Journal of Orthopaedic Research 9594-599 Raven Press, Ltd., New York 0 1991 Orthopaedic Research Society
Comparison of Cortical Bone and Serum Concentrations of Clindamycin Achievable by Direct Local Infusion and Intravenous Administration Steven C. Budsberg, *James M. Gallo, ?Clifford E. Starliper, ?Emmett B. Shotts, and ?John Brown Department of Small Animal Medicine, College of Veterinary Medicine, "Department of Pharmaceutics, College of Pharmacy, and ?Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, U.S.A.
Summary: Tibia1 cortical bone and serum concentrations of clindamycin were compared using two drug delivery methods in dogs. An implantable drug pump, used to continuously infuse clindamycin directly into the cortical bone, was compared with clindamycin administered i.v. Dosage for the direct continuous infusion was 4 mglkglday, and 44 mglkglday for the i.v. bolus regimen. Serum concentrations of clindamycin were significantly higher during i.v. bolus administration when compared with those achieved during pump infusion (p < 0.05). However, tibia1 cortical bone concentrations were significantly higher during pump infusion than were those achieved by i.v. bolus. When examining serum and bone clindamycin concentrations over 21 days of direct local infusion, there was no significant difference in concentrations between sampling days within each tissue (p > 0.05). Furthermore, there were significantly greater concentrations of clindamycin in the cortical bone than in the serum at each sampling period (p < 0.05). Results indicate that delivery of clindamycin to canine bone by implantable drug pump achieve significantly higher bone concentrations than i.v. bolus administration of the drug at higher dosages. Direct infusion also can sustain high concentrations in cortical bone without increasing systemic concentrations of clindamycin. Key Words: Clindamycin-Bone concentrations-Direct infusion.
of Paris, direct irrigation-suction units, isolated perfusion of the affected limb, and fibrin-antibiotic complexes (2-7,9,12,15,16,20). Recently, Perry et al. (21-24) successfully treated refractory cases of osteomyelitis using implantable percutaneously refillable pumps that directly and continuously infused antibiotics into the osteomyelitic lesions. Limited data exist regarding antimicrobial distribution and bone antimicrobial concentrations achieved by direct local infusions into the bone (2124). Furthermore, there are no data comparing bone concentrations of antimicrobial agents delivered by
The ability of antimicrobial agents to penetrate and distribute into ischemic infected bone has long been questioned (14,19,25,26,28,29). Consequently, experimental and clinical studies have examined several methods of delivering antibiotics to osteomyelitic lesions. These methods have included antibiotic-impregnated methylmethacrylate or plaster Received April 18, 1990; accepted December 18, 1990. Address correspondence and reprint requests to Steven C. Budsberg, D.V.M., Department of Small Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, CA 30602, U.S.A.
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DIRECT INFUSION OF CLINDAMYCIN INTO BONE this method with those obtained by a systemic dosage regimen. However, the use of direct pump infusion appears promising because it allows, (a) the delivery of unlimited amounts of the desired antimicrobial agent at a constant infusion rate, (b) changes in the dose of the antimicrobial agent or even the antimicrobial agent itself without surgical intervention, (c) reduction in the duration of hospitalization, and (d) a minimum in systemic toxicity due to low systemic antimicrobial concentrations (21-24). The purpose of this study was to determine the concentrations of clindamycin in normal canine tibial cortical bone and serum obtained by continuous local infusion by an implantable pump, and to compare these concentrations to clindamycin concentrations in tibial cortical bone and serum achieved by a multiple-dose i.v. bolus regimen. In addition, we determined the tibial cortical bone and serum concentrations of clindamycin during 3 weeks of continuous local infusion of the drug. Finally, the study evaluated tibial cortical bone concentrations of clindamycin at different sampling sites in relationship to the position of the outflow catheter site. MATERIALS AND METHODS Animals Fifteen adult mixed-breed dogs (30-35 kg) were used in the study. Inclusion criteria included normal findings on physical examination, complete blood count, serum chemistries, urine analysis, and radiographs of the tibias. Experimental Design Twelve dogs were randomly placed into two groups of six dogs and alternated between therapy regimens. Initially, group 1 dogs received clindamycin by i.v. bolus injections. Concurrently, group 2 dogs received clindamycin through pumps implanted in their flanks, with the outflow catheter from the pump placed randomly into the cortical bone of the tibia. Bone biopsy samples (full thickness) from the tibia and serum samples were obtained on days 3 , 7 , and 21 after initiation of clindamycin infusion. After 1 month and documentation that clindamycin was eliminated, dogs were switched to the other regimen using the opposite tibia for bone biopsy and assay. Three dogs had pumps implanted in their flanks
595
and were infused for 3 , 7 , and 14 days at which time the entire tibia was collected and assayed at 5-mm increments, both proximal and distal to the outflow catheter site. I.V. Bolus Protocol Jugular and cephalic vein catheters were placed in each dog, with patency of each catheter maintained with heparinized saline. Each dog was given clindamycin phosphate (Cleocin, Upjohn Pharmaceuticals, Kalamazoo, Michigan, U.S.A.) i.v. at 11 mglkg every 6 hours for 3 days through the cephalic catheter. One hour after the 10th dosage, each dog was anesthetized and a full-thickness cortical bone biopsy sample was taken from the mid-diaphyseal region of the tibia with a Michelle trephine. A whole blood sample was obtained simultaneously from the jugular catheter. Pump Protocol After routine anesthetic induction and maintenance on an inhalant anesthetic, an implantable drug pump (INFUSAID model 400, Shiley INFUSAID Inc., Norwood, Massachusetts, U.S.A.) filled with sterile water was inserted into an S.C. pouch in the caudal dorsal abdominal region (flank). Dogs were then randomized to either right or left tibia for placement of the outflow infusion catheter. The catheter was passed S.C. down the medial thigh to the level of the mid-tibia. A single longitudinal incision was made in the periosteum. Stay sutures were placed in the edges of the periosteum to minimize trauma during manipulation. A hole was drilled into the medial cortex of the mid-diaphyseal region and the outflow catheter was placed in the hole. The periosteum was then closed over the catheter, with simple interrupted sutures. A single suture was placed in the skin at the level of the catheter insertion to serve as a marker. Routine closure of the flank and tibial incisions was performed. Seven days postimplantation, the sterile water was removed and the infusion of clindamycin was initiated. Concurrently, radiographs of the tibia were taken to verify proper placement of the outflow catheters. A clindamycin dosage of 4 mglkglday was administered by the pump. Animals were closely monitored daily for any clinical signs of local inflammation. The dogs were anesthetized on days 3 , 7 , and 21, and bone biopsy samples were obtained with a
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Michelle trephine. The biopsy samples were collected approximately 1 cm proximal and 1 cm distal to the catheter site. The biopsy site was alternated at each sampling period and the position of the biopsy site on the bone was varied to avoid any inflammatory reaction from a previous biopsy. At each sampling period, a serum sample was taken concurrently from the jugular vein. After completion of this portion of the study, each dog was anesthetized for removal of the infusion pump. The three dogs that were only infused at the assigned day (3, 7, and 14) were killed with an overdose of sodium pentobarbital and the entire infused tibia was collected. Sample Preparation Bone marrow was removed from each bone sample and washed in sterile 0.9% saline solution until all visible blood was removed. The periosteum and bone marrow were removed in the three dogs in which the entire tibia was collected. The infused tibia was then cut into 5-mm sections starting at the outflow catheter site extending both proximal and distal for 3 cm. All samples were then frozen at - 70°C until processing. Clindamycin was eluted from the bone as previously described (18,19) with modifications. Briefly, each bone sample was weighed, frozen in liquid nitrogen, and crushed into a powder. Depending on the weight of the sample, 600-1,OOO p1 of 0.1 M phosphate buffer, pH 8.0, was added to each sample. The mixture was vigorously agitated for 40 minutes, incubated at 4°C for 15 hours, and finally centrifuged for 10 minutes at 1,500 rpm. Aliquots (100 pl) of the supernatant were used in the assay procedure. Serum samples were also prepared as previously described (1,8,11). Whole blood was allowed to clot at 4°C for 2 hours, at which time the serum was removed and stored at -70°C until it was assayed. Aliquots (100 p1) of serum were used in the assay. Antibiotic Assay Clindamycin concentrations were measured by microbiological cylinder plate assay for serum and bone samples as previously described (1,8,11, 18,19). Standard curves were derived using clindamycin hydrochloride [Clindamycin HCL (lot no. 531 PY), Upjohn], canine serum, and a canine bonebuffer mixture identical to that made with each sample. Minimum measurable concentrations of clin-
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damycin were 0.3 pglml for serum and 0.4 pg/g for bone homogenate . Data Analysis All data were logarithmically transformed before analysis. When data are presented in text or tables, they appear as geometric means. Differences in serum and bone concentrations between i.v. bolus and pump infusion of clindamycin at 3 days were analyzed with a two-factor analysis of variance. If this value was significant, means of interest were compared using a Tukey test (p < 0.05). Differences in serum and bone concentrations between days 3, 7, and 21 during direct pump infusion were also analyzed with a two-factor analysis of variance. If this statistic was significant, means of interest were compared using a Tukey test (p < 0.05). Data on site of biopsy in relationship to outflow catheter placement (proximal or distal) was analyzed with a two-factor analysis of variance. If the statistic was significant, means of interest were compared with a Tukey test (p < 0.05). The mean bone/serum concentration was calculated by the following formula: mean cortical bone concentrationlmean serum concentration x 100. RESULTS
Ten dogs completed the cross-over study. One dog from each group was removed during the pump infusion phase of the project. The outflow catheter of one dog became displaced and the second dog had a tibial fracture through one of the biopsy sites. There was no clinical evidence of inflammation related to the direct infusion of clindamycin in any of the dogs. The mean (n = 10) serum clindamycin concentration 1 hour after the 10th dose of the i.v. bolus regimen was 8.7 rfr 0.2 pg/ml. The concurrent mean tibial bone concentration was 2.4 rfr 0.2 pg/g (Table 1). The mean serum clindamycin concentration at a similar time (day 3) during continuous pump infusion was 0.8 rfr 0.2 pg/ml, with concurrent mean tibial cortical bone concentrations of 10.5 k 2.2 pg/g. Mean serum concentrations of clindamycin were significantly higher during the i.v. bolus regimen when compared with those achieved during pump infusion. Conversely, mean tibial cortical bone concentrations were significantly higher during pump infusion than those measured after i.v. bolus administration of the drug.
DIRECT INFUSION OF CLINDAMYCIN INTO BONE TABLE 1. Serum and bone concentrations of clindamycin on day 3 of administration
Serum (pg/ml) Bone (Pg/g) Mean bonekerum ratio (%)
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TABLE 3. Tibia1 bone concentrations divided into sampling site in relationship to outflow catheter
i.v. Bolus (44 mg/kg/day)
Direct bone infusion (4 mg/kg/day)
8.7 2 0.2 2.4 2 0.2
0.8 ? 0.2" 10.5 2 2.2'
31.4
1,312.5
Day 3
Day 7
Day 21
Proximal (Pdd Distal
12.0 2 3.0
11.7 2 3.9
6.9 2 1.5
(Pdd
8.7 2 0.5
7.9 2 4.3
All values are means 2 SD based on n All values are means 2 standard deviation (SD) based on n = 10. " Significantly lower serum concentrations (p < 0.05). Significantly higher cortical bone concentrations (p < 0.05).
'
There were no significant differences in mean serum concentrations of clindamycin between days 3, 7, and 21 during pump infusion (Table 2). Similarly, there were no significant differences in mean cortical bone concentrations of clindamycin between the sampling days. There were, however, significantly greater mean concentrations of clindamycin in the bone than in the serum at each sampling period. The position of the biopsy site (proximal or distal to the outflow catheter) did not have a significant effect on the mean concentration of clindamycin in the sample (Table 3). Furthermore, the day of bone sampling had no significant effect on the concentration of clindamycin at each biopsy site. The three tibias which were samples, 3 centimeters above and below the outflow catheter site, revealed a gradient of cortical concentrations of clindamycin in both directions. Distally, the mean clindamycin concentration in the 5-mm section adjacent to the outflow catheter was 165.7 ? 1.5. In the section from 6 to 10 mm ( 1 cm), the mean concentration decreased to 7.6 3.8, and further decreases were seen in the third section (1.1-1.5 cm distal from the outflow catheter) with a mean clindamycin concentration of 4.2 2 5.1. From 1.5 to 3 cm, the concentrations remained fairly constant at a mean of 1 . 1 k 0.4. Proximal to the outflow catheter a similar gradient was detected with a mean concentration in the first 5-mm section of 8.4 ? 2.9,
*
TABLE 2. Serum and bone Concentrations over the 21-day infusion period at 4 mglkglday Day 3
Day 7
0.8 2 0.2 10.5 i- 2.2"
0.9 2 0.2 9.6 2 1.8"
0.8 ? 0.2 8.4 2 2.3"
Day 21
1,312.5
1,006.7
1,050.0
~~
Serum (pg/ml) Bone (Pdg) Mean bone/serum ratio (%)
____~
All values are means 2 SD based on n = 10. " Significantly greater bone concentration than serum concentrations (p < 0.05).
=
9.8
?
2.0
5.
and a mean concentration of 2.9 2 1.4 in the 6- to 10-mm (1-cm) section. From 1.1 to 3 cm (total of four sections) the mean concentration was 1.1 2 0.6. DISCUSSION
The first objective of this study was to evaluate whether an implantable pump delivery system could achieve effective concentrations in cortical bone for a given dosage regimen. The results of the study indicate that direct infusion of clindamycin at 4 mg/kg/day into the medial tibial cortex produces high concentrations of the drug in the cortical bone. The concentrations achieved greatly exceed the minimum inhibitory concentration measured during in vitro antibiotic sensitivity of the common Grampositive cocci, including Staphylococcus aureus (13,17,27). These concentrations were three to four times higher than those reported in cortical bone using much higher parenteral dosages (10,18,27). There was no clinical indication of tissue inflammation or toxicity to direct infusion of clindamycin. Thus, this form of drug delivery can achieve acceptable drug concentrations in bone. Cortical bone and serum concentrations of clindamycin achieved by direct bone infusion were compared with those obtained after i.v. administration in the second portion of this study. Direct infusion of clindamycin into the tibial cortex, at one tenth the i.v. regimen, yielded significantly greater bone concentrations of the drug than did the repetitive i.v. bolus regimen. Further, direct local infusion produced serum concentrations that were significantly lower than those produced by the i.v. regimen. These findings are similar to data reported for direct infusion of gentamicin into the rabbit femur (23). The mean bone/serum concentration ratio achieved by the i.v. bolus regimen (26.4%) was consistent with the results of previous studies using i.v. and intramuscular routes of administration for clindamycin (10,18,27). Direct infusion of the drug,
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even at lower dosages, increased this ratio about 50 times above the i.v. regimen. Because there were marked differences in the mean boneherum concentration ratios between the two treatment regimens, some of the drug appears to be sequestered within the cortical bone before it enters the systemic circulation when given by direct infusion. A third objective of the study was to determine where this form of antimicrobial delivery could maintain effective concentrations of the drug in the target tissue. Bone concentrations of clindamycin on day 21 of infusion were indistinguishable from those on the third day of infusion. Because this method of drug delivery makes it possible to maintain the high bone concentrations for a sustained period of time at 1 cm from the outflow catheter, it may be useful for the treatment of ischemically infected bone. Furthermore, the serum concentrations remained constantly low throughout the study; thus, drug toxicity potential is less than that during an i.v. regimen. These findings are in contrast to data reported on antibiotic-impregnated methylmethacrylate, where the rate of diffusion of the antimicrobial agent from the cement into the bone is governed by numerous characteristics of the cement and the drug (5). For both impregnated methylmethacrylate and Plaster of Paris the diffusion of the antimicrobial agent varies, with the greatest movement occurring during the first few postoperative days (5,7). Samples of bone were taken either proximal or distal to the outflow catheter to address potential concerns that the biopsy sampling site might effect the concentration of clindamycin in the sample. There was no significant difference between these sites. Our inability to obtain bone samples precisely 1 cm either proximal or distal to the outflow catheter may account for some of the large standard deviation in the cortical bone concentrations of clindamycin reported. Thus, these findings may not accurately reflect a concentration gradient that may be present along a very small portion of the cortical bone. In an attempt to address this concern, three dogs were sampled 3 cm above and below the infusion site in the tibia. The results suggest a gradient within the cortical bone that could explain the large variations seen in other dogs. The gradient decreases markedly about 1.0 cm from the outflow catheter both proximally and distally. Because the Michelle trephine has a 3-mm radius, it is readily apparent why such large variations exist when attempting to sampling at 1 cm. These results also
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indicate that precise positioning of sample sites in future studies is essential for elucidating the concentrations of an antimicrobial agent directly infused into bone. Acknowledgment: This work was supported in part by grants from the University of Georgia Research Foundation and the Veterinary Medical Experiment Station, College of Veterinary Medicine, Athens, Georgia, U.S.A.
REFERENCES 1. Arret B, Johnson DP, Kirshbarum A: Outline of details for microbiological assays of antibiotics: second revision. J Pharm Sci 60:1689-1692, 1971 2. Ascherl R, Stemberger A, Lechner F, et al.: Treatment of chronic osteomyelitis with a collagen-antibiotic compound-preliminary report. Unfallchirurgie 12: 125-127, 1986 3. Braun A, Gussbacher A, Heine WD: Fibrin-antibiotic complex: laboratory investigation and clinical results. In: Uhthoff HK, ed. Current Concepts of Infections in Orthopedic Surgery. Ber1in:Springer-Verlag, 1985 4. Buchholz HW, Elson RA, Heiner K: Antibiotic-loaded acrylic cement: current concepts. Clin Orthop 190:%, 1984 5. Calhoun JH, Mader JT: Antibiotic beads in the management of surgical infections. A m J Surg 157:443-449, 1989 6. Compere EL. Metzger WI, Mitra RW: The treatment of pyogenic bone and joint infections by closed irrigation with a non-toxic detergent and one or more antibiotics. J Bone Joint Surg 49A:614, 1967 7. Dahners LD, Funderburk CH: Gentamicin-loaded plaster of paris as a treatment of experimental osteomyelitis in rabbits. Clin Orthop 219::278-282, 1987 8. DeHaan RM, Metzler CM, Schellenberg D, VandenBosch WD, Masson EL: Pharmacokinetic studies of clindamycin hydrochloride in humans. Int J Clin Pharmacol Ther Toxicol 6: 105-1 19, 1972 9. Dillon RS: Successful treatment of osteomyelitis and soft tissue infections in ischemic diabetic legs by local antibiotic injections and the end-diastole pneumatic compression boot. Ann Surg 204:643-649, 1986 10. Dombusch K, Carlstrom A, Hugo H, Lidstrom A: Antibacterial activity of clindamycin and lincomycin in human bone. J Antimicrob Chemother 3: 153-160, 1977 1 1 . Edberg SC: The measurement of antibiotics in human body fluids: techniques and significance. In: Lorian V, ed. Antibiotics in Laboratory Medicine. 2nd ed, Baltimore: Williams & Wilkins, 1986:381476 12. Fitzgerald RH: Experimental osteomyelitis: description of a canine model and the role of depot administration of antibiotics in the prevention and treatment of sepsis. J Bone Joint Surg 65A:371-380, 1983 13 Geddes AM, Bridgwater FA, Williams DH, Oon J, Grimshaw GJ: Clinical and bacteriological studies with clindamycin. Br Med J 2:703, 1970 14. Hall BB, Fitzgerald RH: The pharmacokinetics of penicillin in osteomyelitic canine bone. J Bone Joint Surg 65-A:526532, 1983 15. Kelly PJ, Martin WJ, Conventry MB: Chronic osteomyelitis. 11. Treatment with closed irrigation and suction. JAMA 213;1843, 1970 16. Mackey D, Varlet A, Debeaumont D: Antibiotic loaded plaster of paris pellets: an in vitro study of a possible method of
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17.
18.
19. 20. 21. 22. 23.
local antibiotic therapy in bone infection. Clin Orthop 167: 263-265, 1982 McGehee RF, Smith CB, Wilcox C, Findland M: Comparative studies of antibacterial activity in vitro and absorption and excretion of lincomycin and clindamycin. Am J Med Sci 256:279-292, 1968 Nicholas P, Meyers BR, Levy RN, Hirschman SZ: Concentration of clindamycin in human bone. Antimicrob Agents Chemother 8:220-221, 1975 Norden CW: Experimental osteomyelitis. 11. Therapeutic trials and measurement of antibiotic levels in bone. J Infect Dis 124565-571, 1971 Organ CH: The utilization of massive doses of antimicrobial agents with isolation perfusion in the treatment of chronic osteomyelitis. Clin Orthop 76:185-189, 1978 Perry CR, Davenport K, Vossen MK: Local delivery of antibiotics via implantable pump in the treatment of osteomyelitis. Clin Orthop 226:222-230, 1988 Perry CR, Pruitt DL: Local administration via an implantable pump for the treatment of chronic osteomyelitis. Hosp Form 23:342-351, 1988 Perry CR, Rice S, Ritterbusch JK, Burdge RE: Local ad-
24.
25. 26. 27.
28. 29.
ministration of antibiotics with an implantable osmotic pump. Clin Orthop 192:284290, 1985 Perry CR, Rice S, Ritterbusch JK, Greenberg RN, Burdge RE, Vossen MK: Antibiotic administration using an implantable drug pump in the treatment of osteomyelitis. Contemp Orthop 10:45-52, 1985 Perry H, Ritterbusch JK, Burdge RE, Perry C R Cefamandole levels in serum and necrotic bone. Clin Orthop 199:28& 283, 1985 Rosin H, Rosin AM, Kramer J: Determination of antibiotic levels in human bone. 1 . Gentamicin levels in bone. Infection 2 : 3 4 , 1974 Schurman DJ, Johnson BL, Finerman G, Amstutz HC: Antibiotic bone penetration: concentrations of methicillin and clindamycin phosphate in human bone taken during total hip replacement. Clin Orthop 11 1:142-146, 1975 Vesci V, Barquet A: Treatment of chronic osteomyelitis by necrectomy and gentamicin PMMA beads. Clin Orthop 159:201-207, 1981 Waldvogel FA, Vasey H: Osteomyelitis: the past decade. N Engl J Med 303:360-370, 1980
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Comparison of Cortical Bone and Serum Concentrations of Clindamycin Achievable by Direct Local Infusion and Intravenous Administration Steven C. Budsberg, *James M. Gallo, ?Clifford E. Starliper, ?Emmett B. Shotts, and ?John Brown Department of Small Animal Medicine, College of Veterinary Medicine, "Department of Pharmaceutics, College of Pharmacy, and ?Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, U.S.A.
Summary: Tibia1 cortical bone and serum concentrations of clindamycin were compared using two drug delivery methods in dogs. An implantable drug pump, used to continuously infuse clindamycin directly into the cortical bone, was compared with clindamycin administered i.v. Dosage for the direct continuous infusion was 4 mglkglday, and 44 mglkglday for the i.v. bolus regimen. Serum concentrations of clindamycin were significantly higher during i.v. bolus administration when compared with those achieved during pump infusion (p < 0.05). However, tibia1 cortical bone concentrations were significantly higher during pump infusion than were those achieved by i.v. bolus. When examining serum and bone clindamycin concentrations over 21 days of direct local infusion, there was no significant difference in concentrations between sampling days within each tissue (p > 0.05). Furthermore, there were significantly greater concentrations of clindamycin in the cortical bone than in the serum at each sampling period (p < 0.05). Results indicate that delivery of clindamycin to canine bone by implantable drug pump achieve significantly higher bone concentrations than i.v. bolus administration of the drug at higher dosages. Direct infusion also can sustain high concentrations in cortical bone without increasing systemic concentrations of clindamycin. Key Words: Clindamycin-Bone concentrations-Direct infusion.
of Paris, direct irrigation-suction units, isolated perfusion of the affected limb, and fibrin-antibiotic complexes (2-7,9,12,15,16,20). Recently, Perry et al. (21-24) successfully treated refractory cases of osteomyelitis using implantable percutaneously refillable pumps that directly and continuously infused antibiotics into the osteomyelitic lesions. Limited data exist regarding antimicrobial distribution and bone antimicrobial concentrations achieved by direct local infusions into the bone (2124). Furthermore, there are no data comparing bone concentrations of antimicrobial agents delivered by
The ability of antimicrobial agents to penetrate and distribute into ischemic infected bone has long been questioned (14,19,25,26,28,29). Consequently, experimental and clinical studies have examined several methods of delivering antibiotics to osteomyelitic lesions. These methods have included antibiotic-impregnated methylmethacrylate or plaster Received April 18, 1990; accepted December 18, 1990. Address correspondence and reprint requests to Steven C. Budsberg, D.V.M., Department of Small Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, CA 30602, U.S.A.
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DIRECT INFUSION OF CLINDAMYCIN INTO BONE this method with those obtained by a systemic dosage regimen. However, the use of direct pump infusion appears promising because it allows, (a) the delivery of unlimited amounts of the desired antimicrobial agent at a constant infusion rate, (b) changes in the dose of the antimicrobial agent or even the antimicrobial agent itself without surgical intervention, (c) reduction in the duration of hospitalization, and (d) a minimum in systemic toxicity due to low systemic antimicrobial concentrations (21-24). The purpose of this study was to determine the concentrations of clindamycin in normal canine tibial cortical bone and serum obtained by continuous local infusion by an implantable pump, and to compare these concentrations to clindamycin concentrations in tibial cortical bone and serum achieved by a multiple-dose i.v. bolus regimen. In addition, we determined the tibial cortical bone and serum concentrations of clindamycin during 3 weeks of continuous local infusion of the drug. Finally, the study evaluated tibial cortical bone concentrations of clindamycin at different sampling sites in relationship to the position of the outflow catheter site. MATERIALS AND METHODS Animals Fifteen adult mixed-breed dogs (30-35 kg) were used in the study. Inclusion criteria included normal findings on physical examination, complete blood count, serum chemistries, urine analysis, and radiographs of the tibias. Experimental Design Twelve dogs were randomly placed into two groups of six dogs and alternated between therapy regimens. Initially, group 1 dogs received clindamycin by i.v. bolus injections. Concurrently, group 2 dogs received clindamycin through pumps implanted in their flanks, with the outflow catheter from the pump placed randomly into the cortical bone of the tibia. Bone biopsy samples (full thickness) from the tibia and serum samples were obtained on days 3 , 7 , and 21 after initiation of clindamycin infusion. After 1 month and documentation that clindamycin was eliminated, dogs were switched to the other regimen using the opposite tibia for bone biopsy and assay. Three dogs had pumps implanted in their flanks
595
and were infused for 3 , 7 , and 14 days at which time the entire tibia was collected and assayed at 5-mm increments, both proximal and distal to the outflow catheter site. I.V. Bolus Protocol Jugular and cephalic vein catheters were placed in each dog, with patency of each catheter maintained with heparinized saline. Each dog was given clindamycin phosphate (Cleocin, Upjohn Pharmaceuticals, Kalamazoo, Michigan, U.S.A.) i.v. at 11 mglkg every 6 hours for 3 days through the cephalic catheter. One hour after the 10th dosage, each dog was anesthetized and a full-thickness cortical bone biopsy sample was taken from the mid-diaphyseal region of the tibia with a Michelle trephine. A whole blood sample was obtained simultaneously from the jugular catheter. Pump Protocol After routine anesthetic induction and maintenance on an inhalant anesthetic, an implantable drug pump (INFUSAID model 400, Shiley INFUSAID Inc., Norwood, Massachusetts, U.S.A.) filled with sterile water was inserted into an S.C. pouch in the caudal dorsal abdominal region (flank). Dogs were then randomized to either right or left tibia for placement of the outflow infusion catheter. The catheter was passed S.C. down the medial thigh to the level of the mid-tibia. A single longitudinal incision was made in the periosteum. Stay sutures were placed in the edges of the periosteum to minimize trauma during manipulation. A hole was drilled into the medial cortex of the mid-diaphyseal region and the outflow catheter was placed in the hole. The periosteum was then closed over the catheter, with simple interrupted sutures. A single suture was placed in the skin at the level of the catheter insertion to serve as a marker. Routine closure of the flank and tibial incisions was performed. Seven days postimplantation, the sterile water was removed and the infusion of clindamycin was initiated. Concurrently, radiographs of the tibia were taken to verify proper placement of the outflow catheters. A clindamycin dosage of 4 mglkglday was administered by the pump. Animals were closely monitored daily for any clinical signs of local inflammation. The dogs were anesthetized on days 3 , 7 , and 21, and bone biopsy samples were obtained with a
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Michelle trephine. The biopsy samples were collected approximately 1 cm proximal and 1 cm distal to the catheter site. The biopsy site was alternated at each sampling period and the position of the biopsy site on the bone was varied to avoid any inflammatory reaction from a previous biopsy. At each sampling period, a serum sample was taken concurrently from the jugular vein. After completion of this portion of the study, each dog was anesthetized for removal of the infusion pump. The three dogs that were only infused at the assigned day (3, 7, and 14) were killed with an overdose of sodium pentobarbital and the entire infused tibia was collected. Sample Preparation Bone marrow was removed from each bone sample and washed in sterile 0.9% saline solution until all visible blood was removed. The periosteum and bone marrow were removed in the three dogs in which the entire tibia was collected. The infused tibia was then cut into 5-mm sections starting at the outflow catheter site extending both proximal and distal for 3 cm. All samples were then frozen at - 70°C until processing. Clindamycin was eluted from the bone as previously described (18,19) with modifications. Briefly, each bone sample was weighed, frozen in liquid nitrogen, and crushed into a powder. Depending on the weight of the sample, 600-1,OOO p1 of 0.1 M phosphate buffer, pH 8.0, was added to each sample. The mixture was vigorously agitated for 40 minutes, incubated at 4°C for 15 hours, and finally centrifuged for 10 minutes at 1,500 rpm. Aliquots (100 pl) of the supernatant were used in the assay procedure. Serum samples were also prepared as previously described (1,8,11). Whole blood was allowed to clot at 4°C for 2 hours, at which time the serum was removed and stored at -70°C until it was assayed. Aliquots (100 p1) of serum were used in the assay. Antibiotic Assay Clindamycin concentrations were measured by microbiological cylinder plate assay for serum and bone samples as previously described (1,8,11, 18,19). Standard curves were derived using clindamycin hydrochloride [Clindamycin HCL (lot no. 531 PY), Upjohn], canine serum, and a canine bonebuffer mixture identical to that made with each sample. Minimum measurable concentrations of clin-
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damycin were 0.3 pglml for serum and 0.4 pg/g for bone homogenate . Data Analysis All data were logarithmically transformed before analysis. When data are presented in text or tables, they appear as geometric means. Differences in serum and bone concentrations between i.v. bolus and pump infusion of clindamycin at 3 days were analyzed with a two-factor analysis of variance. If this value was significant, means of interest were compared using a Tukey test (p < 0.05). Differences in serum and bone concentrations between days 3, 7, and 21 during direct pump infusion were also analyzed with a two-factor analysis of variance. If this statistic was significant, means of interest were compared using a Tukey test (p < 0.05). Data on site of biopsy in relationship to outflow catheter placement (proximal or distal) was analyzed with a two-factor analysis of variance. If the statistic was significant, means of interest were compared with a Tukey test (p < 0.05). The mean bone/serum concentration was calculated by the following formula: mean cortical bone concentrationlmean serum concentration x 100. RESULTS
Ten dogs completed the cross-over study. One dog from each group was removed during the pump infusion phase of the project. The outflow catheter of one dog became displaced and the second dog had a tibial fracture through one of the biopsy sites. There was no clinical evidence of inflammation related to the direct infusion of clindamycin in any of the dogs. The mean (n = 10) serum clindamycin concentration 1 hour after the 10th dose of the i.v. bolus regimen was 8.7 rfr 0.2 pg/ml. The concurrent mean tibial bone concentration was 2.4 rfr 0.2 pg/g (Table 1). The mean serum clindamycin concentration at a similar time (day 3) during continuous pump infusion was 0.8 rfr 0.2 pg/ml, with concurrent mean tibial cortical bone concentrations of 10.5 k 2.2 pg/g. Mean serum concentrations of clindamycin were significantly higher during the i.v. bolus regimen when compared with those achieved during pump infusion. Conversely, mean tibial cortical bone concentrations were significantly higher during pump infusion than those measured after i.v. bolus administration of the drug.
DIRECT INFUSION OF CLINDAMYCIN INTO BONE TABLE 1. Serum and bone concentrations of clindamycin on day 3 of administration
Serum (pg/ml) Bone (Pg/g) Mean bonekerum ratio (%)
597
TABLE 3. Tibia1 bone concentrations divided into sampling site in relationship to outflow catheter
i.v. Bolus (44 mg/kg/day)
Direct bone infusion (4 mg/kg/day)
8.7 2 0.2 2.4 2 0.2
0.8 ? 0.2" 10.5 2 2.2'
31.4
1,312.5
Day 3
Day 7
Day 21
Proximal (Pdd Distal
12.0 2 3.0
11.7 2 3.9
6.9 2 1.5
(Pdd
8.7 2 0.5
7.9 2 4.3
All values are means 2 SD based on n All values are means 2 standard deviation (SD) based on n = 10. " Significantly lower serum concentrations (p < 0.05). Significantly higher cortical bone concentrations (p < 0.05).
'
There were no significant differences in mean serum concentrations of clindamycin between days 3, 7, and 21 during pump infusion (Table 2). Similarly, there were no significant differences in mean cortical bone concentrations of clindamycin between the sampling days. There were, however, significantly greater mean concentrations of clindamycin in the bone than in the serum at each sampling period. The position of the biopsy site (proximal or distal to the outflow catheter) did not have a significant effect on the mean concentration of clindamycin in the sample (Table 3). Furthermore, the day of bone sampling had no significant effect on the concentration of clindamycin at each biopsy site. The three tibias which were samples, 3 centimeters above and below the outflow catheter site, revealed a gradient of cortical concentrations of clindamycin in both directions. Distally, the mean clindamycin concentration in the 5-mm section adjacent to the outflow catheter was 165.7 ? 1.5. In the section from 6 to 10 mm ( 1 cm), the mean concentration decreased to 7.6 3.8, and further decreases were seen in the third section (1.1-1.5 cm distal from the outflow catheter) with a mean clindamycin concentration of 4.2 2 5.1. From 1.5 to 3 cm, the concentrations remained fairly constant at a mean of 1 . 1 k 0.4. Proximal to the outflow catheter a similar gradient was detected with a mean concentration in the first 5-mm section of 8.4 ? 2.9,
*
TABLE 2. Serum and bone Concentrations over the 21-day infusion period at 4 mglkglday Day 3
Day 7
0.8 2 0.2 10.5 i- 2.2"
0.9 2 0.2 9.6 2 1.8"
0.8 ? 0.2 8.4 2 2.3"
Day 21
1,312.5
1,006.7
1,050.0
~~
Serum (pg/ml) Bone (Pdg) Mean bone/serum ratio (%)
____~
All values are means 2 SD based on n = 10. " Significantly greater bone concentration than serum concentrations (p < 0.05).
=
9.8
?
2.0
5.
and a mean concentration of 2.9 2 1.4 in the 6- to 10-mm (1-cm) section. From 1.1 to 3 cm (total of four sections) the mean concentration was 1.1 2 0.6. DISCUSSION
The first objective of this study was to evaluate whether an implantable pump delivery system could achieve effective concentrations in cortical bone for a given dosage regimen. The results of the study indicate that direct infusion of clindamycin at 4 mg/kg/day into the medial tibial cortex produces high concentrations of the drug in the cortical bone. The concentrations achieved greatly exceed the minimum inhibitory concentration measured during in vitro antibiotic sensitivity of the common Grampositive cocci, including Staphylococcus aureus (13,17,27). These concentrations were three to four times higher than those reported in cortical bone using much higher parenteral dosages (10,18,27). There was no clinical indication of tissue inflammation or toxicity to direct infusion of clindamycin. Thus, this form of drug delivery can achieve acceptable drug concentrations in bone. Cortical bone and serum concentrations of clindamycin achieved by direct bone infusion were compared with those obtained after i.v. administration in the second portion of this study. Direct infusion of clindamycin into the tibial cortex, at one tenth the i.v. regimen, yielded significantly greater bone concentrations of the drug than did the repetitive i.v. bolus regimen. Further, direct local infusion produced serum concentrations that were significantly lower than those produced by the i.v. regimen. These findings are similar to data reported for direct infusion of gentamicin into the rabbit femur (23). The mean bone/serum concentration ratio achieved by the i.v. bolus regimen (26.4%) was consistent with the results of previous studies using i.v. and intramuscular routes of administration for clindamycin (10,18,27). Direct infusion of the drug,
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S . C . BUDSBERG ET AL.
even at lower dosages, increased this ratio about 50 times above the i.v. regimen. Because there were marked differences in the mean boneherum concentration ratios between the two treatment regimens, some of the drug appears to be sequestered within the cortical bone before it enters the systemic circulation when given by direct infusion. A third objective of the study was to determine where this form of antimicrobial delivery could maintain effective concentrations of the drug in the target tissue. Bone concentrations of clindamycin on day 21 of infusion were indistinguishable from those on the third day of infusion. Because this method of drug delivery makes it possible to maintain the high bone concentrations for a sustained period of time at 1 cm from the outflow catheter, it may be useful for the treatment of ischemically infected bone. Furthermore, the serum concentrations remained constantly low throughout the study; thus, drug toxicity potential is less than that during an i.v. regimen. These findings are in contrast to data reported on antibiotic-impregnated methylmethacrylate, where the rate of diffusion of the antimicrobial agent from the cement into the bone is governed by numerous characteristics of the cement and the drug (5). For both impregnated methylmethacrylate and Plaster of Paris the diffusion of the antimicrobial agent varies, with the greatest movement occurring during the first few postoperative days (5,7). Samples of bone were taken either proximal or distal to the outflow catheter to address potential concerns that the biopsy sampling site might effect the concentration of clindamycin in the sample. There was no significant difference between these sites. Our inability to obtain bone samples precisely 1 cm either proximal or distal to the outflow catheter may account for some of the large standard deviation in the cortical bone concentrations of clindamycin reported. Thus, these findings may not accurately reflect a concentration gradient that may be present along a very small portion of the cortical bone. In an attempt to address this concern, three dogs were sampled 3 cm above and below the infusion site in the tibia. The results suggest a gradient within the cortical bone that could explain the large variations seen in other dogs. The gradient decreases markedly about 1.0 cm from the outflow catheter both proximally and distally. Because the Michelle trephine has a 3-mm radius, it is readily apparent why such large variations exist when attempting to sampling at 1 cm. These results also
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indicate that precise positioning of sample sites in future studies is essential for elucidating the concentrations of an antimicrobial agent directly infused into bone. Acknowledgment: This work was supported in part by grants from the University of Georgia Research Foundation and the Veterinary Medical Experiment Station, College of Veterinary Medicine, Athens, Georgia, U.S.A.
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