• View in gallery

    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.

  • View in gallery

    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.

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Elution of platinum from carboplatin-impregnated calcium sulfate hemihydrate beads in vitro

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  • 1 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 60801.
  • | 2 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 60801.
  • | 3 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 60801.
  • | 4 Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, IL 60801.
  • | 5 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 60801.

Abstract

OBJECTIVE To characterize the elution of platinum from carboplatin-impregnated calcium sulfate hemihydrate (CSH) beads in vitro.

SAMPLE 60 carboplatin-impregnated CSH beads and 9 CSH beads without added carboplatin (controls).

PROCEDURES Carboplatin-impregnated CSH beads (each containing 4.6 mg of carboplatin [2.4 mg of platinum]) were placed into separate 10-mL plastic tubes containing 5 mL of PBSS in groups of 1, 3, 6, or 10; 3 control beads were placed into a single tube of PBSS at the same volume. Experiments were conducted in triplicate at 37°C and a pH of 7.4 with constant agitation. Eluent samples were collected at 1, 2, 3, 6, 12, 24, and 72 hours. Samples were analyzed for platinum content by inductively coupled plasma–mass spectrometry.

RESULTS The mean concentration of platinum released per carboplatin-impregnated bead over 72 hours was 445.3 mg/L. Cumulative concentrations of platinum eluted increased as the number of beads per tube increased. There was a significant difference in platinum concentrations over time, with values increasing over the first 12 hours and then declining for all tubes. There was also a significant difference in percentage of total incorporated platinum released into tubes with different numbers of beads: the percentage of eluted platinum was higher in tubes containing 1 or 3 beads than in those containing 6 or 10 beads.

CONCLUSIONS AND CLINICAL RELEVANCE Carboplatin-impregnated CSH beads eluted platinum over 72 hours. Further studies are needed to determine whether implantation of carboplatin-impregnated CSH beads results in detectable levels of platinum systemically and whether the platinum concentrations eluted locally are toxic to tumor cells.

Abstract

OBJECTIVE To characterize the elution of platinum from carboplatin-impregnated calcium sulfate hemihydrate (CSH) beads in vitro.

SAMPLE 60 carboplatin-impregnated CSH beads and 9 CSH beads without added carboplatin (controls).

PROCEDURES Carboplatin-impregnated CSH beads (each containing 4.6 mg of carboplatin [2.4 mg of platinum]) were placed into separate 10-mL plastic tubes containing 5 mL of PBSS in groups of 1, 3, 6, or 10; 3 control beads were placed into a single tube of PBSS at the same volume. Experiments were conducted in triplicate at 37°C and a pH of 7.4 with constant agitation. Eluent samples were collected at 1, 2, 3, 6, 12, 24, and 72 hours. Samples were analyzed for platinum content by inductively coupled plasma–mass spectrometry.

RESULTS The mean concentration of platinum released per carboplatin-impregnated bead over 72 hours was 445.3 mg/L. Cumulative concentrations of platinum eluted increased as the number of beads per tube increased. There was a significant difference in platinum concentrations over time, with values increasing over the first 12 hours and then declining for all tubes. There was also a significant difference in percentage of total incorporated platinum released into tubes with different numbers of beads: the percentage of eluted platinum was higher in tubes containing 1 or 3 beads than in those containing 6 or 10 beads.

CONCLUSIONS AND CLINICAL RELEVANCE Carboplatin-impregnated CSH beads eluted platinum over 72 hours. Further studies are needed to determine whether implantation of carboplatin-impregnated CSH beads results in detectable levels of platinum systemically and whether the platinum concentrations eluted locally are toxic to tumor cells.

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.

Figure 1—
Figure 1—

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).

Figure 2—
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

Table 1—

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 (%)
114.93 ± 4.318.54–22.11
230.26 ± 6.3721.27–39.92
343.56 ± 7.2033.04–54.36
662.18 ± 9.6548.18–78.33
1280.99 ± 10.5866.59–102.50
2488.95 ± 9.1774.02–105.14
7292.06 ± 6.4680.62–105.19

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)

Table 2—

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.

GroupMean ± SEM (%)95% CI (%)
1 bead65.0 ± 1.78*60.9–69.1
3 beads63.6 ± 1.78§59.5–67.7
6 beads57.7 ± 1.78*53.6–61.8
10 beads49.5 ± 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

a.

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.

b.

Wedgewood Pharmacy, Swedesboro, NJ.

c.

University of Vermont Instrumentation and Modeling Facility, Burlington, Vt.

d.

Matrix III, US Patent 6391336, Royer Biomedical Inc, Frederick, Md.

e.

Midwest Laboratories Inc, Omaha, Neb.

f.

SPSS, version 23.0, SPSS Inc, Armonk, NY.

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Contributor Notes

Address correspondence to Dr. Phillips (philli@illinois.edu).