Tumor treatment by sustained intratumoral release of cisplatin: Effects of drug alone and combined with radiation

Int. J. Radiation

Oncology

Biol. Phys.. Vol. 39. Nu. 2. pp. 197-504, 1997 G 1907 Elsevirr Science Inc. Printed in the USA. All rightz reserved 03bO-X316/97 El7.00 t .oo

PII s0360-301q97)00331-3

l

Biology Contribution TUMOR

TREATMENT I!Y SUSTAINED INTRATUMORAL RELEASE CISPLATIN: EFFECTS OF DRUG ALONE AND COMBINED WITH RADIATION

OF

DONALD

T. T. YAPP, PH.D., * DAVID K. LLOYD, PH.D., * JULIAN ZHU. PH.D.+ AND SHIRLEY M. LEHNERT, PH.D.*

*Department of Oncology, McGill University and +Department of Chemistry, Universit& de MontrCal, Montrkal, Qubbec, Canada The effect of intratumoral delivery of cispIat.iu to a moose tumor model @IF-l ) by means of a biodewith and without radiation was studied.

with those obtained when cisphth was delivered systemicaNy. Reatal&xWhen dsplstin was delivered by the polymer impktnts, higher levels were present in the tumor for hger time periods (cf. systemic delivery of the drug). For both nonirradlated and h&Wed tumors, tImsa We&d witb the poIymer impiants had significantIy longer tomor growth detays compared to nonimplanted ControIs and

fractionated radiation.

0 1997 Elsevier Science Inc.

Biodegradable polymer, Cisplatin, Intratumoral delivery, Radiation, RIF-1 mouse Tumor model.

INTRODUCTION The toxicities of drugs used in chemotherapy and in conjunction with radiotherapy are frequently a limiting factor in the treatment of cancer. In many cases, the drug dose required for a cure would also cause unacceptably high levels of damage to normal tissue. Cisplatin and etanidazole are two such drugs. Cisplatin has a broad spectrum of activity singly, in combination with other drugs (9) and with radiation [see (7) for review]. Unfortunately the severe, dose-limiting side effects associated with cisplatin ( 14, 18) would prevent the use of optimal drug doses during fractionated radiation treatments ( 10). Similarly, clinical trials with etanidazole used in fractionated radiation treatments have been disappointing due to peripheral neuropathy when the drug was administered over a long treatment schedule (4, 11, 15 ) .

With a delivery system that localizes and releases the drug slowly at the treatment site, however, systemic levels of the drug could be reduced while simultaneously increasing the concentration of drug within the tumor. Any side effects associated with the toxicity of the drug would thus be reduced or eliminated. Such a delivery system would thus allow the tumor to be treated with drugs concentrations that would otherwise not be tolerated if delivered systematically ( 13 ) . This approach has been used previously by Deurloo et al. (5, 6) to deliver cisplatin intratumoralIy using a series of hydrogels whose drug release rates were dependent on the rate of water uptake by the implant. The authors report that drug release was extended as long as 4 days and that drug concentrations in the tumor were increased relative to i.p. administration of the drug (5. 6).

Reprint requests to: Shirley Lehnert, Ph.D., Radiation Oncology, McGill University, Mont&al General Hospital, 1650 Cedar Ave., Mont&al, Qu&ec, Canada H3G lA4. Acknowledgements-This work was supported by the National

Cancer Institute of Canada. The authors wish to thank H. Yeh, Ph.D. for the synthesis of the polymers. Accepted for publication 5 May 1997. 497

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The use of a slow-release implant would also increase the effectiveness of conventional radiotherapy by allowing optimal levels of drugs like etanidazole or cisplatin in the tumor to be sustained during fractionated radiation regimens. This approach could also extend the scope of this modality to tumors resistant to conventional radiotherapy. A polyanhydride/sebacic acid based copolymer [bis(p-carboxyphenoxy)propane-sebacic acid, CPP:SA] has been studied previously as a slow release device ( 1, 16). The polymer’s degradation characteristics ( 16)) the biodistribution (8), and the biocompatibility ( 17, 20) of its degradation products have been reported previously in the literature. In addition, the same polymer has been used to treat intercranial brain tumors with carmustine (BCNU) in humans ( 1). We have used the CPP-SA polymer to fashion biodegradable implants for delivering cisplatin, etanidazole, bromodeoxyuridine ( BrdU) , and tumor necrosis factor in a slow-release manner intratumorally. This method of delivery should ensure that drug levels within the tumor are kept higher and maintained during extended radiation treatments than when delivered via conventional methods. In addition, systemic toxicities should also be minimized or eliminated as the drug is concentrated within the tumor tissue. The drug-containing implants were placed directly into a mouse fibrosarcoma (RIF- 1) implanted subcutaneously in C3H mice (2). This mouse tumor was chosen for the studies because it has been previously studied and because its characteristics (low rate of metastases, grows well without compromising the animal) are ideally suited for our chosen endpoint (2, 21) . In the present article, we report on the in vitro release characteristics of the polymer-cisplatin implant and the effect of delivering cisplatin in high concentrations over an extended period of time to a murine tumor (RIF-1) with and without radiation. The distribution of cisplatin in the mouse following intratumoral implant was also examined, and these results are also reported. METHODS

AND MATERIALS

Radiation The tumors were irradiated with 6oCo gamma rays using a Theratron 780 unit at a dose rate of approximately 1.0 Gy/min. The mice were anesthetized and only the tumor was irradiated, the rest of the body being shielded with 5cm thick lead blocks. The dose levels to the tumor and other parts of the body were measured using thermoluminescent (TLD-100) LiF crystal dosimeters. The dose to the shielded portion of the body was less than 4% of that to the tumor. The dosimeters were calibrated with the 6oCo beam, and their accuracy was found to be within 2-3%. Polymer implant The CPP-SA polymer was synthesized according to the procedure published in the literature ( 16). Briefly, 1,2-

Volume 39, Number 2, 1997

bis(p-carboxyphenoxy)propane (CPP) was first synthesized from p -hydroxybenzoic acid and 1,3-dibromopropane in a basic aqueous medium. The prepolymer of sebacic acid and CPP were then prepared by reaction with acetic anhydride. The final polymer product was prepared from a mixture of the prepolymers (molar ratio of 80:20, SA:CPP) in a polycondensation reaction carried out at ca. 200-200°C in vacua. The crude product was purified by precipitation (CHClJpet. ether) and finally washed with diethyl ether before being dried under vacuum. Cisplatin and Pt analytical standards were obtained from Sigma Chemicals. The polymer and cisplatin ( 17% w/w) were ground together to form a fine, homogeneous yellow powder which was subsequently heated to 80°C and extruded through an Eppendorf Combitip (tip diameter 0.5 mm). Rods with greater diameters ( 1.O and 1.5 mm) were obtained by cutting the tip further along the barrel to obtain the desired diameter before the extrusion process. The resulting polymer-cisplatin rods were cooled at room temperature and stored in a desiccator until required. In vitro degradation Polymer rods containing cisplatin were placed in phosphate-buffered saline (PBS) at 37°C. The solutions containing degraded polymer product were collected daily, and fresh PBS was added to the container. The solutions were then analyzed by flame atomic absorption spectroscopy (FAAS, Perkin-Elmer-3 100) for the presence of Pt. Drug-free polymer rods were also degraded in a similar fashion and analyzed for Pt; as expected, no signals were obtained for these samples. A Pt standard was added to a series of the blank samples to determine if the presence of the polymer affected the Pt signal during the analysis; no anomalous effects were observed. Tissue culture RIF-1 cells were obtained from Dr. R. Hill (Ontario Cancer Institute) and were passaged using standard tissue culture techniques in alpha modification of MEM media supplemented with 10% fetal bovine serum and 1% antibiotic-mycotic (all supplied by Gibco-BRL). Cells were trypsinized, collected by centrifugation, and resuspended in media (4 X lo6 cells/ml) before being injected (50 ~1) subcutaneously into the backs of previously shaved C3H mice (female, 6 weeks old, 20 g, Charles River). Tumors appeared within 10 days and reached a volume of 94- 130 mm3 within 3 weeks. Tumor volumes were calculated from measurements taken at three orthogonal angles using the formula (a X b X c)7r/6. Treatments Treatments were begun when the tumors reached a volume of approximately 100 mm3. Tumor-bearing mice were separated into groups of five for the different treatments. For implant of polymer rods, the mice were anaesthetized (Nembutal, 65 mg/kg), and the surface of the tumor disinfected with ethanol. The skin was then punctured with a

Tumor treatment by intratumoral release of Cisplatin l D. T. T.

hypodermic needle (20 gauge), and the polymer rod inserted into the tumor through the puncture hole. Generally, the rod (8 x 0.5 mm) was divided into three pieces and inserted in three different positions. Systemic administration of cisplatin (7 mg/kg) was by i.p. injection. Mice receiving radiation were irradiated immediately following implantation unless otherwise stated. Fractionated doses (5 x 9.00 Gy) were delivered at 24-h intervals for 5 days beginning immediately after insertion of the polymer rod. Control mice were sham irradiated. Tumor measurements were made three times a week, and the mice were sacrificed when the end-point (4 X initial tumor volume) was reached. Pt levels in the tumor, kidney, and blood Tumor-bearing mice were treated with cisplatin polymer implants or cisplatin injected i.p. and sacrificed at selected intervals. The tumor and kidneys were removed and stored in liquid nitrogen; mouse blood was collected and centrifuged to obtain the plasma which was stored at -20°C. The tissue samples were subsequently homogenized using a Dounce homogenizer and digested in a buffered solution of 0.4% sodium dodecyl sulphate (SDS) and proteinase K (0.4 mg/ml) . The homogenized samples were diluted (85 mg tissue per ml of buffer) prior to analysis for Pt using a Perkin-Elmer 5100 spectrophotometer fitted with a graphite furnace. Plasma samples were analyzed without further workup. RESULTS In vitro degradation Polymer rods of differing diameters (0.5, 1 .O, and 1.5 mm) containing 17% cisplatin by weight were prepared and degraded as described above. The sample aliquots were analyzed for Pt, and the results expressed as a percentage of total Pt found (Fig. 1). The data indicate that drug release is dependent upon the size of the rods; the drug being released fastest from the smallest rods. The major portion of the drug (60%) was released after 24 h and the remainder after 48 h. In contrast, drug release from the rods with diameters of 1 and 1.5 mm was delayed up to 180 h (7.5 days). The fact that the smaller rods are more vulnerable to degradation by the buffer solution suggests that initial degradation at the surface leads to increasingly connected pore structures, and below a certain critical volume the interconnected pore network becomes the dominant pathway for drug loss. A similar model has been proposed by Chiu et al. (3 ) to describe the degradation of poly(dl-lactide-co-glycolide). Tumor treatment studies The effect of cispiatin containing biodegradable polymer implants on tumor growth, specifically tumor growth delay (TGD) were examined with and without radiation. The results of these studies are summarized in Table 1 and shown in Figs. 2 and 3.

YAPP

et al.

499

80 r

u

0.5 x 0.4 mm

-

1 x 0.4 mm

-

1.5x0.4mm

Time (II)

Fig. 1. In vitro degradation of cisplatinum containing polymer rods (17% w/w).

In the absence of radiation (Fig. 2)) implanted biodegradable polymer rods containing 17% cisplatin by weight (average dose, 30 mg/kg) were more effective than systematically delivered cisplatin in delaying tumor growth (TGD of 16.1 and 9.5 days, respectively). The gre&er effectiveness of the implant is presumably due to slow r&ease of the drug in the tumor and the exposure of tumor cells to toxic drug concentrations for a prolonged period of time, which causes more cell kill than does the short drug exposure associated with systemic administration ( 18).

Table 1. Tumor growth delay for RIF- I tumor after treatment with cisplatin and/or radiation

Treatment None Polymer implant Cisplatin (7 m&g, Cisplatin-polymer None Polymer Implant Cisplatin (7 m&g, Cisplatin-polymer None Cisplatin-polymer

IP) (17% w/w) Ip) (17% w/w) (17% w/w)

Tumor growth delay Ifi SD (days) 5.7 f 0.7 6.5 2 0.7 9.5 2 1.7 16.1 -c I.9 10.2 ? 1.8 9.2 +- 0.X 16.8 i: 1.3 26.0 2 3.5 23.1 f 2.5

Radiation None

10.00 Gy

5 x 9.00 Gy

41.0 i- 1.4

Values are the average for 5 mice +- standard deviation.

I. J. Radiation Oncology l Biology l Physics

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16.1

g $

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of the polymer implant in delaying tumor growth indicates the value of this delivery system in maintaining a sensitizing level of drug throughout the period over which radiation was delivered.

(a) 10 6.5

C

Fig. 2. TGD values for tumors treated with cisplatinum systemically or by implant.

When a single dose of radiation ( 10.00 Gy) was given in conjunction with a cisplatin/polymer implant (Fig. 3a), a prolonged delay in tumor growth was obtained when the implant was placed in the tumor 48 h before irradiation (TGD = 26 days). Cisplatin delivered systemically approximately 15-30 min before irradiation resulted in a TGD value of 16.8 days, while tumors treated with 10.00 Gy of radiation alone grew to the defined endpoint by 10.2 days. Blank polymer implants in conjunction with 10.00 Gy did not retard tumor growth significantly (TGD = 9.2 days). Implantation of the cisplatin-containing rod 48 h before irradiation allows a buildup of the drug in tumor tissue, and this level of drug present during irradiation presumably radiosensitized the tumor cells in addition to the combined cytotoxic action for the two modalities. In contrast, the shorter TGD seen when the radiation dose is delivered in conjunction with cisplatin delivered systematically, reflects the lower and shorter lived levels of cisplatin in the tumor during and after irradiation. When fractionated radiation (5 x 9.00 Gy over 5 days) was used in conjunction with the cisplatin implant, the TGD value was 41 days compared with 23 days for control mice, which were treated with radiation only (Fig. 3b).

As in the single dose experiments, the continued presence of cisplatin during the radiation presumably sensitizes the cells to radiation in addition to the additive cytotoxicity of the radiation plus polymer implant. The effectiveness

50

03

r

40 3 3 3 g

30

20

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0 5xQoocGy

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Fig. 3. TGD values for tumors treated with cisplatinum (systemic or implant) plus radiation a) radiation dose 10 Gy b) radiation dose 5 X 9 Gy at 24 hour intervals.

Tumor

treatment

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(a) 6

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Time (h) Fig. 4. Platinum levels in tumor, kidney and blood. a) Cisplatinum given systemically (7mgkg). b) Cisplatinum given by polymer implant.

Cisplatin distribution in tumor-bearing animals The concentration of platinum, measured in tissue samples by atomic absorption spectroscopy (AAS), was used as an indicator of the level of cisplatin present in the mouse tumor. kidney and blood plasma over time when

of Cisplatin

0 D. T. T. Y -2~ rt ul.

50 I

the drug was delivered via the polymer or systemically. It is important to note that AAS only provides information on the total amount of drug present and does not differentiate between the different cisplatin species preseni in free solution or bound to tissue. The results show that, for the tissues investigated. the highest levels of cisplatin were found at 24 h after systemic delivery and that the levels then decreased steadily. These findings are similar to those previously published that reported the elimination of Pt to be biphasic with the first elimination occurring within 90 min followed by a slow decline of Pt levels ( 12, 14). Our data indicate that the drug is essentially cleared from the tumor tissue by 192 h at which time the levels of Pt in the kidney and blood have decreased substantially ( Fig. 4a). Levels of Pt were consistently higher in the kidney than in the tumor. When cisplatin is delivered via the polymeric implant, the levels of Pt in tumor tissue remain constant up to 96 h but increased dramatically by 192 h ( 8 days). Throughout this period, the levels of Pt present in the tumor are consistently higher than those found in the blood or kidney ( Fig. 4b ). The higher Pt concentrations found in tumors with implanted polymer compared to those found in tumors treated with systemic cisplatin indicate the high dose of drug that is delivered over a prolonged period by the implant. The dramatic increase in pt levels by 192 h may be due to the polymer implant having reached the point where drug release is no longer determined by degradation of the polymer but by dissolution and leaking of the drug from what is left of the polymer infrastructure ( 3). The increased concentration of Pt in the tissue is also partly attributable to reduction in the tumor volume (to 70%~of the initial value ). which occurs for tumors treated with the implant. Clearance of Pt from the kidney and blood follows a different pattern when the drug is given systemically than when delivered by the implant (Fig. 5a and b 1. The levels of Pt in the kidney and blood of animals treated systemically reach a maximum at 24 h and then decrease steadily. Following implant, however, drug clearance is delayed with the maximum Pt levels for kidney and blood occurring at 96 and 48 h, respectively. It is possible that the high levels of Pt in the tumor tissue at 192 h as the implant is completely degraded will subsequentiy cause Pt levels in the kidney and blood to rise again as the drug is cleared from the tumor. The Pt levels were calculated both as the total Pt ( yg ) found in the tumor, kidney, and blood (approximate volume of 2 ml) per mg of cispiatin delivered and as the amount of Pt (yg ) per gram of tissue or ml of blood per mg of drug delivered into the animal ( Fig. 6). Both modes of calculation showed that the levels ol’ the drug were consistently higher in the tumors treated with the implant.

DISCUSSION In this study, we have demonstrated the effectiveness of using the biodegradable polymer 1i bis(p-carboxy-

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In vitro data indicates the rate of drug release is dependent on the size of the polymer implant and is decreased for larger implants. The size of the tumors used in this study, however, precluded the use of implants with a diameter greater than 0.5 mm. The kinetics of polymer degradation in the tumor environment, based on Pt measure-

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Implant Systemic

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Volume 39, Number 2, 1997

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phenoxy )propane-sebacic acid, CPP:SA)] to deliver high doses of cisplatin locally and for a prolonged period of time with and without radiation. The tumors treated in this fashion responded more quickly than did those treated systemically (Fig. 7).

24

46

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192

Time (h) Fig. 6. Platinum levels in tumor tissue. a) Expressed as mg Pt/g tumor/pg cisplatinum administered. b) Total pg Pt in the tumor.

Tumor treatment by intratumoral release of Cisplatin 0 D. T. T.

NO treatment Systemtc

CP (7 mghtg)

cp implant 5xeouffiy CP+SxBoocGy

Time (days)

Fig. 7. Tumor growth after cisplatinum and/or radiation treatment.

ments in viva, appeared to be slower than that seen in buffered aqueous solution, and the smallest sized polymer implant in the tumor released drug over a period of at least 1 week. While the rate of drug release in v&-o cannot be directly equated with the rate of drug release in the tumor, in vitro results do indicate that implant size will influence the rate of intratumoral drug release. This has to be borne in mind when treatments of larger tumors with larger sized polymer implants are being designed. The two main advantages of the biodegradable polymer implant used for the delivery of cisplatin to the mouse tumor are the high concentrations of drug attainable and the extended period of time for which it can be maintained in the tumor. The average dose of cisplatin delivered using the implants was 30 mg/kg, which is three times the mean lethal dose for C3H mice (2). Similar high concentrations were tolerated by dogs that were treated with cisplatin released from a D,L-polylactic acid implant ( 19). The delayed release of the drug and localization in the tumor acts to prevent toxic levels being reached in the mouse, whereas a similar dose delivered systematically with a bolus injection would be almost invariably toxic. Results of other experiments indicated that total drug doses higher than 37-40 mg/kg delivered via the polymeric implants are lethal to the mice (data not shown).

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However, the rate of drug release in the tumor is probably responsible for the toxic effect, and this could be slowed down by using a larger implant that would degrade at a slower rate and release the drug over a longer period of time. In addition, a report from Withrow ef al. (22) indicates that the presence of low drug levels in the system may actually be advantageous in the treatment of metastatic disease; the authors report that the sustained release of low levels of systemic cisplatin appears to be beneficial in treating metastatic disease in canine osteosarcoma. The effects of irradiation combined with cisplatin implanted in the tumor appear to be supraadditive. Based on a tumor volume doubling time of 3 days and assuming that tumor volume is proportional to the number of viable clonogens, and that the rate of tumor regrowth is the same as the initial rate of growth, the duration of growth delay can be used to calculate a “surviving fraction” following drug and/or radiation treatment. Results of this type of calculation indicate that the effects of all the treatments in which radiation and cisplatin were combined was greater than the effects that would have resulted from simply additive responses (an additive response would be that produced if the effects of the two treatments were multiplied together). This amplification of the combined effect is least for systemic cisplatin and a single dose of radiation, greater for implant plus a single dose of radiation, and greatest for cisplatin/polymer implant and fractionated radiation. This observation suggests that a high proportion of cell kill is associated with radiosensitization by cisplatin because this would be amplified to a greater extent when multiple radiation doses were used. The advantages of drug delivery by polymer have already been emphasized and are summarized by the results shown in Fig. 7. The dose of cisplatin that can be delivered using the polymer implants is approximately three times greater than that which could be delivered systemically. and the drug is released over an extended period of time. This results in a longer period of growth delay compared to those tumors treated systemically with cisplatin. The polyanhydride-sebacic acid system is extremely flexible. and a wide range of drngs can be incorporated into this polymer system. We are currently using such implants to radiosensitize cells by maintaining high fractions of etanidazole in the tumor during fractionated regimes. In other experiments, we are developing polymer implant systems for the delivery of BrdU and tumor necrosis factor (TFN ) Early results suggest that the polymer system may be as successful for delivery of these compounds as it has proved to be for cisplatin.

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mer of chemotherapy 345: 1008-1012; 1995.

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2. Brown, J. M.; Twentyman, P. R.; Zamvil, S. S. Responseof the RF- 1 tumor in vitro and in C3HKm mice to X-radiation (cell survival. regrowth delay and tumor control ). chemo-

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I. J. RadiationOncology0 Biology 0 Physics therapeutic agents, and activated macrophages. Natl. Cancer Inst. 64:605-611; 1980. Chiu, L. K.; Chiu, W. J.; Cheng, Y.-L. Effects of polymer degradation on drug release-A mechanistic study of morphology and transport properties in 5050 poly (dllactide-coglycolide). Int. J. Pharmacol. 126: 169- 178; 1995. Coleman, C. N.; Noll, L.; Riese, N.; Buswell, L.; Howes, J. R.; Kramer, R. A.; Hurwitz, S. J.; Neben, T. Y.; Grigsby, P. Final report of the PhaseI trial of continuous infusion of etanidazole (SR2508): A Radiation Therapy Oncology Group study. Int. J. Radiat. Gncol. Biol. Phys. 22:527-580, 1992. Deurloo, M. J.; Bohlken, S.; Kop, W.; Lerk, C. F.; Hennink, W.; Bartelink, H.; Begg, A. C. Intratumoral administration of cisplatin in slow-release devices. I. Tumor response and toxicity. Cancer Chemother. Pharmacol. 27:135- 140; 1990. Deurloo, M. J.; Kop, W.; van Tellingen, 0.; Bartelink, H.; Begg, A. C. Intratumoral administration of cisplatin in slowrelease devices: II Pharmacokinetics and Intratumoral distribution. Cancer Chemother. Pharmacol. 27:347-353; 1991. Dewit, L. Combined treatment of radiation and cis-diamminedichloroplatinum( II) : A review of experimental and clinical data. Int. J. Radiat. Oncol. Biol. Phys. 13:403-426; 1987. Domb, A. J.; Rock, M.; Schwartz,J.; Perkin, C.; Yipchuck, G.; Broxup, B.; Villemure, J. G. Metabolic disposition and elimination studies of a mdiolabelled biodegradable polymeric implant in the rat brain. Biomaterials 15681-688; 1994. Douple, E. B. Hill, B. T.; Bellamy, A. S. eds. In: Antitumor drug-radiation interactions. Boca Raton, FL; CRC PressInc.; 1990, pp. 171- 190. Douple, E. B.; Will, M. L.; Jones, E. L. Rotman, M.; Rosenthal, C. J., eds. In: Concomitant continuous Infusion chemotherapy and radiation. Berlin: Springer Verlag; 1985; pp. 191-196. Halberg, F. E.; Cosmatis, D.; Gunderson, L. L.; Noyes, D.; Hanks, G. R.; Buswell, L.; Nargoney, D. M.; Coleman, C. N. A phase I study to evaluate intraoperative radiation therapy and the hypoxic cell sensitizeretanidazole in locally advanced malignancies. Int. J. Radiat. Oncol. Biol. Phys. 28:201-206, 1994. Johnsson, A.; Olsson, C.; Nygren, 0.; Nillson, M.; Seiving, B.; Cavallin-Stahl, E. Pharmacokinetics and tissue distribution of cisplatin in nude mice: Platinum levels and cisplatinDNA adducts. Cancer Chemother. Pharmacol. 37:23-31; 1995.

Volume 39, Number 2, 1997 13. Langer, R. New methods of drug delivery. Science 249:1527-1533; 1990. 14. Landrito, J. E.; Yoshiga, K.; Sakurai, K.; Takada, K. Effects of intralesional injection of cisplatin on squamous cell carcinoma and normal tissue of mice. Anticancer Res. 14:113118; 1994. 15. Lee, D.-J.; Cosmatos, D.; Mar&l, V. A.; Fu, K. K.; Rotman, M.; Cooper, J. S.; Ortiz, G. H.; Beitler, J. J.; Abrams, R. A.; Curran, W. J.; Coleman, C. N.; Wasserman, T. H. Results of an RTOG phase III trial (RTo 85-27) comparing radiotherapy plus etanidazole with radiotherapy alone for locally advanced head and neck carcinomas. Int. J. Radiat. Oncol. Biol. Phys. 32:567-576; 1995. 16. Leong, K. W.; Brott, B. C.; Langer, R. Bioerodible polyanhydrides as drug-carrier matrices. I: Characterization, degradation, and release characteristics. J. Biomed. Mater. Res. 19:941-955; 1985. 17. Leong, K. W.; D’Amore, P.; Marletta, M.; Langer, R. Bioerodible polyanhydrides as drug-carrier matrices. II Biocompatibility and chemical reactivity. J. Biomed. Mater. Res. 20:51-64; 1986. 18. Stewart, D. H.; Molepo, J. M.; Eapen, L.; Monpetit, V. A. J.; Gael, R.; Wong, P. T. T.; Popovic, P.; Taylor, K. D.; Raaphorst, G. P. Cisplatin and radiation in the treatment of tumors of the central nervous system: Pharmacological considerations and results of early studies. Int. J. Radiat. Oncol. Biol. Phys. 28:531-542; 1993. 19. Straw, R. C.; Withrow, S. J.; Douple, E.; Brekke, J. H.; Cooper, M. F.; Schwarz, P. D.; Greco, D. S.; Powers, B. E. Effects of cis-diamminedichloroplatinum (II) released from D,L-polylactic acid implanted adjacent to cortical allografts in dogs. J. Ortho. Res. 12:871-877; 1994. 20. Tamargo, R. J.; Epstein, J. I.; Reinhard, C. S.; Chasin, M.; Brem, H. Brain biocompatibility of a biodegradable, controlled-release polymer in rats. J. Biomed. Mater. Res. 23:253-266; 1989. 21. Twentyman, P. R.; Brown, J. M.; Gray, J. W.; Franko, A. J.; Stoles, M. A.; Kallman, R. F. A new mouse tumor model system (RIF- 1) for comparison of end-point studies.J. Natl. Cancer Inst. 64:595-604; 1980. 22. Withrow, S. J.; Straw, R. C.; Brekke, J. H.; Powers, B. E.; Cooper, M. F.; Ogilvie, G. K.; Lafferty, M.; Jameson, V. J.; Douple, E. B.; Johnson, M. S.; Dernell, W. S. Slow release adjuvant of cisplatin in slow-release devices. Eur. J. Exp. Musculoskel. Res. 4:105- 110; 1995.