccharide factor. J Biol Chem 1996; 271: 28120–28127.

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  • 23

    Tsuzuki H, Tani T, Ueyama H, et al. Lipopolysaccharide: neutralization by polymyxin B shuts down the signaling pathway of nuclear factor kappaB in peripheral blood mononuclear cells, even during activation. J Surg Res 2001; 100: 127–134.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24

    Sanders WE Jr, Sanders CC. Toxicity of antibacterial agents: mechanism of action on mammalian cells. Annu Rev Pharmacol Toxicol 1979; 19: 53–83.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25

    Barton MH, Parviainen A, Norton N. Polymyxin B protects horses against induced endotoxaemia in vivo. Equine Vet J 2004; 36: 397–401.

    • Search Google Scholar
    • Export Citation
  • 26

    MacKay RJ, Clark CK, Logdberg L, et al. Effect of a conjugate of polymyxin B-dextran 70 in horses with experimentally induced endotoxemia. Am J Vet Res 1999; 60: 68–75.

    • Search Google Scholar
    • Export Citation
  • 27

    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.

  • 28

    Green SJ. Oxidative metabolism of murine macrophages.. In: Editorial Board, eds. Current protocols in immunology. New York: Greene Publications Associates & Wiley-Interscience, 1998; 14.5.6–14.5.11.

    • Search Google Scholar
    • Export Citation
  • 29

    Gibaldi M, Perrier D. Noncompartmental analysis based on statistical moment theory.. In: Gibaldi M, Perrier D, eds. Pharmacokinetics. 2nd ed. New York: Marcel Dekker Inc, 1982; 409–417.

    • Search Google Scholar
    • Export Citation
  • 30

    Meyer DS, Harvey JW. Assessment of renal function, urinalysis and water balance.. In: Meyer DS, Harvey JW, eds. Veterinary laboratory medicine. Philadelphia: WB Saunders Co, 1998; 221–235.

    • Search Google Scholar
    • Export Citation
  • 31

    King JN, Gerring EL. Detection of endotoxin in cases of equine colic. Vet Rec 1988; 123: 269–271.

  • 32

    Mollett TA, Elmore RG, Blanchard TL, et al. Effects of intrauterine infusion of Escherichia coli endotoxin in anestrous and steroid treated pony mares. Theriogenology 1985; 23: 597–606.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33

    Jack RS, Fan X, Bernheiden M, et al. Lipopolysaccharide-binding protein is required to combat a murine gram-negative bacterial infection. Nature 1997; 389: 742–745.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34

    Flynn PM, Shenep JL, Stokes DC, et al. Polymyxin B moderates acidosis and hypotension in established, experimental gramnegative septicemia. J Infect Dis 1987; 156: 706–712.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35

    Kunin CM, Bugg A. Recovery of tissue bound polymyxin B and colistimethate. Proc Soc Exp Biol Med 1971; 137: 786–790.

  • 36

    Danner RL, Joiner KA, Rubin M, et al. Purification, toxicity, and antiendotoxin activity of polymyxin B nonapeptide. Antimicrob Agents Chemother 1989; 33: 1428–1434.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37

    Ziv G, Nouws JF, van Ginneken CA. The pharmacokinetics and tissue levels of polymyxin B, colistin and gentamicin in calves. J Vet Pharmacol Ther 1982; 5: 45–58.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38

    Kunin CM, Bugg A. Binding of polymyxin antibiotics to tissues: the major determinant of distribution and persistence in the body. J Infect Dis 1971; 124: 394–400.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39

    Kunin CM. Nephrotoxicity of antibiotics. JAMA 1967; 202: 204–208.

  • 40

    Komiya S, Tachibana M. Target sites of polymyxin B ototoxicity. Eur Arch Otorhinolaryngol 1990; 247: 129–130.

  • 41

    de Silva AJ, Lee C. Neuromuscular and cardiovascular depression produced by prolonged exposure to polymyxin B. Can Anaesth Soc J 1978; 25: 303–306.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42

    Bruckner J, Thomas KC Jr, Bikhazi GB, et al. Neuromuscular drug interactions of clinical importance. Anesth Analg 1980; 59: 678–682.

    • Search Google Scholar
    • Export Citation
  • 43

    Drabick JJ, Bhattacharjee AK, Hoover DL, et al. Covalent polymyxin B conjugate with human immunoglobulin G as an antiendotoxin reagent. Antimicrob Agents Chemother 1998; 42: 583–588.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44

    Larter WE, Corrigan JJ Jr, Fulginiti VA. In vivo effect of polymyxin B on pertussis vaccine. Am J Dis Child 1984; 138: 281–283.

  • 45

    Bosso JA, Liptak CA, Seilheimer DK, et al. Toxicity of colistin in cystic fibrosis patients. DICP 1991; 25: 1168–1170.

  • 46

    Hermsen ED, Sullivan CJ, Rotschafer JC. Polymyxins: pharmacology, pharmacokinetics, pharmacodynamics, and clinical applications. Infect Dis Clin North Am 2003; 17: 545–562.

    • Search Google Scholar
    • Export Citation
  • 47

    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.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48

    Beringer P. The clinical use of colistin in patients with cystic fibrosis. Curr Opin Pulm Med 2001; 7: 434–440.

  • 49

    Nakamura T, Ebihara I, Shoji H, et al. Treatment with polymyxin B-immobilized fiber reduces platelet activation in septic shock patients: decrease in plasma levels of soluble P-selectin, platelet factor 4 and beta-thromboglobulin. Inflamm Res 1999; 48: 171–175.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50

    Tani T, Hanasawa K, Kodama M, et al. Correlation between plasma endotoxin, plasma cytokines, and plasminogen activator inhibitor-1 activities in septic patients. World J Surg 2001; 25: 660–668.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51

    Veenman JN, Dujardint CL, Hoek A, et al. High volume continuous venovenous haemofiltration (HV-CVVH) in an equine endotoxaemic shock model. Equine Vet J 2002; 34: 516–522.

    • Search Google Scholar
    • Export Citation

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