Carboplatin (cis-diammine-1,1-cycobutane dicarboxylate platinum II) is a second-generation platinum-containing chemotherapeutic agent that has been safely used in dogs and cats and is reported to have less severe nephrotoxic and emetogenic effects than cisplatin.1–3 Additionally, recent studies1,4–11 have demonstrated cytotoxicity of carboplatin against appendicular osteosarcoma and oral malignant melanoma in dogs as well as oral and cutaneous squamous cell carcinoma in cats.
The adverse effects associated with platinum-based chemotherapeutics include nephrotoxicosis, myelosuppressive effects such as neutropenia and thrombocytopenia and gastrointestinal effects such as nausea, vomiting, and inappetence.4,12–14 To decrease the incidence of toxicosis and adverse events in treated patients, local, intralesional, and targeted chemotherapeutic protocols for use of cisplatin and carboplatin have been developed to treat local disease.4,12–20 Results of several investigations suggest that direct chemotherapy (ie, intratumoral injection of a single systemic dose of a chemotherapeutic agent) may be associated with short duration of action and substantial uptake into the bloodstream.1,21–23 In contrast, delivery systems allowing sustained, local release of a chemotherapeutic agent for the treatment of local disease offer the advantage of achieving high concentrations at the tumor site with minimal to no systemic toxicosis.12–21,24 In dogs, some sustained-release delivery systems for chemotherapeutic agents have efficacy against appendicular osteosarcoma and nasal tumors (cisplatin) as well as soft tissue sarcomas (carboplatin).15,20,24–27 However, these delivery systems are not commercially available, have been associated with unacceptable regional toxic effects including development of wound dehiscence and local infection, or have resulted in equivocal clinical improvement.15,18,20,24
Carboplatin-impregnated CSH beads are a commercially available drug delivery system for sustained release of carboplatin and can be implanted at sites of grossly evident tumor or of marginal tumor extirpation.22,28 A proven depot for drug release,29–34 CSH is biodegradable, biocompatible, inexpensive, readily available, and sterilizable by γ-irradiation. Published studies22,28 and anecdotal reportsa have shown promising results for cisplatin- and carboplatin-impregnated biodegradable beads as treatment for various tumors in horses and soft tissue sarcomas in dogs. However, to the authors’ knowledge, no published data exist regarding the rate, pattern, and duration of elution of platinum from commercially available carboplatin-impregnated CSH beads.
The purpose of the study reported here was to evaluate whether platinum elutes from carboplatin-impregnated CSH beads in vitro and, if so, to determine the initial pattern of release of platinum. We hypothesized that platinum would elute from carboplatin-impregnated CSH beads into PBS at concentrations greater than the peak plasma concentration reported in dogs following administration of a single IV dose of carboplatin (300 mg/m2 of body surface area).2,4 We also hypothesized that the concentration of platinum in the eluent would be positively associated with the number of carboplatin-impregnated beads placed together in a sample tube.
Materials and Methods
Carboplatin-impregnated beads were created at an accredited compounding pharmacy.b Briefly, a forged metal bead mold with a synthetic polytetrafluoroethylene-based coatingc was used to create chains of uniform, 3-mm-diameter beadsd containing either 4.6 mg of carboplatin (2.4 mg of platinum) with 18.4 mg of CSH or 23.0 mg of CSH (used as a control); both formulations included dextran (at a final concentration of 0.67 mg/bead; added to slow release of the agent).
All beads were formed and evaluated in triplicate (triplicate groups A, B, and C). For each experiment, carboplatin-impregnated CSH beads were placed in individual 10-mL plastic tubes in groups of 1, 3, 6, or 10 with 5 mL of PBSS, and 3 control beads were placed together in another tube with the same volume of PBSS. The tubes were maintained at 37°C and a pH of 7.4 with constant agitation. The eluent was sampled by evacuation of all 5 mL of the PBSS at 1, 2, 3, 6, 12, 24, and 72 hours (with initial placement of the beads in solution considered time 0). The evacuated fluid was replaced with 5 mL of fresh PBSS at each time point. Eluent samples were analyzed for platinum content by inductively coupled plasmamass spectrometry (limit of detection, 0.1 ppm).35,e Beads were monitored for signs of dissolution, including grossly discernible changes to the surface of the bead, opacity of the eluent, or accumulation of particulate matter in the eluent.
Statistical analysis
Distribution of continuous data was evaluated by means of a Shapiro-Wilk test, assessment of skewness and kurtosis, and Q-Q plots. Data for hour 1 through hour 24 were normally distributed; the 72-hour data were not. The non normally distributed data were reported as median, 10th to 90th percentiles, and range. Data that were normally distributed were reported as mean, SD, and minimum-maximum (range). Estimated marginal mean ± SEM data and 95% confidence intervals were reported for normally distributed values over time. Nonnormally distributed data were log-transformed for parametric analysis. A repeated-measures general linear model was used to determine whether there was a difference in platinum concentrations in the eluent fluid over time (within subjects); by triplicate group A, B, or C (between subjects); and by number of beads per tube (between subjects). This was also done for evaluation of the percentage of total incorporated platinum eluted by the beads. A Mauchly test for sphericity was used to evaluate the homogeneity of covariance. Because the homogeneity of covariance was violated, the Greenhouse-Geisser method was used to interpret the results. The total amount of platinum released by each bead was calculated by adding the total milligrams of platinum released over 72 hours and dividing by the number of beads. A 1-way ANOVA was then used to determine whether there was a difference in the amount of platinum released per bead among groups containing different numbers of beads. A commercially available statistical software programf was used to analyze the data. Values of P < 0.05 were considered significant.
Results
There was a significant (P < 0.001) difference in platinum concentrations in the eluents for carboplatin-containing beads over time, with amounts increasing over the first 12 hours and then declining thereafter for all tubes (Figure 1). Control beads did not elute detectable levels of platinum at any time point. There was also a significant (P < 0.001) difference in the total amount of platinum released over 72 hours when results were compared for tubes containing different numbers of carboplatin-impregnated beads, with mean platinum concentrations in the eluent increasing significantly with increasing number of beads per tube (estimated marginal means: 1 bead, 63.4 mg/L; 3 beads, 201.3 mg/L; 6 beads, 377.7 mg/L; 10 beads, 609.3 mg/L). There was no significant (P = 0.974) difference in platinum concentrations of the eluents among triplicates A, B, and C.
Cumulative platinum concentration (mean ± SD of triplicate experiments) in the eluent of carboplatin-impregnated CSH beads placed in groups of 1 (blue bars), 3 (red bars), 6 (green bars), or 10 (black bars) into tubes of PBSS (pH, 7.4) at 37°C. Each bead contained 4.6 mg of carboplatin (2.4 mg of platinum) and 18.4 mg of CSH with dextran. Initial placement of the beads into PBSS was considered time 0; all eluent was removed for analysis and replaced with 5 mL of fresh PBSS at each time point.
Citation: American Journal of Veterinary Research 77, 11; 10.2460/ajvr.77.11.1252
The mean ± SD concentration of platinum released per bead over 72 hours was 445.3 ± 31.5 mg/L (range, 390.0 to 509.1 mg/L). There was no significant (P = 0.488) difference in amount of platinum released per bead when results for all beads over all time points were compared. There was, however, a significant (P < 0.001) difference in the percentage of total incorporated platinum released over time, with the values for all time points significantly different from one another except for the last 2 time points (Table 1). There was also a significant (P = 0.001) difference in the percentage of total incorporated platinum released into tubes with different numbers of beads, with the percentage of platinum eluted significantly higher in tubes with 1 or 3 beads than in tubes with 6 or 10 beads (Table 2). After 12 hours, 89%, 87%, 79%, and 67% of the incorporated platinum had been released in the tubes with 1, 3, 6, and 10 beads, respectively; at 72 hours, 92%, 97%, 91%, and 88% of the incorporated platinum had been released in the tubes with 1, 3, 6, and 10 beads, respectively (Figure 2).
Cumulative percentage (mean ± SD of triplicate experiments) of the total incorporated platinum eluted from carboplatin-impregnated CSH beads into PBSS at predetermined time points (same sample as in Figure I). See Figure I of key.
Citation: American Journal of Veterinary Research 77, 11; 10.2460/ajvr.77.11.1252
Mean and distribution of percentage total incorporated platinum eluted from carboplatin-impregnated CSH beads into PBSS at predetermined time points, irrespective of the number of beads per tube.
| Time (h) | Mean ± SD (%) | Range (%) |
|---|---|---|
| 1 | 14.93 ± 4.31 | 8.54–22.11 |
| 2 | 30.26 ± 6.37 | 21.27–39.92 |
| 3 | 43.56 ± 7.20 | 33.04–54.36 |
| 6 | 62.18 ± 9.65 | 48.18–78.33 |
| 12 | 80.99 ± 10.58 | 66.59–102.50 |
| 24 | 88.95 ± 9.17 | 74.02–105.14 |
| 72 | 92.06 ± 6.46 | 80.62–105.19 |
Carboplatin-impregnated CSH beads were placed in groups of 1, 3, 6, or 10 into tubes of PBSS (pH, 7.4) at 37°C. Each bead contained 4.6 mg of carboplatin (2.4 mg of platinum) and 18.4 mg of CSH with dextran. Each group was tested in triplicate. Initial placement of the beads into PBSS was considered time 0; all eluent was removed for analysis and replaced with 5 mL of fresh PBSS at each time point.
Results for all time points were significantly (P < 0.001) different from each other except for 24 versus 72 hours (P = 0.054)
Cumulative percentage of total platinum content eluted from carboplatin-impregnated CSH beads into PBSS across all time points for the same sample as in Table 1.
| Group | Mean ± SEM (%) | 95% CI (%) |
|---|---|---|
| 1 bead | 65.0 ± 1.78*† | 60.9–69.1 |
| 3 beads | 63.6 ± 1.78‡§ | 59.5–67.7 |
| 6 beads | 57.7 ± 1.78*‡‖ | 53.6–61.8 |
| 10 beads | 49.5 ± 1.78†§‖ | 45.4–53.6 |
Beads were placed in 5 mL of PBSS (pH, 7.4) at 37°C in groups of 1, 3, 6, or 10 for 72 hours.
Values with the same symbols were significantly (P = 0.02) differently.
Values with the same symbols were significantly (P < 0.001) differently.
Values with the same symbols were significantly (P = 0.048) differently.
Values with the same symbols were significantly (P = 0.001) differently.
Values with the same symbols were significantly (P = 0.012) differently.
CI = Confidence interval.
See Table 1 for remainder of key.
Grossly, the beads showed minor changes consistent with dissolution over the study period. By 24 hours, the surface of the beads appeared slightly roughened, and the eluent appeared slightly cloudy on aspiration. These changes had subjectively progressed slightly by 72 hours, but the beads did not completely dissolve and did not disintegrate substantially over the study period.
Discussion
Carboplatin exerts its antitumor effects by binding and cross-linking DNA, resulting in non–cell cycle dependent tumor cell lysis.3,12 It was developed as a safer alternative to cisplatin and is an effective agent against solid tumors in dogs and cats.2,4,5 Specifically, it has been found to be useful in treating osteosarcoma, oral melanoma, and anal sac adenocarcinoma.1,4–10,30,37 Carboplatin is typically administered IV, but it can also be given subcutaneously, intraperitoneally, and intratumorally. The primary dose-limiting toxic effect of the drug is myelosuppression characterized by neutropenia and thrombocytopenia.1,3,4,12–14
The doses of carboplatin administered systemically to dogs and cats are determined on the basis of the maximally tolerated dose (ie, the highest dose associated with an acceptable degree of toxicosis).4 For dogs, the typical dosage is 300 mg of carboplatin/m2 of body surface area, IV, every 21 days. This dose achieves a peak plasma concentration of approximately 80 mg of carboplatin/L (42.1 mg of platinum/L) 4 to 6 hours after IV administration.2,4 In the present study, carboplatin-impregnated beads in all tubes (containing 1, 3, 6, or 10 beads) eluted concentrations of platinum greater than or equal to this peak plasma concentration for ≥ 12 hours, indicating that these beads should be at least as effective locally against tumor cells as a single dose of carboplatin administered IV.
Concerning strategies for local chemotherapy, there have been 2 studies7,38 evaluating the effect of carboplatin on canine tumor cell lines and IC50 of the drug in vitro. Determination of IC50 values for dose-effect testing of drugs has been used to assess efficacy of chemotherapeutic agents in human and veterinary medicine.7,39–41 In the in vitro studies7,38 with canine tumor cell lines, the IC50s for mammary carcinoma, melanoma, and transitional cell carcinoma at 72 hours were found to be between 2.2 and 11.3 mg of carboplatin/L (1.2 to 5.9 mg of platinum/L). In our study, platinum concentrations were greater than these values for the entire 72-hour period in all tubes with carboplatin-impregnated beads except those containing only 1 bead, suggesting that implantation of ≥ 3 beads in a tumor bed may effectively inhibit growth of these tumors. However, the duration for which platinum concentrations must be sustained above a minimal concentration to achieve long-term tumor control is not currently known. The IC50s appeared to be time dependent in the study7 of mammary carcinoma cells, decreasing by > 50% between 24 and 72 hours for carboplatin, indicating that tumor cell susceptibility may increase over time. Additional studies are needed to determine the time period for which such a concentration must be maintained.
In the present study, platinum release increased rapidly during the first 12 hours and declined thereafter. Burst release, a phenomenon of initial rapid release of a compound from a substrate, is characteristic of elution of various compounds from CSH and polymethylmethacrylate beads, with 50% to 90% of total elution often occurring in the first 24 hours.29–31,42,43 Burst release has been attributed to diffusion of the impregnated, hydrophilic compound from the exposed surface of the beads into the surrounding eluent.12,17,32 Subsequent release occurs as a result of diffusion of the compound along the concentration gradient between the center and periphery of the bead.33,34,44 Complete release of the compound occurs upon dissolution of biodegradable substrates such as CSH, and release may continue until complete dissolution has occurred.12,32,33,45–47 However, it is not known whether burst release of a chemotherapeutic agent is desirable for tumor control, and the ideal rate, pattern, and duration of elution of platinum from any substrate have not been determined. The burst effect was diminished and release of compound was more sustained when CSH beads were wrapped with either porcine small intestinal submucosa or biodegradable poly(lactide-co-glycolide).32,48 Additionally, carboplatin poly(l-lactide) microspheres have been dispersed in a thermosensitive biodegradable gel to prevent burst release and to prolong total release.12 It has been suggested that wrapping the beads or dispersing them in gel diminishes the increase in porosity that occurs upon dissolution, thus protecting the CSH construct from further dissolution by the eluent.12,32,48
When all time points were evaluated, there was a significant difference in the percentage of total platinum eluted into tubes containing different numbers of beads. The percentage of total incorporated platinum eluted was significantly higher in tubes that contained 1 or 3 beads than in those that contained 6 or 10 beads, when all time points were compared. This difference was most notable early in the study; 89% and 86%, respectively, of incorporated platinum was eluted into the tubes with 1 and 3 beads during the first 12 hours, as compared with only 79% and 67%, respectively, in the tubes with 6 and 10 beads. This difference in burst release among bead groups may have been attributable to the physical presence of more beads in the tubes with 6 and 10 beads. The beads were placed in 5 mL of PBS within a conical test tube, and beads typically clustered and settled at the base of the tube. As the number of beads per tube increased, less of the total surface area of the beads would be exposed to the surrounding eluent owing to clustering of the beads. As the initial release of compound from a bead is thought to occur via diffusion of the compound from the exposed surface of the bead, a decrease in the exposed surface area of the beads in the tubes with 6 and 10 beads may have resulted in a slower initial release of platinum. It is possible that platinum was released more rapidly from beads in these groups once dissolution of the beads began, equalizing release among all bead groups over the entire study period. Consequently, over the 72-hour period evaluated, the mean ± SD concentration of platinum released per bead was 445.34 ± 31.5 mg/L, with no difference observed in platinum concentration produced per bead regardless of the number of beads in the tube. This information may have added benefit once concentrations of carboplatin necessary for local tumor control and diffusion characteristics in living tissues are known, and the information could be used to help determine how many beads should be implanted into a tumor bed in future clinical research.
Acknowledgments
Supported in part by the Sam and Nancy Lerner Award, University of Illinois Companion Animal Memorial Fund.
The authors declare that there were no conflicts of interest.
Results of this study were presented in part at the Annual Conference of the Veterinary Cancer Society, St Louis, October 2014.
The authors thank Anna Stobnicki for literature searches and assistance with laboratory work.
All beads used in the study were donated by Wedgewood Pharmacy.
ABBREVIATIONS
| CSH | Calcium sulfate semihydrate |
| IC50 | Molar concentration of compound that inhibits specific activity by 50% |
Footnotes
Hess T, Miller J, Fettig A, et al. Treatment of canine subcutaneous soft tissue sarcomas with surgical excision and intra-operative placement of platinum-containing biodegradable beads (abstr), in Proceedings. Annu Conf Vet Cancer Soc 2012;124.
Wedgewood Pharmacy, Swedesboro, NJ.
University of Vermont Instrumentation and Modeling Facility, Burlington, Vt.
Matrix III, US Patent 6391336, Royer Biomedical Inc, Frederick, Md.
Midwest Laboratories Inc, Omaha, Neb.
SPSS, version 23.0, SPSS Inc, Armonk, NY.
References
1. Simcock JO, Withers SS, Prpich CY, et al. Evaluation of a single subcutaneous infusion of carboplatin as adjuvant chemotherapy for dogs with osteosarcoma: 17 cases (2006–2010). J Am Vet Med Assoc 2012; 241:608–614.
2. Gaver RC, George AM, Duncan GF, et al. The disposition of carboplatin in the Beagle dog. Cancer Chemother Pharmacol 1988; 21:197–202.
3. Fox LE. Carboplatin. J Am Anim Hosp Assoc 2000; 36:13–14.
4. Page RL, McEntee MC, George SL, et al. Pharmacokinetic and phase I evaluation of carboplatin in dogs. J Vet Intern Med 1993; 7:235–240.
5. Kisseberth WC, Vail DM, Yaissle J, et al. Phase I clinical evaluation of carboplatin in tumor-bearing cats: a Veterinary Cooperative Oncology Group study. J Vet Intern Med 2008; 22:83–88.
6. Brockley LK, Cooper MA, Bennett PF. Malignant melanoma in 63 dogs (2001–2011): the effect of carboplatin chemotherapy on survival. N Z Vet J 2013; 61:25–31.
7. Simon D, Knebel JW, Baumgartner W, et al. In vitro efficacy of chemotherapeutics as determined by 50% inhibitory concentrations in cell cultures of mammary gland tumors obtained from dogs. Am J Vet Res 2001; 62:1825–1830.
8. Bergman PJ, MacEwen EG, Kurzman ID, et al. Amputation and carboplatin for treatment of dogs with osteosarcoma: 48 cases (1991 to 1993). J Vet Intern Med 1996; 10:76–81.
9. Selmic LE, Burton JH, Thamm DH, et al. Comparison of carboplatin and doxorubicin-based chemotherapy protocols in 470 dogs after amputation for treatment of appendicular osteosarcoma. J Vet Intern Med 2014; 28:554–563.
10. Rassnick KM, Ruslander DM, Cotter SM, et al. Use of carboplatin for treatment of dogs with malignant melanoma: 27 cases (1989–2000). J Am Vet Med Assoc 2001; 218:1444–1448.
11. Théon AP, VanVechten MK, Madewell BR. Intratumoral administration of carboplatin for treatment of squamous cell carcinomas of the nasal plane in cats. Am J Vet Res 1996; 57:205–210.
12. Mittal A, Chitkara D, Kumar N. HPLC method for the determination of carboplatin and paclitaxel with cremophorEL in an amphiphilic polymer matrix. J Chromatogr B Analyt Technol Biomed Life Sci 2007; 855:211–219.
13. Xiong Y, Jiang W, Shen Y, et al. A poly(gamma, l-glutamic acid)-citric acid based nanoconjugate for cisplatin delivery. Biomaterials 2012; 33:7182–7193.
14. Gavini E, Manunta L, Giua S, et al. Spray-dried poly(d,l-lactide) microspheres containing carboplatin for veterinary use: in vitro and in vivo studies. AAPS PharmSciTech 2005; 6. E108–E114.
15. Withrow SJ, Liptak JM, Straw RC, et al. Biodegradable cisplatin polymer in limb-sparing surgery for canine osteosarcoma. Ann Surg Oncol 2004; 11:705–713.
16. Araki H, Tani T, Kodama M. Antitumor effect of cisplatin incorporated into polylactic acid microcapsules. Artif Organs 1999; 23:161–168.
17. Manunta ML, Gavini E, Chessa G, et al. Carboplatin sustained delivery system using injectable microspheres. J Vet Med A Physiol Pathol Clin Med 2005; 52:416–422.
18. Havlicek M, Straw RS, Langova V, et al. Intra-operative cisplatin for the treatment of canine extremity soft tissue sarcomas. Vet Comp Oncol 2009; 7:122–129.
19. Arlt M, Haase D, Hampel S, et al. Delivery of carboplatin by carbon-based nanocontainers mediates increased cancer cell death. Nanotechnology 2010; 21:335101.
20. Venable RO, Worley DR, Gustafson DL, et al. Effects of intratumoral administration of a hyaluronan-cisplatin nanoconjugate to five dogs with soft tissue sarcomas. Am J Vet Res 2012; 73:1969–1976.
21. Dernell WS, Withrow SJ, Straw RC, et al. Adjuvant chemotherapy using cisplatin by subcutaneous administration. In Vivo 1997; 11:345–350.
22. Hewes CA, Sullins KE. Use of cisplatin-containing biodegradable beads for treatment of cutaneous neoplasia in equidae: 59 cases (2000–2004). J Am Vet Med Assoc 2006; 229:1617–1622.
23. Begg AC, Bartelink H, Stewart FA, et al. Improvement of differential toxicity between tumor and normal tissues using intratumoral injection with or without a slow-drug-release matrix system. NCI Monogr 1988; 6:133–136.
24. Dernell WS, Withrow SJ, Straw RC, et al. Intracavitary treatment of soft tissue sarcomas in dogs using cisplatin in a biodegradable polymer. Anticancer Res 1997; 17:4499–4505.
25. Mehl ML, Seguin B, Dernell WS, et al. Survival analysis of one versus two treatments of local delivery cisplatin in a biodegradable polymer for canine osteosarcoma. Vet Comp Oncol 2005; 3:81–86.
26. Straw RC, Withrow SJ, Douple EB, et al. Effects of cis-diamminedichloroplatinum II released from d,l-polylactic acid implanted adjacent to cortical allografts in dogs. J Orthop Res 1994; 12:871–877.
27. Lana SE, Dernell WS, LaRue SM, et al. Slow release cisplatin combined with radiation for the treatment of canine nasal tumors. Vet Radiol Ultrasound 1997; 38:474–478.
28. Bergman NS, Urie BK, Pardo AD, et al. Evaluation of local toxic effects and outcomes for dogs undergoing marginal tumor excision with intralesional cisplatin-impregnated bead placement for treatment of soft tissue sarcomas: 62 cases (2009–2012). J Am Vet Med Assoc 2016; 248:1148–1156.
29. Atilla A, Boothe HW, Tollett M, et al. In vitro elution of amikacin and vancomycin from impregnated plaster of Paris beads. Vet Surg 2010; 39:715–721.
30. Santschi EM, McGarvey L. In vitro elution of gentamicin from plaster of Paris beads. Vet Surg 2003; 32:128–133.
31. Bowyer GW, Cumberland N. Antibiotic release from impregnated pellets and beads. J Trauma 1994; 36:331–335.
32. Seddighi MR, Griffon DJ, Constable PD, et al. Effects of porcine small intestinal submucosa on elution characteristics of gentamicin-impregnated plaster of Paris. Am J Vet Res 2007; 68:171–177.
33. Dacquet V, Varlet A, Tandogan RN, et al. Antibiotic-impregnated plaster of Paris beads. Trials with teicoplanin. Clin Orthop Relat Res 1992; 282:241–249.
34. Rosenblum SF, Frenkel S, Ricci JR, et al. Diffusion of fibroblast growth factor from a plaster of Paris carrier. J Appl Biomater 1993; 4:67–72.
35. US Environmental Protection Agency. Method 6020. Inductively coupled plasma—mass spectrometry. Washington, DC: Environmental Protection Agency, 1994. Available at: www.epa.gov/sw-846/pdfs/6020.pdf. Accessed Sept 14, 2014.
36. Dank G, Rassnick KM, Sokolovsky Y, et al. Use of adjuvant carboplatin for treatment of dogs with oral malignant melanoma following surgical excision. Vet Comp Oncol 2014; 12:78–84.
37. Bennett PF, DeNicola DB, Bonney P, et al. Canine anal sac adenocarcinomas: clinical presentation and response to therapy. J Vet Intern Med 2002; 16:100–104.
38. Knapp DW, Chan TC, Kuczek T, et al. Evaluation of in vitro cytotoxicity of nonsteroidal anti-inflammatory drugs against canine tumor cells. Am J Vet Res 1995; 56:801–805.
39. Sartin EA, Barnes S, Toivio-Kinnucan M, et al. Heterogenic properties of clonal cell lines derived from canine mammary carcinomas and sensitivity to tamoxifen and doxorubicin. Anticancer Res 1993; 13:229–236.
40. Carmichael J, DeGraff WG, Gazdar AF, et al. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity. Cancer Res 1987; 47:943–946.
41. Von Hoff DD, Sandbach JF, Clark GM, et al. Selection of cancer chemotherapy for a patient by an in vitro assay versus a clinician. J Natl Cancer Inst 1990; 82:110–116.
42. Rasyid HN, van der Mei HC, Frijlink HW, et al. Concepts for increasing gentamicin release from handmade bone cement beads. Acta Orthop 2009; 80:508–513.
43. Phillips H, Boothe DM, Shofer F, et al. In vitro elution studies of amikacin and cefazolin from polymethylmethacrylate. Vet Surg 2007; 36:272–278.
44. Seeley SK, Seeley JV, Telehowski P, et al. Volume and surface area study of tobramycin-polymethylmethacrylate beads. Clin Orthop Relat Res 2004;298–303.
45. Streppa HK, Singer MJ, Budsberg SC. Applications of local antimicrobial delivery systems in veterinary medicine. J Am Vet Med Assoc 2001; 219:40–48.
46. Wichelhaus TA, Dingeldein E, Rauschmann M, et al. Elution characteristics of vancomycin, teicoplanin, gentamicin and clindamycin from calcium sulphate beads. J Antimicrob Chemother 2001; 48:117–119.
47. Hayes G, Moens N, Gibson T. A review of local antibiotic implants and applications to veterinary orthopaedic surgery. Vet Comp Orthop Traumatol 2013; 26:251–259.
48. Benoit MA, Mousset B, Delloye C, et al. Antibiotic-loaded plaster of Paris implants coated with poly lactide-co-glycolide as a controlled release delivery system for the treatment of bone infections. Int Orthop 1997; 21:403–408.
