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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Regional limb perfusion (RLP) significantly decreases morbidity and mortality associated with distal limb injuries in horses. There is an urgent need for finding additional effective antimicrobial drugs for use in RLP. In this study, we tested the pharmacokinetics (PK) of chloramphenicol in RLP. Eight horses participated in the study, which was approved by the University Animal Care and Use Committee. The cephalic and the saphenous veins were used to perfuse the limbs. Synovial samples were collected from the metacarpo/metatarsophalangeal (MCP/MTP) joint. The Friedman Test was applied for assessing change in PK concentration over time, for all time points. The Wilcoxon Signed Ranks Test was used to test the difference between PK concentration in joint & serum as well as concentration in joint vs. MIC. The comparison of measurements between measurements taken on hind vs. front legs was carried out using the Mann–Whitney Test. A P-value of 5% or less was considered statistically significant. After RLP, the concentration of chloramphenicol in the synovial fluid of the MCP/MTP joint using either the cephalic or the saphenous vein was initially far above the minimal inhibitory concentration (MIC) of most susceptible pathogens and remained above the MIC for approximately 6 h. The results indicate that performing RLP using the cephalic and saphenous veins enables reaching concentrations of chloramphenicol in the MCP/MTP joint that are well above the MIC of most susceptible pathogens. The chloramphenicol concentrations achieved in the synovial fluid of the MCP/MTP joint in the current study were between 1.5 (MTP) and 7 (MCP) times the MIC of MRSA in horses. These results are encouraging since MRSA infections are becoming far more common, causing considerable morbidity. To the best of our knowledge, this is the first study to evaluate the pharmacokinetics of chloramphenicol following RLP in the horse and the results are positive.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Distal limb injuries involving synovial structures are common in horses, often resulting in a devastating outcome. Regional limb perfusion (RLP) with an antimicrobial drug is a simple and effective method for treating horses with synovial injuries (Rubio-Martinez & Cruz, 2006; Rubio-Martinez et al., 2012). Using RLP enables penetration of the antimicrobial drug into the injured synovial structures and surrounding tissues, (Werner et al., 2003; Rubio-Martinez et al., 2005) at concentrations that are an order of magnitude higher than the minimal inhibitory concentration (MIC). In contrast to systemic drug administration, RLP allows for consistently high concentrations of antimicrobials only at the target tissue (Pille et al., 2005), thereby reducing the development of antimicrobial resistance and increasing the efficacy of treatment (Goldberg & Owens, 2002). In addition, systemic administration of common antimicrobial drugs poses a significant risk for severe colitis, which can be fatal (Cohen & Woods, 1999; McGorum & Pirie, 2010). Local administration requires significantly lower doses than those needed for systemic use. Therefore, local drug delivery systems such as RLP can enable use of newer generation drugs when their systemic use would often be cost prohibitive.

Injury to the distal portion of the limb may result in vascular damage, vascular thrombosis or edema, all associated with local ischemia. This could further limit delivery of antimicrobial agents to sites of infection through the systemic circulation (Knottenbelt, 1997). Most authors describe RLP as an adjunctive treatment to systemic antimicrobial administration (Rubio-Martinez & Cruz, 2006). It has been demonstrated, however, in an early study of iatrogenic-induced septic arthritis model (Whithair et al., 1992) and recently in a clinical retrospective study (Kelmer et al., 2012), that synovial infections can be treated effectively using RLP as a sole antimicrobial therapeutic module. Most studies examining the pharmacokinetic properties of drugs administered by RLP show that using the palmar digital (PD) vein is an efficient mean of delivering therapeutic concentrations of antimicrobial drugs to the distal limb (Murphey et al., 1999; Butt et al., 2001; Werner et al., 2003; Rubio-Martinez et al., 2005; Parra-Sanchez et al., 2006; Errico et al., 2008). Nevertheless, multiple RLP treatments are often needed to resolve persistent infections, and premature termination of RLP is likely to occur due to loss of access to the PD vein (Butt et al., 2001; Scheuch et al., 2002; Mattson et al., 2004). Consequently, the cephalic and saphenous veins have been evaluated, both experimentally and clinically, and proved to be advantageous, alternative routes for performing RLP (Kelmer et al., 2009, 2012). Pharmacokinetic studies examining the disposition of drugs administered by RLP to the synovial structures of the distal portion of the limb of horses using proximal veins are scarce and only the disposition of amikacin and erythromycin was examined (Levine et al., 2010; Kelmer et al., 2013a,b). There is, however, an ever-growing problem of bacterial resistance to the commonly used antimicrobial drugs and there is an urgent need for use of newer and/or more effective ones. One emerging approach to reduce the risk of bacterial resistance is using ‘older’ antimicrobials, that have not been used extensively for decades (Falagas et al., 2008). Chloramphenicol is an old generation, broad spectrum, bacteriostatic antimicrobial drug, with activity against aerobic and anaerobic bacteria, that has excellent penetration to tissues including bone (Summersgill et al., 1982). Our clinical impression is that chloramphenicol is effective for treating persistent and resistant infections, including osteomyelitis, both by RLP and by systemic administration. We hypothesized that using the cephalic and saphenous veins for RLP would enable delivery of high concentrations of chloramphenicol to the distal portion of the thoracic and pelvic limbs.

The objective of this research was to determine the pharmacokinetics (PK) of chloramphenicol used in RLP by measuring metacarpophalangeal or metatarsophalangeal (MCP/MTP) joint fluid concentrations of chloramphenicol after RLP using the cephalic or saphenous vein for drug administration.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Eight mature locally bred horses aged 3–14 years (median 6 years), weighing 376–550 kg (mean 424 kg) participated in the study. Six were geldings, and two were mares. The horses were healthy and sound based on a physical and cursory lameness examination. Horses were randomly assigned to two groups (Cephalic/Saphenous). Each group consisted of four horses, including one mare and three geldings with an average weight of 410 kg. The project was approved by the University Animal Care and Use Committee.

Catheter placement and antimicrobial perfusion

Regional limb perfusion was performed on one randomly selected thoracic or pelvic limb from each horse. Prior to having a catheter inserted, the horse was sedated with intravenous detomidine HCl (0.006 mg/kg, Domosedan; ICE, Gyonggi-do, Korea) and butorphanol tartrate (0.01 mg/kg, Morphasol; aniMedica GmbH, Senden, Germany). Sedation was administered into the jugular vein, ipsilateral to the site of RLP. A 10-cm wide, rubber tourniquet (Esmarch bandage) was placed about 10 cm proximal to carpus/tarsus for perfusion via the cephalic or saphenous vein, respectively, and slightly tightened to occlude venous return and engorge the vein. After sterile site preparation, and subcutaneous local anesthesia (0.5 mL lidocaine-hydrochloride, lidocaine 2%; Hospira, Lake Forest, IL, USA) an over-the-wire catheter (16G, 10 cm; Mila International, Erlanger, KY, USA) was inserted and secured in place. Limbs with catheters in the cephalic and the saphenous veins were perfused with 10 mL 2% mepivacaine HCl (Carbocaine hydrochloride 2%; Hospira) followed by 2 g chloramphenicol diluted with sterile isotonic saline solution to a total perfusion volume of 100 mL. The antimicrobial solution was administered as a constant rate infusion over 5 min, and the tourniquet was removed 30 min after cessation of infusion.

Sample collection

Synovial fluid was collected from the MCP/MTP joint, and blood was collected from the jugular vein immediately before and at 30 min, 2, 6, 12, 24, and 36 h after the end of the infusion. Blood was collected aseptically from the jugular vein by use of a 21G, 40-mm needle from the contra-lateral side to the limb that was perfused. Synovial fluid was collected aseptically by inserting a 21G, 40-mm needle into the depression between the proximal phalanx, the proximal sesamoid bone, and the third metatarsal/metacarpal bones (Bassage & Ross, 2003). Both blood and synovial samples were collected in EDTA tubes, centrifuged at 1000 g for 5 min to remove the cells, and supernatant was collected and stored at −80 °C.

Chloramphenicol analysis

Concentrations of chloramphenicol in serum and in synovial fluid samples were determined using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method utilizing an Agilent 1200 (Agilent Technologies, Waldbronn, Germany) liquid chromatography system (binary pump, degasser, temperature regulated column compartment and autosampler) combined with an ABSciex API 4000 (ABSciex, Toronto, ON, Canada) mass spectrometer.

Drug-free serum/synovial fluid were fortified with chloramphenicol to obtain calibration curves in the range of 0.01–1 mcg/mL in serum and 0.5–50 mcg/mL in synovial fluid. Three replicates of calibration samples were prepared on three different days. The accuracy, as well as intraday and interday precision were calculated for 0.01, 0.1, and 1 mcg/mL in serum and 0.5, 5, and 50 mcg/mL in synovial fluid. These concentrations represent the lower level of quantification, mid-level of quantification. and high level of quantification. The raw results were processed through a specialized PK software (PK Solutions 2.0; Summit Research Services, Montrose, CO, USA).

Statistical analysis

The Friedman test was applied for assessing change in PK concentration over time, for all time points. The Wilcoxon signed-ranks test was used to test the difference between PK concentration in joint & serum as well as concentration in joint versus MIC. The comparison between the groups, with regard to body weight and PK measurements taken on hind versus front legs was carried out using the Mann–Whitney U-test. All tests applied were two-tailed, and a P-value of 5% or less was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The calibration curves in serum and in synovial fluid were linear over the tested range with a correlation coefficient of 0.997 for serum and 0.992 for synovial fluid. The intraday precision for serum at 0.01, 0.1, and 1 mcg/mL was 17.5%, 3.6%, and 6.0%, respectively. The interday precision at these concentrations was 6.4%, 13.4%, and 9.5%, respectively. The intraday precision for synovial fluid at 0.5, 5, and 50 mcg/mL was 15.4%, 1.2%, and 7.3%, respectively. The interday precision at these concentrations was 16.0%, 13.1%, and 9.2%, respectively. The average intraday accuracy for serum at 0.01, 0.1, and 1 mcg/mL was 93%, 86%, and 99%, respectively. The interday accuracy at these concentrations was 107%, 101%, and 98%, respectively. The average intraday accuracy for synovial fluid at 0.5, 5, and 50 mcg/mL was 122%, 108%, and 102%, respectively. The interday accuracy at these concentrations was 102%, 100%, and 104%, respectively.

No difference in body weight was found between the groups (P = 1). Pharmacokinetic data describing concentrations of chloramphenicol measured in the serum or the synovial fluid of the MCP/MTP joint is summarized in Table 1. Comparisons between the forelimbs and the hindlimbs with regards to maximum concentration (Cmax) and area under the curve (AUC) are depicted in Figs 1 and 2, respectively. After RLP, the synovial concentration of chloramphenicol in the MCP ⁄MTP joint using either the cephalic or the saphenous vein was significantly higher than the MIC of Methicillin-resistant Staphylococcus aureus (MRSA; P = 0.012) for up to 6 h. The maximal concentration of chloramphenicol (54 mcg/mL) in the synovial fluid was 10.8 times higher than the MIC of most susceptible pathogens. The chloramphenicol concentration was significantly higher in synovial fluid compared with serum when RLP was performed using either the cephalic or the saphenous vein at time 0.5, 2, 6 (P = 0.012) and 12 (P = 0.018) hours after perfusion. The Cmax of chloramphenicol was significantly higher in synovial fluid compared with serum when RLP was performed using either the cephalic or the saphenous vein (P = 0.012). There was a marked, between-horses variability in synovial fluid concentrations of chloramphenicol at time 0.5 h (SD ±35.7) after administration and variability decreased gradually as concentrations declined over time. In six horses, the maximal synovial concentration (Tmax) was reached at 0.5 h, and in two horses, the maximal concentration was reached at 2 h, thus, on average Tmax was reached at 52.5 min post infusion. The AUC was also significantly higher in the synovial fluid than in the serum when RLP was performed using either the cephalic or the saphenous vein (P = 0.012). The Tmax as well as the half-life (T1/2) were not significantly different between the serum and the synovium (P = 0.414, P = 0.123). No difference was found between the cephalic and saphenous with regard to synovial concentrations either by measuring Cmax (P = 0.462) or by measuring AUC (P = 0.624). Nevertheless, Cmax was more than double after administration into the cephalic vein compared with administration into the saphenous vein (54 vs. 24 mcg/mL) and AUC was 36% higher after the administration into the cephalic vein compared with that into the saphenous vein (162 vs. 119 mcg·h/L). None of the horses showed adverse systemic effects to the procedure or the drugs used during the study.

Table 1. Pharmacokinetical values of chloramphenicol after regional limb perfusion using the cephalic and saphenous veins in standing horses
 MCPMTPSerum
MeanMedianSD n MeanMedianSD n MeanMedianSD n
  1. AUC, area under the curve; MCP, metacarpophalangeal; MTP, metatarsophalangeal.

  2. a

    Significant difference (P ≤ 0.05).

AUC (mcg·h/mL)162.8150.6112.214119.483.8107.9444a2.6a3.22a8
AUC/dose (h/L)0.080.070.0540.050.040.0540a0a0a8
Cmax (mcg/mL)5440.845.5642413.423.4841.2a0.6a1.32a8
Cmax/dose (1/L)0.020.020.0240.0100.0140a0a0a8
Tmax (h)0.870.500.7540.870.500.7541.251.250.808
T1/2 (h)4.174.003.0543.502.352.7546.726.304.398
image

Figure 1. Box Plot and whiskers depicting the Cmax of serum and joint chloramphenicol concentrations after regional limb perfusion to the cephalic and saphenous veins. *Significant difference between Serum and Joint.

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image

Figure 2. Box Plot and whiskers depicting the AUC of serum and joint chloramphenicol concentrations after regional limb perfusion to the cephalic and saphenous veins. *Significant difference between Serum and Joint.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

To our knowledge, this is the first study evaluating the PK of chloramphenicol following RLP in the horse. The results indicate that performing RLP using the cephalic and saphenous veins consistently resulted in concentrations of chloramphenicol in the MCP/MTP joint that were well above the MIC (5 mcg/mL) for most susceptible pathogens (Sisodia et al., 1975). The synovial chloramphenicol concentrations achieved in the MCP/MTP joint were between 3 (MTP) and 6.75 (MCP) times the chloramphenicol MIC of MRSA (MIC90 = 8 mcg/mL) in horses (Rubin et al., 2011). These results are encouraging as MRSA infections are becoming far more common, causing considerable morbidity (Weese et al., 2005). Other emerging multidrug resistance (MDR) bacteria such as extended-spectrum–β Lactamase (ESBL) salmonella, show sensitivity to chloramphenicol in recent years (Takkar et al., 1995; Wasfy et al., 2002). This renders chloramphenicol RLP a potentially efficacious therapy for MDR and other persistent or deep seeded distal limb infections. Because MRSA infection can be transferred from horses to people and vice versa, eradicating these infections rapidly and effectively is necessary from both equine and human health perspective. In humans, chloramphenicol can idiosyncratically cause bone marrow suppression and aplastic anemia and its use is therefore restricted in many countries (Yunis, 1989). However, human ocular medications containing chloramphenicol are extensively used globally and pose minimal health risk (Walker et al., 1998). Thus, it seems that local RLP therapy using chloramphenicol may also pose minimal human health hazard.

In the present study, there was no statistically significant difference in antimicrobial concentrations between the forelimb and the hindlimb joints. Nevertheless, the fact that the forelimb Cmax was twice as high and the AUC was 36% higher compared with the hindlimb is consistent with our previous study (Kelmer et al., 2013ab). In that study, synovial antimicrobial concentrations were significantly higher in the forelimb. It is likely that with a larger sample size, the results of the current study would also show significantly higher antimicrobial concentrations in the forelimb. We suggest two plausible explanations for this potential difference. One is that the tourniquet can compress the vasculature more effectively in the forelimb due to its tubular shape. The other potential cause is that the tissue volume of the hindlimb is higher, thus reducing the final concentration resulting from administration of the same volume of perfusate. Nevertheless, based on this pattern, it may be advisable to increase either the infusion volume or the concentration of the antimicrobial drug in saphenous RLP.

Movement during RLP causes leakage of the perfusate and decreased concentrations of antimicrobial drugs in the target area (Levine et al., 2010). Often a local anesthetic drug such as lidocaine is added to the perfusate to improve the results by decreasing the discomfort and the movement during the procedure. In the current study, mepivacaine was used for that purpose. A recent study found that the addition of lidocaine to the perfusate did not have an effect on the PK of RLP with amikacin (Mahne et al., 2014). As both the antimicrobial and the local anesthetic drugs were different, Mahne et al. study results may not have direct implications to our study.

Several pharmacokinetic studies have shown that following RLP, antimicrobial concentrations are higher in the more distal synovial structures compared with the MCP joint (Murphey et al., 1999; Butt et al., 2001; Rubio-Martinez et al., 2005). Therefore, it appears reasonable that chloramphenicol reached therapeutic concentrations also in the more distal synovial structures. Most traumatic synovial injuries seen in equine practice involve the distal synovial structures, mainly MCP/MTP, digital flexor tendon sheath, distal interphalangeal joint, navicular bursa etc. (Kelmer et al., 2012; Rubio-Martinez et al., 2012). This renders RLP with chloramphenicol potentially effective against most common distal limb synovial traumas. In addition, the concentrations of chloramphenicol in the systemic circulation remained low, which make the use of the drug, according to this method, safe with relation to potential systemic adverse effects. Antimicrobial drug use associated colitis is a severe systemic adverse effect (Cohen & Woods, 1999), and by reaching only minute drug concentrations in the systemic circulation, potentially fatal adverse reaction is unlikely.

The present RLP study produced similar results to our previous studies with amikacin, erythromycin and imipenem (Haziz et al., 2013; Kelmer et al., 2013a,b). It appears that the method is reproducible regardless of the administered antimicrobial agent lending further credit to the efficacy of this technique, using the cephalic/saphenous veins for RLP.

The concentration of chloramphenicol in the synovial fluid remained above the MIC for several hours after the tourniquet was released (t1/2 ~3 h). The t1/2 of chloramphenicol observed in the current study was shorter than the one observed for concentration-dependent antimicrobials such as amikacin (t1/2 = 12 h) when used in a similar RLP setting (Kelmer et al., 2012). A recent study described a 2 h postantibiotic effect for chloramphenicol (Athamna et al., 2004). Considering these PK values, one may contemplate performing chloramphenicol RLP more frequently (e.g. twice daily). Nevertheless, performing RLP in the standing horse requires sedation due to the pain elicited by the application of the tourniquet. Frequent sedations may be detrimental to the horse due to its negative effects on intestinal motility.

One potential drawback of our study was the use of an Esmarch bandage (rubber tourniquet) as opposed to a pneumatic tourniquet. According to a recent study, pneumatic tourniquet placed above the carpus is more effective than a rubber tourniquet (Levine et al., 2010). Nevertheless, the efficacy of an Esmarch tourniquet depends on the exact technique used to apply it, and the force applied during placement. An additional study comparing the efficacy of an Esmarch bandage to that of a pneumatic tourniquet, placed proximal to the carpus/tarsus, may expand our knowledge base and have important clinical implications.

Another limitation of the study is that participating horses were healthy. Horses with injured, inflamed synovial structures are likely to show altered PK (Beccar-Varela et al., 2011). Ideally, additional studies performed with induced, controlled, inflammation will take place and will more accurately represent the clinical situation.

In conclusion, this study demonstrates that chloramphenicol RLP in the veins of the proximal limb is pharmacologically sound and has a potential to be effective as a sole antimicrobial treatment modality for distal limb synovial sepsis in horses.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The study was funded by a grant from the Veterinary Teaching Hospital at the Koret School of Veterinary Medicine, the Hebrew University of Jerusalem.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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