Local, sustained-release chemotherapeutic delivery systems have been developed to decrease the incidence of systemic toxic effects of chemotherapeutic agents while optimizing regional control of nonresectable and incompletely or marginally resected tumors.1–10 However, many of these delivery systems are not commercially available, have resulted in unacceptable regional complications, or have resulted in minimal improvement in clinical outcome.5,8,10,11
Carboplatin-impregnated calcium sulfate hemihydrate beads are a commercially available delivery system for sustained release of the platinum-containing chemotherapeutic agent carboplatin. Calcium sulfate hemihydrate is a proven biodegradable carrier for drug release and causes minimal reaction in tissues.12–18 Studies19,20 have shown promising usefulness of cisplatin-impregnated and CI-CSH beads for treatment of various tumors in horses and soft tissue sarcomas in dogs with negligible local adverse effects, compared with other carriers. Previously, we have described the short-term elution characteristics of CI-CSH beads in an in vitro study.21
Elution is the practice of extracting one material from another by washing it with a solvent. Historically, this process has been used as an in vitro method to predict the effectiveness of antimicrobial-impregnated carriers for sustained release of drugs in infected wounds.15,18,22 Studies evaluating elution characteristics in vitro have most commonly used a sample collection method in which the entirety of the eluent solution is exchanged at each sample collection time.12,15,18,22–35
In humans, the skin and skeletal muscle contain approximately two-thirds of the extracellular fluid volume, combined. Available evidence suggests that interstitial fluid in healthy skin and skeletal muscle is completely exchanged every 24 to 48 hours.36 However, if the tissues are substantially disrupted by inflammation, neoplasia, fibrosis, or surgical intervention, the exchange of fluid is altered.37,38 It is likely that the most commonly used in vitro sample collection method does not adequately mimic the fluid dynamics of these altered in vivo conditions. To our knowledge, no elution studies have taken into account the differences in dynamics of fluid exchange between healthy and disrupted tissues.
The purpose of the study reported here was to characterize the long-term elution of platinum from CI-CSH beads by comparing 2 distinct methods of sample collection: 1 that mimics an environment with rapid and complete fluid exchange, and 1 that mimics an environment where no fluid exchange occurs. We hypothesized that eluent samples obtained by the 2 distinct methods would result in differences in eluent platinum concentrations. An additional objective was to use the platinum concentrations determined in this study to define the minimum and maximum concentrations of platinum that could be expected for elution from CI-CSH beads in any in vivo condition.
Materials and Methods
Sample
All carboplatin-impregnated beads evaluated in this study were created at an accredited compounding pharmacy.a Briefly, a forged metal, polytetrafluoroethylene-coated bead moldb was used to create chains of 3 uniform, 3-mm-diameter beads containing the following per bead: 4.6 mg of carboplatin (2.4 mg of platinum) and 18.4 mg of calcium sulfate hemihydrate with an added retardantc (ie, carboplatin-impregnated [treated] beads]). Control beads contained 23.0 mg of calcium sulfate hemihydrate and an added retardantc (control beads). All beads were formed and evaluated in triplicate for sample collection methods 1 and 2.
Procedures
For the first method, 3 CI-CSH beads were placed in each of a total of 3 (3 measurements/time point; same tubes used for all time points) plastic 10-mL conical tubes containing 5 mL of PBS solution and maintained at 37°C and pH 7.4 with constant agitation. Eluent samples were collected by evacuation of all 5 mL of the PBS solution at 1, 2, 3, 6, 9, and 12 hours, and 1, 2, 3, 6, 9, 12, 15, 18, 22, 26, and 30 days. Then, 5 mL of fresh replacement PBS solution was added back to each tube. Control beads, without carboplatin, were also evaluated by use of this sample collection method.
For the second method, 3 tubes corresponding to each sample collection time point (1, 2, 3, 6, 9, and 12 hours, and 1, 2, 3, 6, 9, 12, 15, 18, 22, 26, and 30 days) were established at time 0, with each of a total of 51 (3 measurements/time point; successive tubes used at each time point) plastic 10-mL conical tubes containing 3 CI-CSH beads and 5 mL of PBS solution. Ambient conditions (temperature, pH, and agitation) were the same as described for method 1. Eluent samples were collected from only the 3 assigned tubes at each time point by evacuation of all 5 mL of the PBS solution. Control beads, without carboplatin, were also evaluated by use of this sample collection method.
Platinum concentrations in samples from method 1 were compared with those of samples from method 2. In addition, platinum concentrations in samples from the first 72 hours of method 1 were compared with those of samples from a previously conducted 72-hour study21 that also used method 1. All eluent samples from method 1 and method 2 were analyzed for platinum concentration by inductively coupled plasma-mass spectrometry (limit of detection: 0.1 ppm).d The previously conducted 72-hour study21 also analyzed eluent platinum concentrations by inductively coupled plasma-mass spectrometry
The median values of platinum concentrations obtained from elution from individual CI-CSH beads from 1 hour to 30 days were calculated by dividing the median platinum concentration (mg/L) eluted from 3 beads by 3 for both sample collection methods. Expected minimum values were derived from method 1, and expected maximum values were derived from method 2.
Statistical analysis
The distribution of the data was evaluated by use of the Shapiro-Wilk test and on the basis of skewness, kurtosis, and Q-Q plots. Data that were normally distributed were reported as the mean, SD, and the minimum and maximum values, whereas nonnormally distributed data were reported as the median, 25th to 75th percentiles, and the minimum and maximum values. Data that were not normally distributed were logarithmically transformed for parametric testing. A general linear model for repeated measures was used to determine whether there was a significant difference in platinum concentrations over time (1, 2, 3, 6, and 12 hours, and 1 and 3 days) and by study (present study vs previous study21). This same statistical test was used to compare methods 1 and 2 over time (1, 2, 3, 6, 9, and 12 hours, and 1, 2, 3, 6, 9, 12, 15, 18, 22, 26, and 30 days). The Mauchly test of sphericity was used. Because sphericity was not found, the Green-house-Geisser test was used to determine within-subjects effects. A commercially available statistical software programe was used to analyze the data. A value of P < 0.05 was used to determine significance.
Results
There was a significant (P < 0.001) change in platinum concentrations over time in method 1 samples during the first 72 hours of testing. No significant (P = 0.762) difference, however, was found in platinum concentrations during the first 72 hours between the previous study21 and method 1 of the present study.
A significant difference was found in platinum concentrations in samples over time for each method and also between methods (P < 0.001; Figure 1). Platinum concentrations from method 2 were significantly (P < 0.001) higher than platinum concentrations from method 1 for all time points, except for hour 1 (P = 0.495).
Graphical representation of platinum concentrations from eluent samples obtained by method 1 and method 2 over time. Boxes indicate the interquartile range between the first and third quartiles, the horizontal line in each box is the median, and whiskers indicate the minimum and maximum values. *Significant (P < 0.05) difference in platinum concentrations between sample collection methods.
Citation: American Journal of Veterinary Research 78, 5; 10.2460/ajvr.78.5.618
Graphical representation of platinum concentrations from eluent samples obtained by method 1 and method 2 over time. Boxes indicate the interquartile range between the first and third quartiles, the horizontal line in each box is the median, and whiskers indicate the minimum and maximum values. *Significant (P < 0.05) difference in platinum concentrations between sample collection methods.
Citation: American Journal of Veterinary Research 78, 5; 10.2460/ajvr.78.5.618
Graphical representation of platinum concentrations from eluent samples obtained by method 1 and method 2 over time. Boxes indicate the interquartile range between the first and third quartiles, the horizontal line in each box is the median, and whiskers indicate the minimum and maximum values. *Significant (P < 0.05) difference in platinum concentrations between sample collection methods.
Citation: American Journal of Veterinary Research 78, 5; 10.2460/ajvr.78.5.618
The percentage of platinum eluted from CI-CSH beads differed significantly over time for each method and also between methods (P < 0.001). The percentage of platinum released from CI-CSH beads over time was significantly (P < 0.01) higher for method 2 samples, compared with method 1 samples, for all time points, except hour 1 (P = 0.487).
The median values of minimum (method 1) and maximum (method 2) platinum concentrations obtained for elution from individual CI-CSH beads from 1 hour to 30 days were calculated (Table 1).
The minimum and maximum platinum concentrations* expected from elution of platinum from individual CI-CSH beads.
| Range of platinum (mg/L) eluted per bead | ||
|---|---|---|
| Time | Minimum | Maximum |
| 1 h | 84.0 | 85.0 |
| 2 h | 84.3 | 114 |
| 3 h | 79.0 | 123 |
| 6 h | 86.0 | 174 |
| 9 h | 65.3 | 195 |
| 12 h | 67.7 | 241 |
| 1 d | 60.0 | 341 |
| 2 d | 5.73 | 450 |
| 3 d | 0.160 | 529 |
| 6 d | 0.0867 | 557 |
| 9 d | 0.0600 | 619 |
| 12 d | 0.0467 | 598 |
| 15 d | 0.0334 | 597 |
| 18 d | 0.0300 | 580 |
| 22 d | 0.0234 | 597 |
| 26 d | 0 | 552 |
| 30 d | 0 | 534 |
Platinum eluted per bead was calculated by dividing the median platinum concentrations eluted from 3 beads by 3 for both methods. Expected minimum values were derived from method 1. Expected maximum values were derived from method 2.
Discussion
Calcium sulfate hemihydrate has been extensively studied as a drug delivery substrate and is a proven depot for drug release, or elution.12–17 Drug elution studies should evaluate species-specific in vivo pharmacokinetics and pharmacodynamics, but inherent to such studies are risks to the species involved and substantial costs.39 Therefore, an emphasis has been placed on in vitro study designs to evaluate drug elution and its variability.12–18,22–35 One consistency among in vitro studies is the use of a sample collection method that requires the complete exchange of an eluent medium such as PBS solution or canine serum.12,15,18,22–35 Although some investigators removed only an aliquot of eluent for drug measurement and replenished the same volume of fresh medium,2,4,40 1 investigative team attempted to mimic in vivo conditions by exchanging a diminishing volume of eluent over the course of the study.13 However, this component of the study design was based solely on a clinical impression that wound effusion diminishes over time, a supposition that does not apply to all wounds.13
Because the most commonly used sample collection method could promote more rapid elution by creating a new concentration gradient at each sample collection time, 2 distinct sample collection methods were used in the present study to evaluate platinum release from CI-CSH beads. Comparing results of the 2 sample collection methods allowed characterization of the long-term elution of platinum from commercially available CI-CSH beads and defined a range of platinum concentrations that could be expected from elution under various in vivo conditions.
Delivery systems allowing sustained, local release of a chemotherapeutic agent for the treatment of neoplasia offer the advantage of achieving high concentrations at a tumor site with minimal risk of systemic toxic effects.2–11,18,22 Elution of substrate-bound drugs in tissue is largely dependent on the distribution of the drug, or concentration gradient, between the substrate and the biological tissue.41 The shape, size, and porosity of the substrate carrier are also important.12–18,22–35 Distribution, absorption, and elimination of the drug are then mediated by the extracellular concentration and the vascularity, lymphatic density, cellular microenvironment, and fluid dynamics of the wound bed.37,38,41–43 Inflammatory, neoplastic, or fibrous infiltrations or surgical intervention could sufficiently alter the tissue environment to result in variation in drug distribution, absorption, and elimination among patients.37,38 In patients with a healthy tissue microenvironment and interstitial fluid dynamics, complete exchange of extracellular fluid might be expected to occur quite frequently, often within hours.37 However, in patients with substantial inflammatory, neoplastic, or fibrous tissue infiltration, little to no exchange of extracellular fluid may occur, or exchange of fluid might occur slowly.37,38
In the present in vitro study, method 1 sample collection involved complete exchange of eluent at every time point to more closely mimic a wound environment where rapid and complete extracellular fluid exchange would be expected. Results of method 1, therefore, represented the minimum concentration of platinum that would be expected to occur in vivo. Method 2 sample collection involved no exchange of eluent to more closely mimic a wound environment where fluid exchange would be expected to be negligible. Therefore, results of method 2 represented the cumulative release of platinum into the surrounding medium over time and the maximum concentration of platinum that would be expected to occur in vivo. Because wounds or tumor beds will vary among dogs and cats in size, vascularity, severity of inflammation, and rate and proportion of wound fluid exchange, the actual concentration of platinum eluted in vivo from CI-CSH beads should fall within the range of these minimum and maximum in vitro values.
Recommended dosages for carboplatin are currently dictated by the systemic maximally tolerated dosage, not by targeted plasma or tissue concentrations.1 Currently, carboplatin chemotherapy for dogs involves a dosage of 300 mg of carboplatin/m2 of body surface area administered IV every 21 days. A single IV dose achieves a peak plasma concentration of approximately 42.1 mg of platinum/L (80 mg of carboplatin/L) 4 to 6 hours following IV administration.1,44 In the present study, platinum concentrations in samples from method 1 were > 1.3 times the peak plasma concentration for up to 24 hours, and platinum concentrations in samples from method 2 were 1.3 to 15 times the peak plasma concentration for up to 30 days.
To our knowledge, only 2 studies have investigated targeted tissue concentrations by evaluating the effect of carboplatin directly on canine tumor cells.45,46 These studies determined the IC50, or the concentration of carboplatin necessary to achieve 50% inhibition of replication of tumor cells in vitro. Determination of IC50 values for dose-response testing of drugs has been used to assess efficacy of carboplatin in human and veterinary studies.45–49 In these studies, the IC50 values for canine mammary carcinoma, melanoma, and transitional cell carcinoma at 72 hours were between 1.2 and 5.9 mg of platinum/L (2.2 and 11.3 mg of carboplatin/L). Although platinum concentrations in samples from method 2 were 13 to 90 times the IC50 values from 1 hour to 30 days, platinum concentrations in samples of method 1 were below IC50 values between 48 and 72 hours. This discrepancy highlights the importance of accurate modeling for in vitro studies. The IC50 values also appear to be time dependent, and the duration for which platinum concentrations must be sustained above some minimal concentration to achieve tumor control is not known.45–49 Further studies are needed to evaluate the interval and longer-term IC50 values of carboplatin for various susceptible tumor types and the ideal rate, pattern, and duration of platinum elution from CI-CSH beads.
In the present study, the minimum and maximum platinum concentrations that could be expected from elution from individual CI-CSH beads from 1 hour to 30 days were calculated. The actual amount of platinum eluted will depend on the physical environment of the wound or tumor bed. The values in the present study were median values of experiments performed in triplicate and were derived from measurement of platinum elution by method 1 sample collection (minimum concentration) and method 2 sample collection (maximum concentration). These measurements may prove clinically useful once IC50 values for local tumor control are known. It should be noted that these concentrations were derived from an in vitro study and are only estimates of concentrations that will be achieved in vivo. The accuracy of these estimations depends on the accuracy with which our sample collection methods modeled the 2 most extreme wound or tumor bed conditions. Further studies are needed to compare the findings of our in vitro study with local tissue concentrations of platinum measured near the beads in vivo.
Method 1 sample collection used in this study was also used in a previous study21 conducted by the authors. The previous study21 evaluated platinum elution from CI-CSH beads from the same manufacturera during 72 hours. When platinum elution measured during the previous study was compared with the first 72 hours of elution in samples collected by method 1 in the present study, there was a significant difference in platinum concentrations over time, but not between measurements of the previous study21 and the present study. This finding indicated precision in sample collection by the authors as well as consistency in total bead platinum concentration among beads with different manufacturer lot numbers.
Significant differences were found in platinum concentration and percentage of platinum eluted from CI-CSH beads between samples obtained by method 1 versus method 2 for nearly all time points. Platinum release did increase sharply in the first 24 hours for method 1 and declined thereafter, a phenomenon known as burst release in elution studies.12–18,22–35 Interestingly, an initial burst of release was not noted for method 2 sample collection, as large proportions of the total incorporated platinum were released until day 9 (hour 216). Subsequent to this time, little additional platinum accumulated in the eluent fluid. This finding raised the question whether the phenomenon of burst release was actually an artifact of sample collection by method 1, because at each time point with this method, a new gradient was created between the bead and freshly added PBS solution containing no drug at all.
Sample collection method significantly affected the concentrations of platinum released from CI-CSH beads at nearly all time points. A sample collection method that involves frequent and complete exchange of eluent fluid may mimic a wound bed environment with normal vascularity, cellularity, and fluid dynamics but may not accurately apply to all clinical situations. Elution profiles of local drug delivery systems obtained by this sample collection method may not be reliably applied to patients. Further studies are needed to compare in vitro elution profiles of drugs with pharmacokinetic and pharmacodynamic profiles obtained from in vivo studies.
Acknowledgments
Results of this study were presented in part at the American College of Veterinary Surgeons Surgical Summit, Nashville, Tenn, October 2015.
ABBREVIATIONS
| CI-CSH | Carboplatin-impregnated calcium sulfate hemihydrate |
| IC50 | Half maximal inhibitory concentration |
Footnotes
Matrix III carboplatin beads, Wedgewood Pharmacy, Swedesboro, NJ.
Bead mold, University of Vermont Instrumentation and Modeling Facility, Burlington, Vt.
Dextran sulfate, Royer Biomedical Inc, Frederick, Md.
Midwest Laboratories Inc, Omaha, Neb.
SPSS, version 23.0, SPSS Inc, Armonk, NY.
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