Toms AD, Davidson D, Masri BA, Duncan CP. (2006) The management of peri-prosthetic infection in total joint arthroplasty. J Bone Joint Surg (Br) 88(2):149â ...
40 The Gentamicin-Vancomycin Spacer: A Pharmacological Study E. Bertazzoni Minelli, A. Benini Department of Medicine and Public Health, Pharmacology Section, University of Verona, Italy
Introduction Gentamicin-loaded spacers are used in two-stage revision prosthesis to deliver high local antibiotic concentrations for the treatment of prosthetic infections [6, 18]. The concentration of antibiotics released largely exceeds the minimum inhibitory and bactericidal concentration of susceptible bacteria. With the emergence of resistant bacteria, the addition to bone cement of two antibiotics potentially synergistic has became a frequent practice [9]. The antibiotics utilised in spacer preparation should be carefully selected and should respond to ideal characteristics, such as wide spectrum of activity against the majority of microorganisms, bactericidal activity, good release from cement at inhibitory concentrations for prolonged periods, low interference with mechanical properties of the cement, high biocompatibility, low hypersensitivity and adverse drug reactions, capacity and maintenance of antimicrobial activity at the site of infection. The combination of two or more antibiotics in PMMA cement should consider the inhibitory [11] or synergistic [12] effects on drug release. In addition, when mixed in combination, antibiotics should maintain their antimicrobial activity and possibly exerting a synergistic antimicrobial effect. The combination of two antibiotics with PMMA may result in modified release kinetics, or in inactivation of drug or in reduced drug release [11, 16]. Consequently, the antimicrobial activity of the mixture may result in an unpredictable effect. The release of vancomycin is modified by the presence of imipenem, and the amount released depends on type of cement [5]. Vancomycin and an aminoglycoside are often combined for their potential synergistic effect in the treatment of severe infections. Vancomycin is a bactericidal glycopeptide antibiotic with a primary spectrum of activity against Gram-positive cocci, such as Staphylococcus aureus – including methicillin-resistant –, Staphylococcus epidermidis, Enterococcus faecalis, etc. The elution characteristics of antibiotic-loaded spacers and their antimicrobial activity in vivo is poorly known. Few data are available for the comparison of spacers made with a single antibiotic or with antibiotic combinations both in vitro and in vivo [2, 7, 8, 10, 14]. In two-stage revision of infected THA, spacers are either hand moulded from bone cement, fabricated in a teflon mould, or preformed. A new spacer has been studied to respond the demand of surgeons and the increasing infection rate due to gentamicin-resistant pathogens. Preformed industrial spac-
40 The Gentamicin-Vancomycin Spacer: A Pharmacological Study
ers incorporating gentamicin and vancomycin made with high porosity resin which provides a long-lasting release were tested. This preformed spacer exhibits adequate properties such as good mechanical resistance and compatibility of drugs in PMMA cement. The release of antibiotics in vitro from this new spacer is described.
Material and Methods Spacers prepared with a high porosity PMMA-based resin (HP Cemex, Tecres Spa, Italy) incorporating gentamicin (1.15 g) and vancomycin (1.15 g) were obtained from a mould under aseptic conditions (sterile chamber). The amount of each antibiotic in the fully formed device is equivalent to a final concentration of 1.9 % (3.8 % total antibiotic). Spacers prepared with the same resin incorporating gentamicin alone (1.15 g) were studied for comparison. The tested spacers were sterile (ethylene oxide). Spacers’ surface area was 122.21 cm2. Elution of Antibiotics As previously described [3], the spacers were immersed in pyrex tubes with 600 ml of phosphate buffer (0.2 M, pH = 8.0, phosphate buffer [PB]) at 37 °C for 30 days. The PB was removed and replaced with the same volume of fresh PB after 24, 48, 72, 240, 480 and 720 hours of immersion. The removed buffer was subdivided into small aliquots and frozen at –24°C. The PB samples from each prosthesis were analysed in the same experiment. Drugs Gentamicin and vancomycin (powder, EP grade) were utilised for the preparation of standards. Stock solutions of antibiotic were prepared in PB. Standard concentrations of antibiotics were processed along with samples. Antibiotic concentrations in eluted samples and standards were processed in parallel using two methods, namely, bioassay and fluorescence polarization immunoassay. Bioassay Concentrations of gentamicin and vancomycin were determined using the standard large-plate agar-well diffusion method [4] according to the NCCLS guidelines [15]. Briefly, Bacillus subtilis spore suspension ATCC 6633 (final concentration 0.02 %) in Isosensitest Agar (Oxoid Unipath Ltd, Basingstoke, England) was used as the testmicroorganism. After overnight incubation at 37°C, the diameters of the inhibition zones were measured. Antibiotics concentrations were determined in relation to the diameters of the inhibition zones yielded by a standard series of known concentrations of antibiotics. All samples and standard concentrations were assayed in duplicate or triplicate. The assay for gentamicin and vancomycin was linear over a range of 0.6 – 20.0 mg/L (R2 = 0.98). The assay for the combination was linear over a range of 1.25 – 40.0 mg/l
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(R2 = 0.99). The between-day coefficients of variation were 0 % and 2.2 % for the highest and lowest concentrations, respectively, for both drugs alone and in combination. Fluorescence Polarisation Immunoassay (FPIA) Concentrations of gentamicin and vancomycin in the PB samples were also determined by FPIA (TDx, Abbott). Vancomycin and gentamicin can be measured separately by the FPIA method even when used in combination. The method was calibrated and applied according to the manufacturer’s recommendations (TDx, Abbott). The lowest measurable level of drug concentration was 0.27 mg/L for gentamicin and 2.0 mg/L for vancomycin [1]. All tests were carried out in duplicate.
Results The release of gentamicin and vancomycin in combination from PMMA spacer is reported in Figure 1. Gentamicin and vancomycin are released from spacer in high amounts, 31.2 mg and 13.1 mg, respectively, in the first 24 hours. This initial peak is followed by a high and constant release over the next days. The amount of gentamicin released from spacer is higher than that of vancomycin. Gentamicin and vancomycin in combination 1:1 are released from spacer in different amounts, 63.2 mg and 29.3 mg, respectively, after 30 days of elution. The ratio of gentamicin to vancomycin in the eluate corresponds to 2:1. The elution of gentamicin (mg 31.2) in presence of vancomycin is higher than that recorded for the elution of 36 30
Antibiotic Release (mg)
354
24 18 12
Gentamicin alone Gentamicin from combination Vancomycin from c ombination
6 6 4 2 0 0
24 48 72 96 120 144 168 192 216 240 240
480
720
Hour s
Fig. 1. Release (mg) of Gentamicin and Vancomycin in combination (G+V 1:1) and Gentamicin alone from PMMA hip spacers. Values of antibiotics release from 72 to 720 hours are extrapolated as mg/day of elution. (FPIA method).
40 The Gentamicin-Vancomycin Spacer: A Pharmacological Study Table 1. Release of gentamicin (G, 1.9 %) and vancomycin (V, 1.9 %) in combination (1:1) from HP-PMMA spacer (HP Cemex ®) and gentamicin (1.9 %) alone evaluated with FPIA and microbiological method. Results determined after 24, 240 and 720 hours of elution. Elution time after
Antibiotics
Methods FPIA Gentamicin
Vancomycin
(mg)
Microbiological
(mg)
Total calculated (mg)
(mg)
24 hours
G alone G+V
20.4 31.2
– 13.1
20.4 44.3
30.6 59.3
10 days
G alone G+V
40.1 46.3
– 21.9
40.1 68.2
51.1 92.6
30 days
G alone G+V
56.8 63.2
– 29.3
56.8 92.5
68.5 117.2
Table 2. In vitro activity of gentamicin and vancomycin in combination against multiresistant clinical isolates. Checker-board method [13].
* Oxacillin Resistant ° Methicillin Resistant R = Resistant I = Intermediate FIC e 0.5 Synergism S FIC = 1 Additivity A FIC & 2 Antagonism Ant
Vancomycin + Gentamicin Strain
S. epidermidis (8/28) S. haemolyticus (8/28) * S. haemolyticus (82/26) S. haemolyticus (70/26) * S. epidermidis (137/25) ° S. hominis (126/26) S. aureus (3A10) S. aureus (9A28) E. coli (7A27) P. aeruginosa (4/28)
FIC Index
1.00 1.00 1.00 1.00 0.50 1.02 0.15 0.48 0.25 0.12
A A A A A A S S S S
MIC (mg/L) Vancomycin
Gentamicin
2.5 1.25 1.25 1.25 2.5 1.25 2.5 1.25 156.25 1250
R R R R R I I 1.25 5 5
gentamicin alone (mg 20.2) in the first 24 hours, thereafter the release of gentamicin shows similar values. The determination with microbiological method allows to evaluate a good antibacterial activity, represented by higher values in comparison with those determined by immunoenzymatic method at any time considered (Table 1). The antimicrobial activity of the combination is higher than expected from the sum of amounts of gentamicin and vancomycin determined separately (59.3 mg versus 44.3 mg calculated amount by FPIA at 24 hours, respectively). Gentamicin and vancomycin are released from spacers in bactericidal amounts in the first ten days of elution, maintaining high bioactivity. In the following period (20 days) the release (extrapolated as mg/day) corresponds to 1.4 – 1.6 mg/day after 30 days of elution (Fig. 2). Considering the spacer area, this equals 11.5 – 13.1 µg per cm2, an amount which is highly inhibitory against multi-resistant clinical isolates. The antimicrobial activity of the combination is higher than gentamicin alone (Figure 2 and Table 1). This effect is defined as summatory or synergistic as shown in Table 2, according to FIC method [13].
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Novel Applications and Perspectives 80 70 60
Antibiotic Release (mg)
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50 40 30 20
Gentamicin alone
10
Gentamicin - Vancomycin combination
10 8 6 4 2 0 0 24 48 72 96 120 144 168 192 216 240 240
480
720
Hours Fig. 2. Release (mg) of Gentamicin and Vancomycin in combination (1:1) and of Gentamicin alone from PMMA hip spacers. Values of antibiotics release from 72 to 720 hours are extrapolated as mg/day of elution. (Microbiological method)
Discussion and Conclusions The release kinetics of the combination show that: i- high initial peak is followed by sustained constant release of both antibiotics; ii- the release profile of gentamicin and vancomycin is superimposable; iii- gentamicin release seems enhanced by the presence of vancomycin. The antibiotics maintain their bactericidal activity once they have been released from cement and show synergistic effect in these experimental conditions. Anagnostakos et al [2] demonstrated a synergism between vancomycin and gentamicin against MRSA and S. epidermidis, and autonomy against E. faecalis and S. aureus. The antimicrobial activity is maintained at the site of infection. Kelm et al [10] showed that spacers loaded with gentamicin and vancomycin (1 g + 4 g / 80 g PMMA), can release both antibiotics in different amount ratio exhibiting an inhibitory activity in vitro for two weeks after removal. These data seem to confirm the potential efficacy of such combination in vivo in two-stage revision infection treatment. These results are in agreement with Anagnostakos et al [2] where the combination vancomycingentamicin (ratio 2:1) shows superior percentage elution of gentamicin compared to vancomycin and good antimicrobial activity against Gram-positive microorganisms. In this study vancomycin showed a peak concentration on day 2 of elution. Differently, the release of both gentamicin and vancomycin from the preformed tested spacer showed peak concentrations on day 1. Gentamicin confirms very favourable release characteristics from bone cement. Further investigations are warranted in order to determine if an optimal ratio of gentamicin to vancomycin is needed to obtain inhibitory synergistic effect. Moreover,
40 The Gentamicin-Vancomycin Spacer: A Pharmacological Study
the optimal concentrations of both antibiotics in spacer cement should be defined ranging from 1.25 % to 10 % [2, 10, 16, 17]. Compatibility with cement, interference with polymerization process and mechanical resistance are limitations for the use of vancomycin at too high concentrations. The industrial preparation seems to overcome the limitations derived from handmade spacers [3, 11, 17] such as the variability depending on hand-made mixture and preparations. The use of preformed spacers may be advantageous in terms of standardization of the device characteristics, mechanical resistance, uniform antibioticcement mixing and reproducible antibiotic release. In conclusion, the new preformed spacer loaded with gentamicin and vancomycin exhibits in vitro favourable properties and release characteristics similar to those described for PMMA cements. The combination shows inhibitory activity against microorganisms responsible for prosthetic infections. Acknowledgements We thank Chiara Caveiari for her excellent technical assistance.
References 1. Abbott Laboratories Diagnostic Division. (1996). TDx FLx™ system assay I. North Chicago, IL; Abbott Laboratories 2. Anagnostakos K, Kelm J, Regitz T et al (2005) In vitro evaluation of antibiotic release from and bacteria growth inhibition by antibiotic-loaded acrylic bone cement spacers. J Biomed Mater Res B Appl Biomater. 72(2):373 – 378 3. Bertazzoni Minelli E, Benini A, Magnan B et al (2004) Release of gentamicin and vancomycin from temporary human hip spacers in two-stage revision of infected arthroplasty. J Antimicrob Chemother 53(2):329 – 334 4. Bertazzoni Minelli E, Caveiari C, Benini A (2002). Release of antibiotics from polymethylmethacrylate (PMMA) cement. J Chemother 14(5):64 – 72 5. Cerretani D, Giorgi G, Fornara P et al (2002) The in vitro elution characteristics of vancomycin combined with imipenem-cilastatin in acrylic bone-cements: a pharmacokinetic study. J Arthroplasty. 17(5):619 – 626 6. Cohen J (1999) Management of chronic infection in prosthetic joints. In “Infectious Disease” (Armstrong D. and Cohen J. Eds). Mosby, London. Vol. I, Section 2 – p 46.1 – 46.6 7. Gonzales Della Valle A, Bostrom M, Brause B et al (2002). Effective bactericidal activity of tobramycin and vancomycin eluted from acrylic bone cement. Acta Orthop Scand 72(3): 237 – 240 8. Holtom PD, Warren CA, Greene NW et al (1998) Relation of surface area to in vitro elution characteristics of vancomycin-impregnated polymethylmethacrylate spacers. Am J Orthop 27(3):207 – 210 9. Joseph TN, Chen AL, and Di Cesare PE. (2003). Use of antibiotic-impregnated cement in total joint arthroplasty. J Am Acad Orthop Surg 11(1):38 – 47 10. Kelm J, Regitz T, Schmitt E et al (2006) In vivo and in vitro studies of antibiotic release from and bacterial growth inhibition by antibiotic-impregnated polymethylmethacrylate hip spacers. Antimicrob Agents Chemother 50(1):332 – 335 11. Klekamp J, Dawson JM, Haas DW et al (1999) The use of vancomycin and tobramycin in acrylic bone cement: biochemical effects and elution kinetics for use in joint arthroplasty. J Arthroplasty 14(3):339 – 346 12. Kuechle DK, Landon GC, Musher DM et al (1991) Elution of vancomycin, daptomycin, and amikacin from acrylic bone cement. Clin Orthop Rel Res 264:302 – 308
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Novel Applications and Perspectives 13. Lorian V (1996) Antibiotic in laboratory medicine. Fourth edition. Williams & Wilkins Ed., Baltimore 14. Masri BA, Kendall RW, Duncan CP et al (1994) Two-stage exchange arthroplasty using a functional antibiotic-loaded spacer in the treatment of the infected knee replacement: the Vancouver experience. Semin Arthroplasty 5(3):122 – 136 15. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility testing for bacteria that grow aerobically, 5th ed. Approved standard. NCCLS document M7-A5. National Committee for Clinical Laboratory Standards, Wayne, PA 2000 16. Neut D, de Groot EP, Kowalski RS et al (2005) Gentamicin-loaded bone cement with clindamycin or fusidic acid added: biofilm formation and antibiotic release. J Biomed Mater Res A 73(2):165 – 170 17. Takahira N, Itoman M, Higashi K et al (2003) Treatment outcome of two-stage revision total hip arthroplasty for infected arthroplasty using antibiotic-impregnated cement spacer. J Orthop Sci 8(1):26 – 31 18. Toms AD, Davidson D, Masri BA, Duncan CP. (2006) The management of peri-prosthetic infection in total joint arthroplasty. J Bone Joint Surg (Br) 88(2):149 – 155
