Introduction
In horses, traumatic wounds to the limbs are common, frequently involve synovial structures, and can limit the athletic career and life of the animal.1,2,3 For horses with a wound at the distal aspect of a limb (distal limb), antimicrobial administration by regional limb perfusion is simple and allows therapeutic drug concentrations to be achieved locally with minimal risk for adverse systemic effects.4,5,6,7
Administration of a drug by regional limb perfusion requires use of a tourniquet to compress the blood vessels proximal to the area being infused to slow distribution of the perfusate into the systemic circulation and away from the target region. An Esmarch tourniquet with a width > 10 cm is the most commonly used type of tourniquet in clinical equine practice.4,5,6,7,8 The effectiveness of a tourniquet is measured by its ability to prevent leakage of the perfusate into the systemic circulation. Multiple studies have been conducted to evaluate the most effective method to perform IVRLP in standing horses and have focused on tourniquet type (ie, pneumatic vs rubber) and width,8 site of injection,9,10 volume of perfusate,11,12 and the effect of exsanguination of the target region prior to drug injection.13 In horses, therapeutic antimicrobial concentrations in synovial structures of the distal limb have been achieved following IVRLP of antimicrobials with the tourniquet left in place for 25 to 30 minutes.5,8,14,15,16
Aminoglycosides are concentration-dependent antimicrobials; thus, their bactericidal efficacy increases as the Cmax:MIC ratio increases.5 In human patients with gram-negative sepsis, the efficacy of systemic aminoglycoside administration is maximized when a Cmax:MIC ratio between 8:1 and 10:1 is achieved.17,18 Successful treatment of infections caused by pathogens with high aminoglycoside MICs requires attaining high drug concentrations at the site of infection.19 Therefore, the goal of aminoglycoside administration is to optimize the Cmax of the drug at the site of infection by use of the highest possible nontoxic dose.4,18
In a 2018 study,20 it took 15 minutes for amikacin to reach its Cmax in the synovial fluid of the DIPJ of horses following IVRLP of the drug into the ipsilateral cephalic vein, and the Cmax did not increase beyond 15 minutes despite maintenance of the tourniquet. Shortening the duration that the tourniquet is left in place following IVRLP might decrease the discomfort for the horse and the extent of sedation required to perform the procedure in a standing animal.
In many studies11,15,20,21,22,23 involving IVRLP of drugs to horses, the perfusate was injected into the cephalic vein of a forelimb. In only a few studies7,9,10,24,25 was the perfusate injected into the saphenous vein of a hind limb. The purpose of the study reported here was to determine the tmax of amikacin in the synovial fluid of the tarsocrural joint following IVRLP of 2 g of the drug into the saphenous vein. We hypothesized that the tmax of amikacin in the tarsocrural joint would be < 30 minutes, which is the usual duration that a tourniquet is left in place following IVRLP in clinical practice.
Materials and Methods
Animals
The study protocol was reviewed and approved by the University of California-Davis Institutional Animal Care and Use Committee. Seven horses (3 Thoroughbreds and 4 Quarter Horses) were selected for study enrollment from the research herd at the University of California-Davis Center for Equine Health. The ages of the horses ranged from 7 to 14 years (mean, 10 years), and body weights ranged from 534 to 646 kg (mean, 563 kg). The horses were not lame when observed at a walk and had no evidence of vascular abnormalities, tarsocrural joint effusion, or obvious lesions of the hind limbs as determined on the basis of results of physical and lameness examinations. Horses were individually housed in stalls (approx 3.5 × 3.5 m) and monitored twice daily during the sampling period and for 1 day after collection of the last sample for evidence of joint swelling, localized swelling and sensitivity at the injection site, and lameness when walked in a straight line and in circles to the left and right. Following that initial period, the horses were closely monitored by facility caretakers for an additional 5 days.
IVRLP
For each horse, a hind limb was randomly selected for IVRLP by use of an online randomization tool.a The horse was sedated with detomidine hydrochlorideb (0.01 mg/kg, IV) and butorphanol tartratec (0.01 mg/kg, IV). In the hind limb designated for IVRLP, a tibial nerve block was performed as described26 with 20 mL of 2% mepivacaine hydrochlorided to reduce the discomfort associated with tourniquet application and arthrocentesis for synovial fluid sample collection. Additional sedation was administered if the horse exhibited signs of discomfort (shifting of weight, lifting of the limb, pawing, or trying to walk away) during the IVRLP procedure.
For the hind limb designated for IVRLP, hair was clipped from the skin overlying and surrounding the saphenous vein, and the skin was aseptically prepared for injection. A 10-cm-wide Esmarch tourniquet was applied to the limb 10 cm proximal to the point of the tarsus (hock) with a roll of gauze placed over the saphenous vein underneath the tourniquet. To standardize tourniquet placement, the same investigator (JEN) applied the tourniquet to all horses. A 22-gauge, 2.5-cm-long cathetere was placed in the saphenous vein below the tourniquet. Two grams of amikacin sulfatef was diluted with physiologic saline (0.9% NaCl) solution to yield a volume of 60 mL, which was infused over 3 minutes through the catheter in the saphenous vein by means of extension tubing and a peristaltic pump.g After the infusion was completed, the catheter was removed, and a pressure bandage was placed over the catheter site. The tourniquet was removed approximately 30 minutes later after the last tarsocrural synovial fluid sample was collected.
Sample collection
From each horse, a blood sample (approx 2 to 3 mL) was collected by jugular venipuncture into a blood collection tube that contained lithium heparin (lithium heparin tubes) as an anticoagulant at 5 (T5), 10 (T10), 15 (T15), 20 (T20), 25 (T25), 29 (T29; ie, 1 minute before tourniquet release), and 31 (T31; ie, 1 minute after tourniquet release) minutes after completion of the IVRLP to assess tourniquet efficacy. Additionally, a synovial fluid sample (0.5 mL) was aseptically collected from the tarsocrural joint by arthrocentesis via a dorsal approach either lateral or medial to the saphenous vein (depending on which side the joint pouch was most discernable) at T5, T10, T15, T20, and T25 and 30 minutes after completion of the IVRLP (T30). Synovial fluid samples were placed into lithium heparin tubes immediately after collection. A light bandage consisting of soft roll gauze secured with elastic tape was applied around the hock immediately after collection of the T30 synovial fluid sample and was removed 24 hours later. Each horse was administered phenylbutazoneh (2 g [approx 4 mg/kg], IV) after sample collection was completed.
Plasma and synovial fluid analysis
Immediately after completion of sample collection, all blood and synovial fluid samples were centrifuged at 1,700 × g for 5 minutes. The supernate from each sample was harvested, placed in a plastic cryotube, and stored frozen at −80°C until analysis. The amikacin concentration in each sample was determined by use of a fluorescence polarization immunoassayi (kinetic interaction of microparticles in solution as measured by changes in light transmission) as described.12 All analyses were performed at the Biochemical Laboratory of the William R. Pritchard Veterinary Medical Teaching Hospital at the University of California-Davis.
Data analysis
For each horse, the amikacin concentrations in both plasma and synovial fluid were plotted over time, and the Cmax and tmax were determined by visual evaluation of those plots. The Cmax and tmax for amikacin in plasma and synovial fluid were summarized for the study population and reported as the median (range).
Results
No complications were observed following the IVRLP injections and serial arthrocenteses or in association with tourniquet application for 30 minutes. One horse was excluded from all data calculations because we were unable to collect an adequate volume of synovial fluid for analysis (at least 0.3 mL) from the tarsocrural joint at multiple sample collection times. We were unable to attain an adequate volume of synovial fluid for analysis at 1 sample collection time from 2 other horses (at T5 for one horse and T25 for the other). Thus, the median values for T5 and T25 represented data from only 5 horses.
The median tmax was 25 minutes (range, 20 to 30 minutes), and the median Cmax was 450.5 µg/mL (range, 304.7 to 930.7 µg/mL). The median (range) amikacin concentrations in plasma and synovial fluid samples over time were summarized (Table 1). The synovial fluid amikacin concentration was ≥ 160 µg/mL (ie, 10 times the amikacin MIC [16 µg/mL] for many fairly resistant equine pathogens; therapeutic concentration) at T20 in all horses. All 6 horses included in the analyses had detectable (albeit low) concentrations of amikacin in the systemic circulation prior to tourniquet removal at T30.
Median (range) amikacin concentration in plasma and tarsocrural synovial fluid samples over time following IVRLP of 2 g of the drug into the saphenous vein of a randomly selected hind limb for 6 healthy nonlame adult horses.
| Time after completion of IVRLP (min) | Plasma amikacin concentration (μg/mL) | Synovial fluid amikacin concentration (μg/mL) |
|---|---|---|
| 5 | 1.3 (0–3.8) | 99 (5.1–210)* |
| 10 | 2.2 (0–4.2) | 104.5 (32.7–400.8) |
| 15 | 2.1 (0.6–3.7) | 190.3 (80.3–310.8) |
| 20 | 3.1 (1–7.3) | 366.3 (160–930.7) |
| 25 | 3.2 (0.9–7.4) | 392.7 (304.7–716.1)* |
| 29 | 3 (0.8–6.4) | — |
| 30 | — | 403.7 (284.9–722.4) |
| 31 | 9.2 (0.9–19.9) | — |
Represents the median (range) for only 5 horses because an adequate volume of synovial fluid for analysis could not be obtained from 1 horse at this sample acquisition time.
— = Not calculated because this sample type was not collected at this time.
Discussion
Results of the present study indicated that the median tmax for amikacin in the tarsocrural joint of horses following IVRLP was 25 minutes (range, 20 to 30 minutes). The median synovial fluid amikacin concentration did not increase substantially between T25 (392.7 µg/mL) and T30 (403.7 µg/mL), which suggested that tourniquet application for 25 minutes after completion of IVRLP was sufficient for the Cmax of amikacin to be achieved in the tarsocrural joint and supported the current recommendation to leave the tourniquet in place for 20 to 30 minutes after IVRLP in horses.
Most studies8,9,13,20,21,27 that evaluated IVRLP of amikacin in the distal limbs of horses involved injection of the drug into the cephalic vein of a forelimb, and only a few studies7,9,24,25 involved injection of the drug into the saphenous vein of a hind limb. In one of those studies,9 the synovial fluid amikacin concentration (363 µg/mL) achieved in the distal hind limb 30 minutes after pneumatic tourniquet application and injection of the drug into the saphenous vein was 91 times the amikacin MIC (4 µg/mL) for common susceptible equine bacterial pathogens. However, synovial fluid samples were not collected prior to 30 minutes after IVRLP in that study,9 and the amikacin Cmax may have occurred prior to that sampling time. In another study25 in which IVRLP was performed in anesthetized (ie, laterally recumbent) horses, the mean Cmax (701.8 µg/mL) for amikacin in the tibiotarsal joint was achieved at the completion of the infusion, which was approximately 30 minutes after it was begun (ie, the tmax was approx 30 minutes).
In the present study, the median Cmax and tmax for amikacin in tarsocrural synovial fluid samples were 450.5 µg/mL and 25 minutes, respectively, following IVRLP of the drug in the saphenous vein of standing horses. The Cmax for amikacin in synovial fluid was ≥ 160 µg/mL (ie, therapeutic concentration) at T20 in all horses. That finding suggested that, in most horses, leaving the tourniquet in place for 20 minutes after IVRLP may be sufficient to achieve adequate concentrations of amikacin in the tarsocrural joint for effective treatment of many common, more resistant bacterial pathogens. Limiting the amikacin concentration in synovial tissues may be clinically important. Results of a recent in vitro study28 suggest that amikacin has a dose-dependent cytotoxic effect on equine articular cells. In that study,28 the dose of amikacin that resulted in the death of 50% of cells in culture was 0.8 mg/mL (800 µg/mL) for synoviocytes and 0.31 mg/mL (310 µg/mL) for chondrocytes. Those doses fall within the amikacin Cmax range (304.7 to 930.7 µg/mL) observed for the horses of the present study. Although further in vivo research is necessary, it is possible that the amikacin concentration achieved in the distal limb joints of horses following IVRLP may be toxic to articular cells. Factors that warrant further investigation include the extent of amikacin penetration into articular cartilage in vivo and whether the adverse effects of amikacin differ substantially between healthy and diseased cartilage. Until the potential detrimental effects of amikacin on in vivo articular tissues have been elucidated, it may be advisable to administer the drug by IVRLP rather than by intra-articular injection because IVRLP results in lower drug concentrations in the joint.
The Cmax of amikacin in the tarsocrural synovial fluid varied substantially among the horses of the present study, and that finding was consistent with results of other studies11,15,20,21,22 in which amikacin was administered by regional limb perfusion in horses. The variation in the synovial fluid amikacin Cmax for the horses of the present study might have been caused by a loss of perfusate from the target region owing to variations in the position of and pressure applied by the tourniquet or differences in the amikacin dose-to-body weight ratio among horses. Although the same investigator applied the tourniquet to all horses, some variation in placement among horses was unavoidable, which could have altered blood flow and amikacin distribution in the tissues of the target region. We chose to use a 10-cm-wide Esmarch (rubber) tourniquet in the present study because tourniquets of that width and type are commonly used in clinical practice. All horses of the present study had detectable concentrations of amikacin in the systemic circulation prior to removal of the tourniquet. In another study20 in which amikacin was administered by IVRLP in a cephalic vein, 5 of 7 horses had detectable concentrations of the drug in the systemic circulation prior to tourniquet removal, which was attributed to partial failure of the tourniquet. Interestingly, the 2 horses of that study20 that had undetectable systemic amikacin concentrations prior to tourniquet removal (ie, the horses without partial tourniquet failure) had the highest amikacin Cmax in synovial fluid samples obtained from the DIPJ. We speculated that anatomic differences between the forelimbs and hind limbs of horses, such as the larger diameter and more conical shape of the upper portion of the hind limb, might make it challenging to completely occlude blood flow from the distal hind limb with tourniquet placement, resulting in low and variable drug concentrations in the systemic circulation following IVRLP.
For the horses of the present study, we chose to use a peristaltic pump to instill the amikacin perfusate over 3 minutes in an attempt to limit the infusion-induced increase in hydrostatic pressure within the saphenous vein immediately distal to the tourniquet, thereby decreasing the amount of drug that leaked into the systemic circulation and improving drug distribution into the tarsocrural joint (ie, maximize the amikacin Cmax in synovial fluid). We diluted the amikacin with physiologic saline solution to yield a perfusate volume of 60 mL because that is the volume of perfusate routinely used for IVRLP in our hospital. The therapeutic concentration of amikacin (160 µg/mL) was achieved in the synovial fluid for all horses of the present study, compared with only 3 of 7 horses11 and 6 of 7 horses20 of other studies in which the same perfusate volume of amikacin was administered by IVRLP in a cephalic vein. However, it is important to note that synovial fluid samples were obtained from the metacarpophalangeal joint11 and DIPJ20 for the horses of those other studies and the tarsocrural joint for the horses of the present study, so the results of the 3 studies are not directly comparable.
It may not be common for a peristaltic pump to be used for drug installation during IVRLP in clinical practice. We used a peristaltic pump rather than manual drug instillation in the present study to minimize the potential for human error and variability in amikacin infusion. The fact that adequate concentrations of amikacin were achieved in synovial fluid samples following use of a peristaltic pump for IVRLP in the present study, as well as a previous study20 conducted in our laboratory, emphasized the importance of instilling the drug slowly over 2 to 3 minutes during IVRLP to minimize hydrostatic pressure in the vessel immediately distal to the tourniquet and leakage of the drug into the systemic circulation.
Measurement of the amikacin concentration in the synovial fluid of the tarsocrural joint following IVRLP of the drug might not be representative of the amikacin concentration achieved in the other joints of the distal hind limb. In multiple pharmacokinetic studies,5,9,16,22 synovial drug concentrations varied among joints of the distal limbs of horses following regional perfusion of an antimicrobial. Thus, for the horses of the present study, the amikacin concentration achieved in the other joints of the distal hind limb might have been similar to or perhaps greater than that observed in the tarsocrural joint.
The present study was not without limitations. The study population was small, and there was substantial variability among horses in regard to the amikacin concentration in both plasma and synovial fluid samples. Enrollment of a larger number of horses in the study might have offset or minimized some of the variation in the amikacin concentration data. Additionally, all horses evaluated in this study were healthy with no evidence of hind limb lameness or joint disease; therefore, the findings may not accurately reflect the Cmax and tmax of amikacin achieved in the plasma or tarsocrural synovial fluid of horses with inflammatory conditions such as synovial sepsis or trauma. Results of another study15 suggest that the pharmacokinetics and pharmacodynamics of amikacin differ between healthy and inflamed joints, with inflammation resulting in an increase in Cmax and decrease in tmax. Difficulty collecting an adequate volume of synovial fluid for analysis from the tarsocrural joint was another limitation of the present study. Data from 1 horse were excluded from all calculations because we were unable to collect an adequate volume of synovial fluid for analysis at multiple sample acquisition times. An inadequate volume of synovial fluid for analysis was obtained at T5 for 1 horse and at T25 for 1 horse, which decreased the number of horses that contributed to the median synovial fluid values at those times. We chose to collect synovial fluid samples from the tarsocrural joint because it was deemed safer than collection of samples from more distal joints, the samples could be obtained without manipulating the limb, and any movement during arthrocentesis would not affect the efficacy of the tourniquet.
Findings of the present study indicated that, for healthy adult horses, leaving the tourniquet in place for 20 to 25 minutes after IVRLP of amikacin into a saphenous vein was sufficient to allow peak drug concentrations to be achieved in the tarsocrural joint. The median Cmax of amikacin achieved in the tarsocrural synovial fluid samples (450.5 µg/mL; range, 304.7 to 930.7 µg/mL) for the horses of this study far exceeded the target therapeutic concentration (160 µg/mL; ie, 10 times the MIC [16 µg/mL]) for most common resistant equine pathogens. Thus, IVRLP of amikacin into a saphenous vein should be effective for treatment of infections of the distal hind limbs of horses, although further research involving horses with such infections is necessary.
Acknowledgments
Supported by Katie Doyle and Richard Cunningham.
Abbreviations
| Cmax | Maximum drug concentration |
| DIPJ | Distal interphalangeal joint |
| IVRLP | IV regional limb perfusion |
| MIC | Minimum inhibitory concentration |
| tmax | Time to maximum drug concentration |
Footnotes
Random.org. Randomness and Integrity Services Ltd, Dublin, Ireland. Available at: www.random.org. Accessed Mar 25, 2019.
Dormosedan, Zoetis, Florham Park, NJ.
Torbugesic, Fort Dodge Animal Health, Overland Park, Kan.
Carbocaine-V, Zoetis, Florham Park, NJ.
BD InstyteTM, Becton Dickinson Infusion Therapy Systems Inc, Sandy, Utah.
TEVA Parenteral Medications Inc, Irvine, Calif.
Minipuls 3, Gilson Inc, Middleton, Wis.
VetOne, Boise, Idaho.
Kinetic Interaction of Microparticles in Solution (KIMS) screening assay, Cobas c311 analyzer, Roche Diagnostics GmBH, Mannheim, Germany.
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