Increasing tourniquet number has no effect on amikacin concentration within the radiocarpal joint in horses undergoing intravenous regional limb perfusion

Thomas C. Bergstrom William R. Prichard Veterinary Medical Teaching Hospital, University of California-Davis, Davis, CA

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Isabelle Kilcoyne Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA

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K. Gary Magdesian Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA

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Jorge E. Nieto Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA

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Abstract

OBJECTIVE

To determine whether IV regional limb perfusion (IVRLP) performed in the cephalic vein with a wide rubber tourniquet (WRT) applied proximal and distal to the carpus results in a higher peak concentration (Cmax) of amikacin in the radiocarpal joint (RCJ), compared with the Cmax for IVRLP using a single WRT proximal to the carpus.

Animals

7 healthy adult horses.

Procedures

Horses underwent IVRLP using standing sedation with 2 g of amikacin sulfate diluted to 60 mL by use of saline (0.9% NaCl) solution in the cephalic vein with 2 different tourniquet techniques; proximal WRT (P) and proximal and distal WRT (PD). Synovial fluid was collected from the RCJ at 5, 10, 15, 20, 25, and 30 minutes after IVRLP. Tourniquets were removed after the 30-minute sample was collected. Blood samples from the jugular vein were collected at 5, 10, 15, 20, 25, 29, and 31 minutes after IVRLP. Amikacin concentration was quantified by a fluorescence polarization immunoassay. Median peak concentration (Cmax) of amikacin and time to maximum drug concentration (Tmax) within the RCJ were determined.

Results

Median peak concentration in the RCJ was 1331.4 μg/mL with technique P and 683.1 μg/mL with technique PD. Median Tmax occurred at 30 minutes with technique P and 25 minutes with technique PD. No significant (Cmax, P = 0.18; Tmax, P = 0.6) difference in amikacin Cmax or Tmax between techniques was detected.

Clinical Relevance

Placement of 2 WRTs offers no advantage to a single proximal WRT when performing IVRLP to deliver maximal amikacin concentrations to the RCJ using IVRLP.

Abstract

OBJECTIVE

To determine whether IV regional limb perfusion (IVRLP) performed in the cephalic vein with a wide rubber tourniquet (WRT) applied proximal and distal to the carpus results in a higher peak concentration (Cmax) of amikacin in the radiocarpal joint (RCJ), compared with the Cmax for IVRLP using a single WRT proximal to the carpus.

Animals

7 healthy adult horses.

Procedures

Horses underwent IVRLP using standing sedation with 2 g of amikacin sulfate diluted to 60 mL by use of saline (0.9% NaCl) solution in the cephalic vein with 2 different tourniquet techniques; proximal WRT (P) and proximal and distal WRT (PD). Synovial fluid was collected from the RCJ at 5, 10, 15, 20, 25, and 30 minutes after IVRLP. Tourniquets were removed after the 30-minute sample was collected. Blood samples from the jugular vein were collected at 5, 10, 15, 20, 25, 29, and 31 minutes after IVRLP. Amikacin concentration was quantified by a fluorescence polarization immunoassay. Median peak concentration (Cmax) of amikacin and time to maximum drug concentration (Tmax) within the RCJ were determined.

Results

Median peak concentration in the RCJ was 1331.4 μg/mL with technique P and 683.1 μg/mL with technique PD. Median Tmax occurred at 30 minutes with technique P and 25 minutes with technique PD. No significant (Cmax, P = 0.18; Tmax, P = 0.6) difference in amikacin Cmax or Tmax between techniques was detected.

Clinical Relevance

Placement of 2 WRTs offers no advantage to a single proximal WRT when performing IVRLP to deliver maximal amikacin concentrations to the RCJ using IVRLP.

Introduction

Intravenous regional limb perfusion (IVRLP) is a common procedure in equine practice used to deliver high concentrations of an antimicrobial to infected regions in the distal limb.13 This procedure consists of placement of a tourniquet proximal to the region being treated, followed by injection of an antimicrobial mixed with isotonic fluids into the venous circulation distal to the tourniquet.2,4 Increased hydrostatic pressure within the venous vasculature and the antimicrobial concentration gradient in the distal limb are thought to result in diffusion of the antimicrobial into surrounding tissues,57 thus delivering therapeutic concentrations of antimicrobials to the affected area.3,8 The most commonly used antimicrobials in IVRLP are aminoglycosides, but the use of other concentration and time-dependent antimicrobials has been evaluated.3,915 Given its relative ease to perform and ability to achieve therapeutic levels of antimicrobials in infected structures, IVRLP is a cornerstone in the treatment of septic orthopedic conditions of the distal limb.16

Aminoglycosides are concentration-dependent drugs; therefore, a higher ratio of maximum drug concentration (Cmax) to minimum inhibitory concentration (MIC) is associated with a greater bactericidal effect.17,18 In the treatment of gram-negative sepsis in human medicine, the efficacy of systemic aminoglycoside administration is maximized with a Cmax:MIC ratio between 8:1 and 10:1.17,18 The MIC for susceptible pathogens is considered ≤ 4 ug/mL, 8 ug/mL is considered intermediate susceptibility, and ≥ 16 ug/mL is considered resistant for amikacin.19 Therefore, concentrations of 40 μg/mL for susceptible bacteria, 80 μg/mL for intermediate bacteria, and 160 μg/mL for bacteria with an MIC ≤ 16 μg/mL are considered therapeutic. The goal of treatment with aminoglycosides should be optimization of peak concentrations by use of the highest possible nontoxic dose.

Vascular isolation of the area to be perfused is achieved with a tourniquet to compress the blood vessels proximal to the area being perfused. In a clinical setting the most commonly used tourniquet is an Esmarch wide (> 10-cm) rubber tourniquet (WRT).15,16,2022 The effectiveness of the tourniquet is measured by its ability to prevent leakage of the perfusate into the systemic circulation. Many studies7,2024 have examined different variables associated with the IVRLP, including type and width of tourniquet (ie, pneumatic vs rubber), volume of perfusate, effect of general anesthesia, previous exsanguination, and time of tourniquet application to determine the most efficacious way to perform intravenous regional limb perfusions.

Previous studies25,26 have demonstrated that therapeutic levels of amikacin can be achieved in the radiocarpal joint (RCJ) using IVRLP in the cephalic vein. In an effort to increase Cmax in the RCJ, the use of IVRLP with 2 tourniquets to further isolate the carpus from systemic circulation has been evaluated.25 In that study,25 low-volume IVRLP using 1 g of amikacin was performed with a pneumatic tourniquet placed proximal to the carpus and a WRT distal to the carpus. This 2-tourniquet technique resulted in significantly higher amikacin concentrations in the RCJ, compared with results of IVRLP by use of a single proximal pneumatic tourniquet. When the 2-tourniquet technique was used, all horses in the previously mentioned study25 exceeded the target concentration of 160 μg/mL of amikacin 30 minutes after IVRLP. In a field setting, the use of a pneumatic tourniquet may not be practical. To the authors knowledge, the use of 2 WRTs for IVRLP has not been reported in the scientific literature and the effect on Cmax and time to maximum drug concentration (Tmax) has not been evaluated.

The objective of the present study is to determine whether IVRLP performed with 2 WRTs, 1 applied proximal and 1 applied distal to the carpus, would result in higher concentrations of amikacin sulfate in the RCJ, compared with results of IVRLP when a single WRT is applied proximal to the carpus. We hypothesized that IVRLP of the cephalic vein using 2 WRTs would result in a different Cmax and Tmax of amikacin in the RCJ than IVRLP with 1 WRT.

Materials and Methods

Animals

Seven healthy adult horses (5 Thoroughbreds, 1 warmblood, and 1 American Quarter Horse) with a median age of 9 years (range, 6 to 13 years) and median weight of 573 kg (range, 486 to 604 kg) were enrolled in the study. All horses were geldings and owned by the University of California-Davis, Center for Equine Health. The study took place from March 2, 2021, to April 15, 2021. Horses were free from lameness at the walk and had no evidence of vascular abnormalities, carpal joint effusion, or obvious pathology of the forelimb based on physical and lameness examinations. Horses were housed in stalls and monitored during the sampling period and for 24 hours following the last sample collection, particularly in regard to joint swelling, localized swelling at the site of perfusion, or lameness at a walk in a straight line and in circles to the left and right. After this initial period, horses were monitored closely by caretakers for a further 5 days before being turned out to pasture. The protool was approved by the University California-Davis Institutional Animal Care and Use Committee.

Intravenous regional limb perfusion

Horses received IVRLP with 2 g of amikacin sulfate diluted to 60 mL with saline (0.9% NaCl) solution under sedation while standing by use of 1 of 2 techniques: 1) a single proximal WRT (technique P) placed 10 cm proximal to the accessory carpal bone or 2) proximal and distal WRTs (technique PD) with 1 WRT tourniquet placed at the same location as for technique P and 1 additional WRT tourniquet placed at the level of the midmetacarpus.

For each horse, the choice of forelimb and technique was randomized by use of an online randomization tool (Randomness and Integrity Services Ltd). The second technique was performed on the opposite forelimb following a minimum 2-week washout period.

Horses were sedated in standing stocks with a mixture of detomidine hydrochloride (0.01 mg/kg, IV) and butorphanol tartrate (0.01 mg/kg, IV) administered in the left jugular vein. Additional sedation was administered and recorded during the procedure if the horse demonstrated discomfort (shifting weight, lifting the limb, pawing, or walking). Unilateral median, ulnar, and medial cutaneous antebrachial nerve blocks were performed with 30 mL of 2% mepivacaine hydrochloride in total to desensitize the medial aspect of the limb to allow catheter insertion into the cephalic vein and reduce the discomfort of the tourniquets. The selected cephalic vein was clipped and aseptically prepared for injection. The lateral and dorsal aspect of the carpus were also aseptically prepared for synoviocentesis without clipping.

For both techniques a 10-cm WRT (Skylar Instruments) was placed approximately 10 cm proximal to the accessory carpal bone. One roll of gauze was placed over the cephalic vein underneath the tourniquet. The WRT was wrapped at least 10 turns and applied by the same investigator (IK) for each perfusion. For technique PD, in addition to the proximal WRT, a distal WRT was applied in the midmetacarpus with a roll of gauze over the medial and lateral aspect of the limb and was wrapped ten turns by the same investigator (IK).

Following placement of the tourniquets, the IV perfusate was injected through the cephalic vein distal to the proximal tourniquet using a 22 gauge by 2.5-cm catheter (Becton Dickinson Infusion Therapy Systems Inc) over 3 minutes by the same investigator (TCB). The injection of perfusate was manually timed with the use of a stopwatch. After infusion, the catheter was removed and a pressure bandage was placed over the injection site. The tourniquets were removed after the 30-minute synovial fluid sample was obtained. The procedure was then repeated on the contralateral limb with the alternative tourniquet number after the washout period of at least 14 days.

Sample collection

Synovial fluid (0.5 mL) was collected in lithium heparin tubes aseptically by arthrocentesis of the ipsilateral RCJ at 5 (T5), 10 (T10), 15 (T15), 20 (T20), 25 (T25), and 30 (T30) minutes following completion of the perfusion. Joint fluid was collected from the RCJ from the lateral palmar approach. If joint fluid was not obtained from this location, arthrocentesis was performed from the dorsal aspect of the RCJ with the carpus in mild flexion. Additionally, a blood sample (2 to 3 mL) was collected by jugular venepuncture from each horse and placed in a liquid heparin tube prior to IVRLP (T0) and at T5, T10, T15, T20, T25, 1 minute before tourniquet release (T29), and 1 minute following tourniquet release (T31).

Following collection of the last sample, horses were administered 2 g (4 mg/kg) of phenylbutazone IV. A 2- to 3-cm strip of diclofenac ointment was placed over the cephalic vein following IVRLP as described in a previous study.27 A light bandage consisting of soft roll gauze secured with an elastic adhesive bandage (3M) was applied to the carpal region after the final collection of joint fluid and maintained for 24 hours. After 24 hours, horses were examined for lameness and any abnormalities of the cephalic vein following IVRLP. Following that initial period, the horses were closely monitored by facility caretakers for an additional 5 days.

During the IVRLP period, any movements were graded using a previously described system.28 Movements were considered minor movements if there was a decrease in weight bearing that included the hoof maintaining contact with the ground or major movements if the entire hoof lifted off the ground. Major and minor movements were recorded for the 0- to 10-, 10- to 20-, and 20- to 30-minute time periods. Each time the carpus was flexed for synovial fluid collection from the dorsal aspect of the joint, this was recorded as a major movement.

Plasma and synovial fluid analysis

Immediately after completion of sample collection, blood and synovial fluid samples were centrifuged at 1,700 X g for 5 minutes. The supernatant from each sample was harvested and placed in a 1-mL cryotube (Fisher Scientific). Following centrifugation, samples were stored at –80°C until analysis. Amikacin concentrations in each sample were measured using fluorescence polarization immunoassay (Roche Diagnostics GmBH). All analyses were performed at the Biochemical Laboratory of the William R. Pritchard Veterinary Medical Teaching Hospital at the University of California-Davis.

Target concentrations are based on previous research and the Clinical and Laboratory Standards Institute Subcommittee on Veterinary Antimicrobial Susceptibility Testing’s reported MIC for common orthopedic pathogens. The Clinical and Laboratory Standards Institute reports that the MIC for susceptible bacteria is ≤ 4 μg/mL and 8 μg/mL for bacteria with intermediate susceptibility.19 A concentration of aminoglycosides 8 to 10 times the MIC is considered therapeutic.3,17,18 Using this information, the target concentration is ≥ 160 μg/mL for pathogens with an MIC of ≤ 16 μg/mL. To ensure appropriate antimicrobial levels to treat bacterial with an MIC of ≤ 16 μg/mL, 160 μg/mL was chosen as the target concentration.

Data analysis

Data was evaluated for normality with the Shapiro-Wilk test using commercial statistical software (SPSS Statistics version 27.0; IBM Corp). Concentration values were not normally distributed and were log(10) transformed. Data were reported as median, range, mean and 95% CIs. The Cmax and Tmax for each horse was determined for both techniques by visual inspection of median data for the amikacin concentration in synovial fluid. To detect differences between systemic and synovial amikacin concentrations between tourniquet techniques, blood and synovial amikacin concentrations were analyzed by use of 2-way repeated-measures ANOVA (SPSS Statistics for Window version 19.0; IBM Corp). The Cmax and Tmax values were further analyzed with the Wilcoxon signed rank test. Differences in movement between groups were also analyzed using 2-way repeated-measures ANOVA (SPSS Statistics for Window version 19.0; IBM Corp). For all analyses, significance was set at values of P < 0.05.

Results

No systemic or local complications were observed following IVRLP in any of the horses. Median systemic and synovial amikacin sulfate concentrations at the various sampling points were compiled (Tables 1 and 2). With technique P, all horses reached the target minimum synovial concentration (160 μg/mL) necessary to treat infections caused by organisms with an MIC of ≤ 16 μg/mL. With technique PD, 6 of 7 horses reached the target minimum synovial concentration and the seventh horse reached 153.4 μg/mL (Supplementary Table S1). No significant (P = 0.14; power = 0.31) difference in synovial fluid–amikacin concentrations were found between techniques; however, synovial amikacin concentrations were higher in 6 of 7 horses with technique P (Supplementary Table S1). Median plasma concentrations of amikacin did not differ significantly (P = 0.22; power = 0.21) between techniques P and PD at any time point; however, in 6 of 7 horses systemic amikacin concentrations were higher with technique PD when the tourniquet was in place.

Table 1

Median, range, mean, and 95% CI of the mean concentration of amikacin (μg/mL) in synovial fluid samples from the radiocarpal joints of 7 healthy university-owned horses 5 (T5), 10 (T10), 15 (T15), 20 (T20), 25 (T25), and 30 (T30) minutes after cephalic vein IV regional limb perfusion (IVRLP) with the use of a single tourniquet proximal to the carpus (technique P) or the combined use of a tourniquet placed proximal and a tourniquet placed distal to the carpus (technique PD) between March 2, 2021, and April 15, 2021.

Time Technique P synovial concentration of amikacin sulfate (μg/mL) Technique PD synovial concentration of amikacin sulfate (μg/mL)
Median (range) Mean (95% CI) Median (range) Mean (95% CI)
T5 39.5 (3.4–420.2) 129.1 (12.3–245.9) 31.9 (<0.6–188.1) 68.3 (9.1–127.5)
T10 262.3 (22.1–4620.8) 900.0 (0–2134.0) 184.8 (5.6–603.9) 190.7 (38–343.4)
T15 341.6 (36.8–4236.8) 950.7 (0–2050.5) 146.3 (11.8–816.8) 231.0 (22.7–439.3)
T20 420.8 (133.7–915.2) 438.2 (225.7–651.2) 401 (99–1643.4) 681.6 (205.7–1157.5)
T25 435.6 (159.5–985.6) 536.3 (393.0–770.6) 643.5 (69.3–1762.2) 755.4 (247.6–1263.2)
T30 1331.4 (161.7–1990.4) 1208.3 (667.4–1749.2) 683.1 (73.1–1480) 705 (333.3–1076.7
Table 2

Median, range, mean, and 95% CI of the mean plasma concentration of amikacin sulfate (μg/mL) in blood samples obtained from the jugular veins of the horses described in Table 1 before IVRLP (T0) and then at T5, T10, T15, T20, and T25; 1 minute before tourniquet release (T29); and 1 minute after tourniquet release (T31), stratified by technique (P vs PD).

Time Technique P plasma concentration of amikacin sulfate (μg/mL) Technique PD plasma concentration of amikacin sulfate (μg/mL)
Median (range) Mean (95% CI) Median (range) Mean (95% CI)
T5 8.7 (0–23.4) 12.5 (9.9–15.1) 22.5 (7.0–24.7) 18.5 (13.5–23.5)
T10 7.5 (0.7–22.9) 10.1 (4.9–15.3) 19.1 (6.7–21.6) 16.3 (12.2–20.4)
T15 6.8 (1.2–19.3) 9.1 (4.8–13.4) 16.7 (6.2–18.9) 14.4 (11.0–17.8)
T20 6.0 (1.1–17.4) 8.1 (4.3–11.9) 14.7 (5.8–17.5) 13.1 (10.1–16.1)
T25 5.6 (1.5–16.6) 7.6 (4.0–11.2) 13.1 (5.5–16.0) 11.8 (9.1–14.5)
T29 5.2 (1.6–15.2) 7.1 (3.8–10.9) 12.7 (5.3–14.9) 11.2 (8.7–13.7)
T31 15.8 (8.6–22.2) 15.9 (12.1–19.7) 15.4 (8.7–18.5) 14.3 (11.9–16.7)

Median Cmax for technique P was 1,331.4 μg/mL (range, 246.4 to 4620.8 μg/mL) and for technique PD was 683.1 μg/mL (range, 153.4 to 1762.2 μg/mL). Although no statistically significant (P = 0.18) difference in Cmax was detected between techniques P and PD, the Cmax was higher in 6 of 7 horses with technique P than with technique PD. Median Tmax occurred at T30 (range, 10 to 30 minutes) with technique P and at T25 (range, 10 to 30 minutes) with technique PD (P = 0.6). With technique P, Tmax occurred at T30 in 4 of 7 horses (Supplementary Table S1) and the target concentration (160 μg/mL) was reached in 6 of 7 by T15. The seventh horse reached the target concentration by T25. With technique PD, median Tmax occurred at T25 minutes and 6 of 6 horses that reached the target concentration did so by T20.

Movement in horses was not significantly (major movement, P = 0.64; minor movement, P = 0.52) different between techniques P and PD (Supplementary Tables S2 and S3).

Discussion

In the present study, both IVRLP techniques were successfully completed in all horses, and no complications were noted during the postprocedure monitoring period. Contrary to our hypothesis, the results did not demonstrate that median Cmax is different between technique P (1331.4 μg/mL) and technique PD (683.1 μg/mL; P = 0.18). Interestingly, technique P resulted in lower systemic amikacin levels achieved in 6 of 7 horses than those achieved with technique PD, suggesting less systemic amikacin leakage when using 1 tourniquet resulting in more amikacin available to diffuse into the synovial structures. We hypothesize that increased hydrostatic pressure within the venous vasculature caused by the presence of 2 tourniquets resulted in maximum venous pressure at the proximal WRT, leading to tourniquet failure and subsequent leakage of amikacin into the systemic circulation. Maximum hydrostatic pressure and subsequent leakage of perfusate under the tourniquet have been previously suggested to cause decreased delivery of therapeutic medication to synovial structures in both human and equine medicine.29,30 Movement of the limb due to increased noxious stimulus from 2 tourniquets is another possible explanation, as movement has been suggested to result in tourniquet failure.20,28,31 However, the difference was not statistically significant (major movement, P = 0.64; minor movement P = 0.52) in major or minor movement between techniques P and PD, indicating the presence of 2 tourniquets was not more painful or resulted in more movement than the use of 1 tourniquet.

The median Tmax for technique PD occurred slightly earlier at T25, compared with technique P at T30. This finding was consistent with our hypothesis, although the difference was not statistically significant (P = 0.6). A previous study26 evaluated Tmax in the RCJ following IVRLP with 1 tourniquet and found mean Tmax occurred at 15 minutes (range, 10 to 20 minutes). Contrary to these findings, the present study found that with technique P, the Tmax occurred at T30 in 4 of 7 horses. The reason for the prolonged Tmax with technique P is unclear, but 6 of 7 horses reached the target concentration (160 μg/mL) by T15 and the seventh horse reached the target concentration by T25. With technique PD, the median Tmax occurred at T25 minutes, and all 6 horses that reached the target concentration of 160 μg/mL did so by T20. Other studies21,32 have found Tmax in the tarsocrural joint occurred at 25 minutes and at 15 minutes in the distal interphalangeal joint. However, in the latter study,21 the tourniquet was removed at 20 minutes, meaning Tmax may have occurred at a later time if the tourniquet had been left in place for longer. Regardless, the data from the present study provide further evidence that when performing IVRLP to treat the RCJ, the tourniquet should be maintained in place for 20 minutes at least if possible.

Although technique P resulted in greater synovial amikacin concentrations than technique PD in 6 of 7 horses, our results were not statistically significant (P = 0.14). Our statistical power was low for this comparison (0.31) due to a small group size and a large SD. It is possible a type II error (failure to detect a significant difference) exists. Similar to previous studies,79,21,2426,28,29 the present study demonstrated a wide variability in synovial amikacin concentrations achieved using IVRLP. This variability is likely due to a number of factors, including differences in individual patient drug metabolism, different amikacin dose-to-body weight ratios, and leakage of amikacin around the WRTs. To minimize these effects, all horses were of similar age, breed, and size, and the same investigators applied the WRTs and performed the perfusions. Despite attempts to control for these factors, there were large variations in the synovial fluid–amikacin concentrations. Regardless of the wide variations in synovial amikacin concentrations and Cmax, the findings of the study suggest that technique PD offers no advantage to technique P when attempting to maximize Cmax. More research is warranted to fully evaluate the individual differences in horses that create such wide variations in Cmax to provide data that can be used to more effectively produce consistent results following IVRLP.

In a clinical setting, the high levels of variability in synovial amikacin concentrations following IVRLP are problematic for numerous reasons. First, therapeutic drug monitoring of synovial fluid is not feasible in equine patients; therefore, synovial amikacin concentration and success of the IVRLP procedures must be based on clinical judgment. Second, recent research has demonstrated that amikacin has a dose-dependent cytotoxic effect in vitro at concentrations of 0.8 mg/mL (800 μg/mL) for synoviocytes and 0.31 mg/mL (310 μg/mL) for chondrocytes.33 However, the present studies33 evaluating the cytotoxicity of amikacin are performed in vitro, and it is not known whether there is significant penetration of amikacin into articular cartilage in vivo. Due to potential detrimental effects of amikacin in the joint, it is possible that IVRLP, compared with intra-articular injections, may result in fewer detrimental effects since the concentrations are lower. Again, more research is necessary to determine whether either intra-articular injection or IVRLP with amikacin has a more detrimental effect on articular cartilage in vivo.33

Previous studies20,25 have evaluated the success of IVRLP with a different tourniquet type and position on the limb. To the authors knowledge, only 1 previous study25 has evaluated the effect of tourniquet number on amikacin concentrations and found significantly higher amikacin concentrations in the RCJ using 2 tourniquets. In that study,25 a proximal pneumatic tourniquet and distal WRT were used with a low volume perfusate (6 mL) to perform IVRLP, and all horses exceeded the target therapeutic concentration of 160 μg/mL. The present study used a high volume (60 mL) with 2 WRTs, and 6 of 7 horses reached amikacin levels above 160 μg/mL by the end of the 30-minute sampling period. In that 2017 study,25 only 1 g of amikacin was used to achieve amikacin synovial concentrations (range, 217 to 1,070 μg/mL) similar to those in the present study (153.4 to 1,762.2 μg/mL). It is plausible that using a lower volume with the 2-tourniquet IVRLP technique may result in less systemic leakage and higher synovial fluid–amikacin concentrations, compared with results in the present study.

Interestingly, higher synovial concentrations in that study25 were noted at the 30-minute time point, compared with those at the 15-minute time point, indicating Cmax occurred later, similar to the present study. It should be noted, however, that differences in tourniquet type (pneumatic vs WRT) and sampling time points between studies make direct comparisons difficult.

The Cmax:MIC ratio has long been regarded as the primary pharmacokinetic-pharmacodynamic index of clinical efficacy for aminoglycosides due to their concentration-dependent killing and has been frequently used when studying9,10,21,26 intravenous regional limb perfusion with amikacin in horses. However, it has been recently been reported in the literature34,35 that area under the plasma concentration-versus-time curve (AUC):MIC ratio may be a more reliable indicator of bacterial killing and clinical efficacy for these agents. Evaluation of available literature suggests that an AUC/MIC ratio of 30 to 50 for aminoglycoside therapy may be optimal when targeting noncritically ill immunocompetent patients with low-bacterial gram-negative burden; however, an AUC:MIC ratio target of 80 to 100 may be more prudent when treating critically ill patients with high-bacterial burden infections. Whether Cmax:MIC or AUC:MIC ratios are better predictors of efficacy also likely varies with the specific bacterial agent. How this would apply to a 30-minute regional limb perfusion, as compared with AUC for plasma concentrations of aminoglycosides over 24 hours, requires study. In addition, only the AUC from time point 5 through 30 minutes were calculated as a 0 time point was not obtained, and synovial fluid was not sampled beyond the 30-minute time point. Since amikacin concentrations persist in the synovial fluid for up to 36 hours36 and we measured synovial amikacin concentrations for up to 30 minutes, the AUC would be markedly underestimated. Finally, the much higher concentrations of amikacin achieved with RLP, compared with results for systemic administration, are likely associated with unique and not well-studied pharmacodynamics. Whether Cmax:MIC or AUC:MIC ratios are optimal for RLP remains to be determined.

Limitations of this study include the small sample size in combination with large SDs in the data, resulting in a low statistical power. This small sample size increased the difficulty in identifying statistically significant differences in the Cmax and synovial amikacin concentrations with technique P and PD. Another limitation is that IVRLP was performed manually over 3 minutes with a stopwatch. To further standardize the instillation of the perfusate and ensure that the amount of systemic amikacin lost was minimized, a syringe pump could have been used for the antibiotic infusion.21,32 Another limitation is the effect of repeated arthrocentesis and resulting blood contamination of synovial amikacin concentrations. It is conceivable that with repeated arthrocentesis, trauma to the joint results in blood contamination of the synovial fluid, which may alter the concentration of amikacin present. The authors are unaware of studies evaluating the effect of repeated arthrocentesis and subsequent hemarthrosis on synovial amikacin concentrations during IVRLP. Future studies could include RBCs to try and ascertain whether blood contamination does indeed affect the synovial concentrations achieved. Another limitation of this study is that AUC was not calculated because we did not measure synovial concentrations beyond the 30-minute time point. Finally, the development of a pharmacokinetics-pharmacodynamics model to analyze all available data may have helped evaluate the effect of subject and interoccasion variability.

The findings of this study indicated that a high-volume IVRLP with technique PD does not increase Cmax in the RCJ when compared with technique P. Additionally, there is more systemic leakage with technique PD, which likely contributed to Cmax being approximately half that with technique P. In conclusion, the present study demonstrates that the use of 2 tourniquets for high-volume IVRLP in the cephalic vein, in comparison to IVRLP with a single tourniquet proximal to the carpus, reduces the Cmax of amikacin sulfate in the RCJ and only marginally reduces the Tmax of amikacin sulfate in the RCJ. In clinical practice, the use of 1 proximally applied WRT is sufficient for completion of IVRLP.

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org

Acknowledgments

Supported by the Center for Equine Health with funds provided by the State of California pari-mutuel fund and contributions by private donors. The authors declare that there were no conflicts of interest.

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