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    Figure 1—

    Schematic depiction of the bead pattern (top row) and photographs of agar gels with implanted CI-CSH beads (bottom row). Grid lines in the schematic drawing and on the agar gels are 1 cm apart.

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    Figure 2—

    Schematic depiction of the 3-D shells (shells 1 to 5) that were spaced 1 cm apart and radiated from the center of each 10 × 10 × 10-cm agar block. The agar entirely comprising each shell was homogenized for analysis.

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    Figure 3—

    Mean ± SD platinum concentration in shells 1 to 5 at 24 (A), 48 (B), and 72 (C) hours after implantation of CI-CSH beads (1 [white diamonds], 3 [black squares], 6 [white triangles], or 10 [black circles]) into the agar gel. At each time point, platinum concentrations were highest in shell 1 (within 1 cm of the implanted bead) and lowest in shell 5 (5 cm from the implanted bead), and an increase in the number of beads implanted resulted in a higher concentration of platinum.

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    Figure 4—

    Diffusion of platinum into each of 5 shells at 24 (A) and 72 (B) hours after implantation of CI-CSH beads into the agar gel. Implantation of 1 CI-CSH bead resulted in release of platinum at concentrations greater than the IC50 for 5 FISAS cell lines (13.2 mg of platinum/L at 24 hours and 6.6 mg of platinum/L at 72 hours)27 for a distance of 2 cm. Notice that the addition of beads to achieve totals of 3, 6, and 10 C-I CSH beads extended effective diffusion to only 3 cm from the center of the bead pattern at 24 hours but to 3, 4, and 5 cm from the center of the bead pattern at 72 hours.

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Simulation of spatial diffusion of platinum from carboplatin-impregnated calcium sulfate hemihydrate beads by use of an agarose gelatin tissue phantom

Heidi PhillipsDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Elizabeth A. MaxwellDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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David J. SchaefferDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Timothy M. FanDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Abstract

OBJECTIVE To characterize spatial release of platinum from carboplatin-impregnated calcium sulfate hemihydrate (CI-CSH) beads by use of an agarose tissue phantom.

SAMPLE 3-mm-diameter beads (n = 60) containing 4.6 mg of carboplatin (2.4 mg of platinum)/bead.

PROCEDURES 18 L of 1% agarose was prepared and poured into 36 containers (10 × 10 × 10 cm), each of which was filled half full (0.5 L/container). After the agarose solidified, 1, 3, 6, or 10 CI-CSH beads were placed on the agar in defined patterns. An additional 36 blocks of agar (0.5 L/block) were placed atop the beads, positioning the beads in the center of 1 L of agar. The experiment was replicated 3 times for each bead pattern for 24, 48, and 72 hours. At these times, representative agarose blocks were sectioned in the x-, y-, and z-planes and labeled in accordance with their positions in shells radiating 1, 2, 3, 4, and 5 cm from the center of the blocks. Agarose from each shell was homogenized, and a sample was submitted for platinum analysis by use of inductively coupled plasma–mass spectroscopy.

RESULTS Platinum diffused from CI-CSH beads at predicted anticancer cytotoxic concentrations for 2 to 5 cm.

CONCLUSIONS AND CLINICAL RELEVANCE Results provided information regarding the spatial distribution of platinum expected to occur in vivo. Agarose may be used as a diffusion model, mimicking the characteristics of subcutaneous tissues. Measured platinum concentrations might be used to guide patterns for implantation of CI-CSH beads in animals with susceptible neoplasms.

Abstract

OBJECTIVE To characterize spatial release of platinum from carboplatin-impregnated calcium sulfate hemihydrate (CI-CSH) beads by use of an agarose tissue phantom.

SAMPLE 3-mm-diameter beads (n = 60) containing 4.6 mg of carboplatin (2.4 mg of platinum)/bead.

PROCEDURES 18 L of 1% agarose was prepared and poured into 36 containers (10 × 10 × 10 cm), each of which was filled half full (0.5 L/container). After the agarose solidified, 1, 3, 6, or 10 CI-CSH beads were placed on the agar in defined patterns. An additional 36 blocks of agar (0.5 L/block) were placed atop the beads, positioning the beads in the center of 1 L of agar. The experiment was replicated 3 times for each bead pattern for 24, 48, and 72 hours. At these times, representative agarose blocks were sectioned in the x-, y-, and z-planes and labeled in accordance with their positions in shells radiating 1, 2, 3, 4, and 5 cm from the center of the blocks. Agarose from each shell was homogenized, and a sample was submitted for platinum analysis by use of inductively coupled plasma–mass spectroscopy.

RESULTS Platinum diffused from CI-CSH beads at predicted anticancer cytotoxic concentrations for 2 to 5 cm.

CONCLUSIONS AND CLINICAL RELEVANCE Results provided information regarding the spatial distribution of platinum expected to occur in vivo. Agarose may be used as a diffusion model, mimicking the characteristics of subcutaneous tissues. Measured platinum concentrations might be used to guide patterns for implantation of CI-CSH beads in animals with susceptible neoplasms.

Localized sustained-release chemotherapeutic delivery systems have been developed to decrease the incidence of systemic toxic effects of chemotherapeutic agents while achieving high concentrations at tumor sites and optimizing regional control of non-resectable and incompletely or marginally resected tumors.1–10 Localized administration of cisplatin and carboplatin has been evaluated,4,5,7,8,11–16 and cisplatin and carboplatin sustained-release delivery systems have proven efficacious against appendicular osteosarcomas,5,16 nasal tumors,15 and soft tissue sarcomas8,14 of dogs. However, these delivery systems are not commercially available, have unacceptable local toxic effects (eg, wound dehiscence and infection), or have resulted in equivocal clinical improvement. Given these limitations, there remains a clinical need for the development and validation of localized drug delivery strategies for improving the management of certain cancers in companion animals.

Carboplatin-impregnated calcium sulfate hemihydrate beads are available commercially as a delivery system for sustained release of carboplatin and are intended for implantation at sites of gross tumor or marginal tumor extirpation. Calcium sulfate hemihydrate is a proven depot for drug release and is biodegradable, biocompatible, and inexpensive and can be sterilized.10,12,17–24 Several uncontrolled clinical case series have revealed promising results regarding the efficacy of cisplatin-impregnated calcium sulfate hemihydrate beads and CI-CSH beads against various tumors in horses,12 dogs,25 and other species.26 Antitumor effects were observed in these animals without clinically important local or systemic toxic effects, but there was a lack of studies conducted to characterize the elution rate and pattern of platinum release from CI-CSH beads at the time those animals received treatment.

This informational void was addressed by 2 in vitro studies23,24 conducted by our research group, which revealed that platinum elutes from CI-CSH beads for 22 to 30 days at concentrations significantly higher than those achieved after systemic administration of carboplatin. Additionally, our research group27 and others28–30 have reported that the concentration of platinum released from CI-CSH beads sufficiently achieves anticancer cytotoxic concentrations against various neoplasms, including injection site–associated sarcomas of cats. Despite these important in vitro findings supporting the potential clinical use of CI-CSH, additional sophisticated modeling systems are required to more accurately recreate drug release kinetic profiles for CI-CSH within localized tumor microenvironments.31–33

Various strategies have been used to predict in vivo drug release from sustained-release drug formulations.34,35 After medications are injected or implanted SC or IM in mammals, they encounter an extracellular matrix that behaves more like gel than bulk fluid.36–39 Therefore, hydrogels have been proposed as methods for evaluating local drug diffusion owing to mechanical properties similar to those of the extracellular matrix comprising the subcutaneous and muscular tissues.32,40

Agarose is a hydrogel consisting of a linear polysaccharide material, and it has a variety of scientific applications. At relatively low concentrations in aqueous solution, agarose solidifies to form a gel at room temperature. Solidified agarose gel forms a 3-D structure with pore sizes similar to those encountered in physiologic tissues.41–43 Agarose phantoms also mimic several other mechanical properties of mammalian tissue (eg, volume of distribution, density, and permeability) crucial for determining drug diffusion.44–46 As a result, agarose gels provide realistic conditions for characterizing diffusion of drugs administered via SC or IM routes and are a proven surrogate for mammalian tissue. Agarose tissue phantoms have been evaluated most extensively in neuropharmacological experiments, whereby concentrations of agarose from 0.6% to 2% have infusion pressure profiles similar to those of the mammalian brain.45–50 A concentration of 0.6% to 1% was found to most closely mimic characteristics of mammalian soft tissues.46,51–53

An in vivo investigation of cisplatin-impregnated beads inserted subcutaneously in horses with dermal or subcutaneous neoplasms revealed that cisplatin diffused to a radial distance of approximately 1.5 cm from the site of implantation and that platinum concentrations gradually decreased as distance from a bead increased.a On the basis of results of that study,a the manufacturers of cisplatin- and carboplatin-impregnated beads have recommended that beads be spaced 2 cm apart when implanted in clinically affected animals. However, to the authors' knowledge, no controlled studies have been conducted to evaluate diffusion characteristics of CI-CSH beads and substantiate this recommendation, nor has the spatial release of platinum from CI-CSH beads been determined for small animals.

The primary objective of the study reported here was to characterize the spatial release of platinum from CI-CSH beads by use of an agarose gel tissue phantom. A secondary goal was to generate translationally relevant data that could be used to guide development of the optimal pattern or patterns for CI-CSH bead implantation to be used in tumor-bearing companion animals, including cats with injection site–associated sarcomas.

Materials and Methods

Sample

Carboplatin-impregnated calcium sulfate hemihydrate beadsb were created at an accredited compounding pharmacy.c Briefly, a forged metal bead moldd was used to create 3-mm-diameter beads. Each of the CI-CSH beads contained 4.6 mg of carboplatin (2.4 mg of platinum), 18.4 mg of calcium sulfate hemihydrate, and a retardant.e

Procedures

Thirty-six liters of 1% agarose gel was prepared; each liter of gel was created by adding 1 L of distilled water to 10 g of agarose powder.f The resulting slurry was heated to boiling and stirred by use of a rotary hot plate until the agarose powder was completely dissolved. The aqueous 1% agarose solution was allowed to cool slightly for 5 minutes. Then 500 mL of the solution was poured into each of 36 plastic containers; each container was 10 × 10 × 10 cm and was filled only half full. The 0.5-L blocks of agarose gel were allowed to cool to room temperature (20° to 25°C) and solidify. Once each gel had solidified, 1, 3, 6, or 10 CI-CSH beads were placed on the surface of the 36 agar blocks in 4 defined patterns (Figure 1). Because the manufacturer of CI-CSH beadsc recommended that the beads be placed 2 cm apart when implanted in tissue, the 4 defined patterns were created by symmetric placement of the beads spaced 2 cm apart in all directions. When only 1 bead was evaluated, it was placed centrally within an agar gel block. When 3, 6, or 10 beads were evaluated, they were placed as centrally as possible within an agar block while a distance of 2 cm was maintained between beads. Thirty-six additional 0.5-L blocks of agarose gel were created. These were placed atop the other 36 blocks and beads, effectively positioning the beads within the center of a 1-L agar block. Blocks and beads were maintained at room temperature. The experiment was replicated 3 times for each of the 4 bead patterns for time periods of 24, 48, and 72 hours. At these times, representative agarose blocks were sectioned in the x-, y-, and z-planes to create 1-cm cubes, which were labeled according to their positions in shells radiating 1, 2, 3, 4, and 5 cm from the center of each 1-L block (Figure 2). All beads were removed from the agar.

Figure 1—
Figure 1—

Schematic depiction of the bead pattern (top row) and photographs of agar gels with implanted CI-CSH beads (bottom row). Grid lines in the schematic drawing and on the agar gels are 1 cm apart.

Citation: American Journal of Veterinary Research 79, 6; 10.2460/ajvr.79.6.592

Figure 2—
Figure 2—

Schematic depiction of the 3-D shells (shells 1 to 5) that were spaced 1 cm apart and radiated from the center of each 10 × 10 × 10-cm agar block. The agar entirely comprising each shell was homogenized for analysis.

Citation: American Journal of Veterinary Research 79, 6; 10.2460/ajvr.79.6.592

Agarose from each shell was homogenized and a sample submitted for platinum analysis by means of inductively coupled plasma–mass spectroscopy. Homogenization of the agar cubes comprising each shell served to minimize the effects of gravity on the measured spatial distribution of platinum. An additional sample was obtained from each shell for the replicates at 24, 48, and 72 hours and analyzed as a quality control measure. These additional samples were evaluated for platinum content and compared with content of the original samples from each shell to determine whether there were any differences in platinum concentration. This comparison served as an evaluation of the complete homogenization of each shell and a confirmation that the original samples were representative.

Statistical analysis

A 1-compartment model for theophyllineg was adapted by adding a code for clearance, absorption rate, and elimination rate. Estimates for some pharmacokinetic parameters (eg, clearance, absorption rate, and elimination rate) from the basic 1-compartment model were verified by use of a pharmacokinetic statistical program.h The full parameterization of the 1-compartment model was used to analyze multivariate interactions among the experimental variables. Values were considered significant at P < 0.05.

Results

The CI-CSH beads released platinum predictably in the agarose tissue phantom (Table 1). There were no significant differences in platinum concentrations among replicates and between the original samples and the quality control samples, which indicated a high degree of precision in preparation of all agarose gels, sectioning of gels, homogenization of the agarose comprising individual shells, and performing the platinum analysis. At all time points, platinum concentration decreased in each individual shell as the distance of the shell from the CI-CSH bead or beads increased (Figure 3). Comparison within shells over time (24, 48, and 72 hours) revealed that there was an overall decrease in the platinum concentration in the innermost shells and an increase in platinum in the outermost shells over time. Evaluation of results for 1 bead at 24 hours revealed that the platinum concentration was highest in shell 1 (within 1 cm of the centrally positioned bead; 128 mg/L) and decreased precipitously in shell 2 (1 to 2 cm from the centrally positioned bead; 27 mg/L). The addition of more beads to the agar blocks resulted in an increase in platinum concentrations in all shells. Platinum diffused into each of the 5 shells after implantation of CI-CSH beads into the agarose gels (Figure 4). A range of the estimates of pharmacokinetic parameters determined by use of a 1-compartment model for platinum concentrations measured 24, 48, and 72 hours after implantation of 1, 3, 6, and 10 CI-CSH beads in agarose gels was summarized (Table 2).

Table 1—

Mean ± SD platinum concentration measured in each of 5 shells at various times after implantation of 1, 3, 6, and 10 CI-CSH beads in agarose gels.

  Platinum concentration (mg/L)
Time (h)Shell1 bead3 beads6 beads10 beads
245< 0.100 ± 00.178 ± 0.031.153 ± 0.101.633 ± 0.41
 40.182 ± 0.081.876 ± 0.517.133 ± 1.6511.800 ± 0.46
 32.470 ± 0.859.607 ± 1.5027.670 ± 2.9941.430 ± 4.88
 227.030 ± 12.5743.970 ± 12.55101.200 ± 26.85117.400 ± 46.94
 1128.300 ± 25.42167.300 ± 55.94187.700 ± 7.77196.700 ± 49.52
 Total158.1222.9324.9369.0
4850.895 ± 1.260.930 ± 0.203.127 ± 0.256.330 ± 1.13
 41.810 ± 0.814.010 ± 1.0211.100 ± 1.6520.400 ± 2.17
 33.397 ± 1.5814.400 ± 1.8026.000 ± 4.9245.270 ± 6.11
 212.480 ± 6.0236.900 ± 6.0855.800 ± 7.3690.500 ± 11.99
 136.270 ± 24.6466.170 ± 22.1096.500 ± 9.17100.400 ± 27.89
 Total54.85122.4192.5262.9
72
 50.382 ± 0.041.697 ± 0.43*4.240 ± 0.36*9.677 ± 0.38*
 41.243 ± 0.12*4.810 ± 0.64*10.120 ± 0.23*17.700 ± 1.71*§
 34.073 ± 0.72*10.560 ± 1.07*19.100 ± 2.10*§43.300 ± 5.74*§
 28.897 ± 3.12*21.330 ± 3.76*§48.500 ± 4.40*§64.070 ± 12.83*§
 132.300 ± 26.6*§50.530 ± 7.02*§79.400 ± 13.02*§95.370 ± 74.88*§
 Total46.9088.93161.4230.1

Shells 1 through 5 radiated 1, 2, 3, 4, and 5 cm from the center of each 1-L block, respectively.

Platinum concentrations measured in the present study would be expected to be effective against transitional cell carcinoma of dogs.28

Platinum concentrations measured in the present study would be expected to be effective against melanoma of dogs.28

Platinum concentrations measured in the present study would be expected to be effective against injection site–associated sarcoma of cats.27

Platinum concentrations measured in the present study would be expected to be effective against mammary gland carcinoma of dogs.29

Figure 3—
Figure 3—

Mean ± SD platinum concentration in shells 1 to 5 at 24 (A), 48 (B), and 72 (C) hours after implantation of CI-CSH beads (1 [white diamonds], 3 [black squares], 6 [white triangles], or 10 [black circles]) into the agar gel. At each time point, platinum concentrations were highest in shell 1 (within 1 cm of the implanted bead) and lowest in shell 5 (5 cm from the implanted bead), and an increase in the number of beads implanted resulted in a higher concentration of platinum.

Citation: American Journal of Veterinary Research 79, 6; 10.2460/ajvr.79.6.592

Figure 4—
Figure 4—

Diffusion of platinum into each of 5 shells at 24 (A) and 72 (B) hours after implantation of CI-CSH beads into the agar gel. Implantation of 1 CI-CSH bead resulted in release of platinum at concentrations greater than the IC50 for 5 FISAS cell lines (13.2 mg of platinum/L at 24 hours and 6.6 mg of platinum/L at 72 hours)27 for a distance of 2 cm. Notice that the addition of beads to achieve totals of 3, 6, and 10 C-I CSH beads extended effective diffusion to only 3 cm from the center of the bead pattern at 24 hours but to 3, 4, and 5 cm from the center of the bead pattern at 72 hours.

Citation: American Journal of Veterinary Research 79, 6; 10.2460/ajvr.79.6.592

Table 2—

Range of the estimates of pharmacokinetic parameters by diffusion time and number of beads (2.4 mg of platinum/bead).

Time (h)No. of beadsClearance (L/kg/cm)Ka (h−1)Ke* (h−1)
2410.0075–0.01192.409–4.475NA
 30.0183–0.03701.841–2.775NA
 60.0335–0.03951.121–1.636NA
 100.0424–0.07401.029–1.105NA
 AllNANA1.593
4810.0158–0.05591.636–13.132NA
 30.0431–0.06461.252–2.263NA
 60.0505–0.06961.335–1.522NA
 100.0765–0.08170.720–1.250NA
 AllNANA1.000
7210.0344–0.06401.116–2.404NA
 30.0335–0.06661.064–2.482NA
 60.0558–0.07730.885–1.322NA
 100.0643–0.12470.490–1.111NA
 AllNANA1.335

Estimates for these pharmacokinetic parameters were determined by use of a 1-compartment model for platinum concentrations measured at various times after implantation of 1, 3, 6, and 10 CI-CSH beads in agarose gels and verified with noncompartmental analysis software.h

Value reported is the mean.

Ka = Absorption rate constant. Ke = Elimination rate constant. NA = Not applicable.

Discussion

To mimic the subcutaneous tissue environment, the release of platinum from CI-CSH beads into an agarose gel tissue phantom was measured under static conditions as a function of time and space. Mechanisms that may affect drug transport in in vitro situations include migration, convection, and diffusion.32 For the conditions of the present study, the mass transport of platinum in the surrounding environment was affected mainly by diffusion, which is a critical transport process in subcutaneous tissues. Diffusion is a mass transfer phenomenon that causes the distribution of a chemical species to become more uniform in space as time passes.31,32,50 At all time points, concentrations of platinum diminished with increasing distance from the center of the bead pattern and agar block. Evaluation of all bead configurations revealed that concentrations were highest within 1 to 2 cm of the center of the block (shells 1 and 2) and diminished substantially in shells 4 and 5 located 4 to 5 cm from the center of the block.

Exposure time also had a substantial effect on platinum release from the beads. Interestingly, the platinum concentrations increased with increasing exposure time in the outer shells (shells 4 and 5) and diminished with increasing exposure time in the inner shells (shells 1 and 2). This phenomenon was evident for all bead patterns and indicated that platinum was not continuously released from the beads over the entire 72-hour period. At some time < 72 hours, platinum release from the beads likely ceased or substantially slowed, and the platinum began moving from shells 1 and 2 toward shells 4 and 5. This observation may be akin to the burst phenomenon observed in classical elution assessments, wherein an initial burst of drug release occurs in the first 24 hours of elution and is followed by a sharp decline in release thereafter.22,23,54–58 With regard to localized carboplatin treatment for susceptible tumors, this observation indicated that the greatest impact on tumor cell death may be expected to occur in the first 24 to 72 hours after implantation.

An unexpected finding was that platinum concentrations at 48 and 72 hours were substantially less than those obtained at 24 hours. If platinum were to accumulate over each additional time period, it would be expected that accumulations at 48 hours would be greater than accumulations at 24 hours and that accumulations at 72 hours would be greater than those at 48 hours. Given that platinum was found to move from the inner to the outer shells over time and that the concentrations of platinum in the outermost shell (shell 5) were > 0 mg/mL, platinum may have been lost by diffusion beyond 5 cm and out of the agar block. Agar blocks were contained within 10 × 10 × 10-cm plastic containers. A mild to moderate amount of aqueous solution was noted in the inter-face between the agar block and the walls of the container at the time of sample collection. It is possible that platinum that diffused out of the agar block was concentrated in this aqueous solution. The aqueous solution was not evaluated for platinum content. However, the solution may have mimicked a sink effect seen physiologically wherein drug that diffuses into the subcutaneous space is removed by the vascular and lymphatic circulations.31–33

Few strategies have been used in human and veterinary medicine to predict in vivo drug release from sustained-release drug formulations.31–35 The most frequently used in vitro methods have been for evaluation of elution or release of a drug into a bulk fluid reservoir, often with agitation.22,31,32,54 Bulk fluids used most commonly include PBS solution and serum or plasma. Examples of reservoirs include vials, beakers, or dialysis bags, and sample withdrawal has been performed most often by complete exchange of the fluid eluent at predetermined time points22,23,31,32,54; concentrations in the bulk solution then were measured as a function of time. The net effect of complete exchange of eluent medium and agitation is to assure that a steep concentration gradient is maintained between the surface of the bead and the surrounding bulk fluid at all times.31,32 Such an effect does not reliably simulate the conditions beads are likely to be exposed to in in vivo situations because wounds or tumor beds differ among dogs and cats with regard to size, vascularity, severity of inflammation, and rate and proportion of wound fluid exchange. To address this concern, our research group conducted a study24 to evaluate 2 methods for sampling of bulk fluid to characterize elution of platinum from CI-CSH beads (1 that involved complete eluent exchange at every time point and 1 that involved no eluent exchange). It was concluded that the resultant concentrations represented the minimum and maximum range of platinum release possible from CI-CSH beads, and it was suggested that the actual platinum release in vivo would be between the minimal and maximal concentrations reported in that study.24 Interestingly, release of platinum from CI-CSH beads into an agarose medium for 24 and 72 hours in the present study resulted in concentrations within the range of the previously reported minimum and maximum values, as was suggested would occur in an actual wound or tumor bed.24

A purported benefit of the agarose tissue phantom diffusion method for assessing drug release is that it more accurately simulates the extracellular matrix and release conditions of subcutaneous tissues in a live animal.31–33,50,59 Agarose is a linear polysaccharide that forms a 3-D porous structure containing mostly water but that has measured pore sizes similar to those encountered in physiologic soft tissues.31–33,36 Cooling the polysaccharide molecules results in formation of double helices that aggregate to form fiber networks similar to the collagen matrix of the extracellular space.39 Agarose hydrogels, although simplistic, have been widely validated as in vitro methods that can be used to predict the in vivo behavior of various drug formulations.31–33,43,60

The IC50 (ie, concentration of carboplatin necessary to achieve 50% inhibition of replication of tumor cells or tumor cell death in vitro) has been used to assess efficacy of carboplatin in human and veterinary studies.27,61,62 The IC50 of tumors, including transitional cell carcinoma,28 melanoma,28 and mammary gland carcinoma29 of dogs, at 72 hours after administration reportedly is 1.2, 1.2, and 16.9 mg of platinum/L, respectively. Recently, our research group found that carboplatin has a significant dose- and time-dependent inhibitory effect on the viability of FISAS cells in culture and induces apoptosis in most cells by 72 hours after drug administration.27 The overall mean IC50 of carboplatin for 5 FISAS cell lines was found to be 13.2 mg of platinum/L at 24 hours and 6.6 mg of platinum/L at 72 hours.27 An objective of the present study was to derive the ideal pattern of bead implantation for local treatment of FISASs. The data reported here indicated that by 24 hours, 1 CI-CSH bead released platinum for a distance of 2 cm at concentrations greater than the IC50 for FISASs. The addition of beads to achieve totals of 3, 6, and 10 CI-CSH beads extended effective diffusion to a distance of only 3 cm from the center of the bead pattern and agar block. By 72 hours, 1 CI-CSH bead also released platinum for a distance of 2 cm at concentrations greater than the IC50 for FISASs. However, by 72 hours, the addition of beads to achieve totals of 3, 6, and 10 CI-CSH beads extended the sphere of effective diffusion to 3, 4, and 5 cm from the center of the bead patterns and agar blocks, respectively. This finding indicated that for tumors or extirpated tumor beds with radii of 3, 4, and 5 cm, implantation of 3, 6, and 10 beads in the selected patterns, respectively, should provide diffusion of a sufficient amount of platinum to result in 50% inhibition of tumor cell growth or cell death over the entire affected area. For tumors with a radius < 2 cm, local tumor control may be achieved by implantation of only 1 CI-CSH bead immediately following excision of the primary tumor. Additionally, minimally invasive treatment of recurrent tumors may be achieved by simple implantation of CI-CSH beads without tumor resection or by marginal excision of larger tumors with concurrent bead implantation.

Limitations of the present study included the nature of the agarose gel. In studies conducted by our research group, hydrogels consisting of an agarose concentration of 1% have been used to mimic the diffusion characteristics of subcutaneous tissues. In the present study, markings (1-cm grid) were imprinted on the agar blocks to improve the precision for sectioning of shells; however, the semisolid agar occasionally collapsed during sectioning, and some blocks were softer than others. Although agarose powder was precisely measured by use of a gram scale and distilled water was collected in 1-L beakers, water may have been lost to evaporation during boiling in some cases, which would have changed the consistency of the agar among blocks. Although no significant differences were detected in the measured platinum concentrations, such inconsistencies could have affected the platinum concentrations. Also, to minimize effects of gravity on the measured spatial distribution of platinum, agar comprising each shell was homogenized and processed collectively, and a representative sample was obtained from each homogenized shell for analysis. However, as the number of the shell increased, the volume of agarose gel increased, and this may have impacted our ability to completely homogenize the agar entirely comprising each shell.

Ideally, in vitro systems are tested at physiologic temperature and pH. Given the large physical scale of the experimental containers, maintenance of the agar blocks at body temperature was not possible. Agar was created with distilled water at a pH of 7.0, as previously described.31–33,43,47,50 However, the use of PBS solution at a pH of 7.4 as a solvent for the agarose powder may permit testing at physiologically relevant pH.

Finally, exposure times of 24, 48, and 72 hours do not necessarily correspond to the amount of time that carboplatin concentrations might be maintained locally in vivo. Additional factors, including tumor blood flow, drug metabolism, and host and tumor immune responses, may alter concentrations and efficacy of chemotherapeutic agents.

For the study reported here, platinum was released from CI-CSH beads into agarose as a function of 3-D space and time, and recommendations for number of beads and pattern of bead placement relative to tumor bed size can be made. However, the in vivo fate of platinum that diffuses from CI-CSH beads is expected to be more complex than for the method used in the present study and would involve metabolism, cellular uptake, and degradation.31 Although widely validated as a method for evaluation of drug diffusion, agarose gel is a simplistic, inert, nonperfused medium and cannot mimic the complexity of actual in vivo conditions.31–33,43,47,50 In small animals with tumors, growth and excision of a neoplasm is expected to result in loss of tissue organization and aberrant behavior of cellular components and extracellular tissues of the subcutaneous space. Further studies are necessary to determine whether platinum release into an in vitro agarose tissue phantom is predictive of in vivo release and to affirm dosing recommendations for beads used in clinical settings.

Acknowledgments

Supported by the American College of Veterinary Surgeons Foundation and Wedgewood Pharmacy.

The authors have no conflicts of interest to declare.

Presented in part at the Society of Veterinary Soft Tissue Surgery Annual Meeting, Whitefish, Mont, June 2017.

ABBREVIATIONS

CI-CSH

Carboplatin-impregnated calcium sulfate hemihydrate

FISAS

Feline injection site–associated sarcoma

IC50

Half maximal inhibitory concentration

Footnotes

a.

Marble GM, Sullins KE. A biodegradable matrix for cisplatin to treat equine skin neoplasia (abstr), in Proceedings. 10th Annu Am Coll Vet Surg Symp 2000;29:469.

b.

Matrix III carboplatin beads, provided by Wedgewood Pharmacy, Swedesboro, NJ.

c.

Wedgewood Pharmacy, Swedesboro, NJ.

d.

Bead mold, Instrumentation and Modeling Facility, Instrumentation and Technical Services (ITS) Department, Office of the Vice President for Research, University of Vermont, Burlington, Vt.

e.

Dextran sulfate, Royer Biomedical Inc, Frederick, Md.

f.

Granulated agar, Fischer Scientific Co LLC, Pittsburgh, Pa.

g.

SAS, version 9.4, SAS Institute Inc, Cary, NC.

h.

Phoenix WinNonlin, version 5.0, Pharsight Corp, Mountain View, Calif.

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Contributor Notes

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