Interstitial chemotherapy of experimental brain tumors: comparison of intratumoral injection versus polymeric controlled release

Interstitial chemotherapy with controlled release polymers is a clinical adjunct in the management of malignant gliomas. The need for polymer to release the chemotherapeutic drug rather than simply injecting the drug into the tumor warrants further investigation. Therefore, we compared the effects of direct intralesional injection of carmustine (BCNU) and 4-hydroperoxycyclophosphamide (4HC) into the rat brain tumor bed with those from the same agents delivered via controlled release polymers implanted intracranially. Treatment was initiated on the fifth day after intracranial implantation of 9L gliosarcoma into male rats; two doses of each drug were injected intratumorally, representing either the amount of drug typically releasedin vivo from polymer during the first 24 h, or the maximal drug loaded on each polymer. Control rats were treated with empty polymers. We found that the median lifespan was extended in the groups of rats treated with intratumoral injection of BCNU (23% and 36% for 1 mg and 2 mg doses), and 271% with BCNU-impregnated polymer. Similar results were found with intratumoral 4HC (21% and 36% for 0.1 mg and 2 mg injection doses), and 121% with 4HC-impregnated polymer. Overall survival after intraneoplastic injections, however, was not statistically significantly different from that of control rats (p > 0.05). Furthermore, improvement in survival was not consistent, and some animals subjected to 4HC injection died early in the course of treatment. Polymeric treatment resulted in statistically significant prolongation of survival, compared to control rats (p < 0.001 for both BCNU and 4HC). We conclude that direct intralesional injection of BCNU and 4HC is less effective than controlled release via polymers for the treatment of 9L gliosarcoma in the rat model.     

Local Delivery of Minocycline and Systemic BCNU have Synergistic Activity in the Treatment of Intracranial Glioma

Minocycline, a tetracycline derivative, has been shown to inhibit tumor angiogenesis through inhibitory effects on matrix metalloproteinases. Previous studies have shown this agent to be effective against a rodent brain tumor model when delivered intracranially and to potentiate the efficacy of standard chemotherapeutic agents. In the present study, the in vivo efficacy of intracranial minocycline delivered by a biodegradable controlled-release polymer against rat intracranial 9L gliosarcoma was investigated to determine whether it potentiates the effects of systemic 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Minocycline was incorporated into the biodegradable polymer polyanhydride poly[bis(p-carboxyphenoxy)propane-sebacic acid] (pCPP:SA) at a ratio of 50:50 by weight. The release kinetics of minocycline from the polymer were assessed. For the efficacy studies, female Fischer 344 rats were implanted with 9L glioma. Treatment with minocycline delivered by the pCPP:SA polymer at the time of tumor implantation resulted in 100% survival in contrast to untreated control animals that died within 21 days. Treatment with the minocycline-polymer 5 days after tumor implantation provided only modest increases in survival. The combination of intracranial minocycline and systemic BCNU extended median survival by 82% compared to BCNU alone (p < 0.0001) and 200% compared to no treatment (p < 0.004). We conclude that local intracranial delivery of minocycline from biodegradable controlled-release polymers inhibits tumor growth and may have clinical utility when combined with a chemotherapeutic agent.     

Dose escalation of carmustine in surgically implanted polymers in patients with recurrent malignant glioma: a New Approaches to Brain Tumor Therapy CNS Consortium trial.

PURPOSE: This New Approaches to Brain Tumor Therapy CNS Consortium study sought to determine the maximum-tolerated dose (MTD) of carmustine (BCNU) that can be implanted in biodegradable polymers following resection of recurrent high-grade gliomas and the systemic BCNU exposure with increasing doses of interstitial BCNU. PATIENTS AND METHODS: Forty-four adults underwent tumor debulking and polymer placement. Six patients per dose level were studied using polymers with 6.5%, 10%, 14.5%, 20%, and 28% BCNU by weight. Toxicities were assessed 1 month after implantation by a safety monitoring committee to determine whether subsequent escalations should occur. Nine additional patients were studied at the MTD to confirm safety. BCNU blood levels were obtained before and after polymer implantation. RESULTS: No dose-limiting toxicities were identified at the 6.5%, 10%, or 14.5% dose levels, although difficulties with wound healing, seizures, and brain edema were noted. At the 20% dose, these effects seemed more prominent, and six additional patients were treated at this dose and tolerated treatment well. Three of four patients receiving the 28% polymers developed severe brain edema and seizures, and accrual to this cohort was stopped. Nine additional patients received 20% polymer, confirming this as the MTD. Maximum BCNU plasma concentrations with the 20% loaded polymers were 27 ng/mL. Overall median survival was 251 days. CONCLUSION: The MTD of BCNU delivered in polymer to the surgical cavity is 20%. This polymer provides five times more BCNU than standard commercially available BCNU polymers and results in minimal systemic BCNU exposure. Additional studies are needed to establish the efficacy of high-dose BCNU polymers.     

Systemic BCNU enhances the efficacy of local delivery of a topoisomerase I inhibitor against malignant glioma

Purpose To investigate the ability of systemically delivered BCNU to enhance the activity of either systemically delivered irinotecan (CPT-11) or locally delivered camptothecin from a biodegradable polymer for treatment of an intracranial 9L gliosarcoma.Methods We used a single systemic dose of BCNU on treatment day 1 in combination with systemic doses of CPT-11 on treatment days 1–5 and 8–12 against an intracranial rat 9L gliosarcoma model implanted into female Fischer 344 rats. We also used the same systemic dose of BCNU given on treatment day 1, followed by a local dose of a 20% loaded camptothecin biodegradable polymer implanted on the same day.Results Two doses of CPT-11 (10 and 60 mg/kg) were delivered systemically against intracranial 9L. Neither dose showed an increase in survival compared to controls (P>0.2 for 10 mg/kg and P=0.17 for 60 mg/kg). Systemic delivery of CPT-11 (10 mg/kg per day) in combination with systemic BCNU (15 mg/kg) did not show a significant effect on survival compared to systemic BCNU alone (P>0.2), even at the maximally tolerated systemic dose of CPT-11 (60 mg/kg per day; P=0.06). The combination of systemic BCNU (15 mg/kg) and intracranial delivery of camptothecin (20% loaded polymer), however, significantly extended survival compared to systemic BCNU alone (P<0.001) and compared to intracranial delivery of camptothecin alone (P=0.01).Conclusions In a 9L gliosarcoma model, systemic delivery of CPT-11 showed no benefit in survival when delivered alone or in combination with systemic BCNU, because CPT-11 is unable to cross the blood–brain barrier in cytotoxic levels. When cytotoxic levels of a topoisomerase I inhibitor are delivered directly to the brain tumor via a biodegradable polymer, however, the systemic delivery of the alkylating agent BCNU significantly enhances the antitumor effects of camptothecin in a 9L gliosarcoma model.     

Implantable biodegradable polymers for IUdR radiosensitization of experimental human malignant glioma

Purpose: The potential of halogenated pyrimidines for the radiosensitization of human malignant gliomas remains unrealized. To assess the role of local delivery for radiosensitization, we tested a synthetic, implantablebiodegradable polymer for the controlled release of 5-iodo-2-deoxyuridine (IUdR) both in vitro and in vivo and the resultant radiosensitizationof human malignant glioma xenografts in vivo.Materials and methods: In vitro: To measure release, increasing (10%, 30%, 50%) proportions (weight/weight) of IUdR in the polyanhydride [(poly(bis(p-carboxyphenoxy)-propane) (PCPP) :sebacic acid (SA) (PCPP : SA ratio 20 : 80)] polymer discs were incubated (1 ml phosphate-buffered saline, 37° C). The supernatant fractions were serially assayed using high performance liquid chromatography. To measure modulation of release,polymer discs were co-loaded with 20 Ci 5-125-iodo-2-deoxyuridine (125-IUdR) and increasing (10%, 30%, or 50%) proportions of D-glucose. To test radiosensitization, cells (U251 human malignant glioma) were sequentially exposed to increasing (0 or 10 M) concentrations of IUdR and increasing (0, 2.5, 5.0, or 10 Gy) doses of acute radiation. In vivo: To measure release, PCPP : SA polymerdiscs having 200 Ci 125-IUdR were surgically placed in U251 xenografts (0.1—0.2 cc) growing in the flanksof nude mice. The flanks were reproducibly positioned over a collimated scintillation detector and counted. To measure radiosensitization, PCPP : SApolymer discs having 0% (empty) or 50% IUdR wereplaced in the tumor or contralateral flank. After five days, the tumors were acutely irradiated (500 cGy × 2 daily fractions).Results: In vitro: Intact IUdR was released from the PCPP : SA polymer discs in proportion to the percentage loading. After 4 days the cumulative percentages of loaded IUdR that were released were 43.7 $plusmn; 0.1, 70.0 ± 0.2, and 90.2 ± 0.2 (p < 0.001 ANOVA) for the 10, 30, and 50% loadings. With 0, 10, 30,or 50% D-glucose co-loading, the cumulative release of 125-IUdR from PCPP : SA polymers was 21, 70, 92, or 97%(p < 0.001), respectively, measured 26 days after incubation.IUdR radiosensitized U251 cells in vitro. Cell survival (log₁₀) was – 2.02 ± 0.02 and – 3.68± 0.11 (p < 0.001) after the 10 Gy treatment and no (control) or 10 M IUdR exposures, respectively. In vivo: 125-IUdR Release: The average counts (log₁₀ cpm ± SEM) (hours after implant) were 5.2 ± 0.05 (0.5), 4.3 ± 0.07 (17), 3.9 ± 0.08 (64), and 2.8 ± 0.06 (284). Radiosensitization: Afterintratumoral implantation of empty polymer or intratumoral 50%IUdR polymer, or implantation of 50% IUdR polymers contralateral to tumors, the average growth delays of tumors to4 times the initial volumes were 15.4 ± 1.8, 20.1 + 0.1,and 20.3 + 3.6 (mean + SEM) days, respectively (p = 0.488one-way ANOVA). After empty polymer and radiation treatments,no tumors regressed and the growth delay was 31.1 + 2.1 (p = 0.046 vs. empty polymer alone) days. After implantation of50% IUdR polymers either contralateral to the tumors orinside the tumors, followed by radiation, tumors regressed; growth delays to return to the initial average volumes of 14.0+ 3.6 or 24.2 + 0.2 (p < 0.01) days, respectively.Conclusions: Synthetic, implantable biodegradable polymers hold promise for the controlled release and local delivery ofIUdR for radiosensitization of gliomas.     

Interstitial delivery of carboplatin via biodegradable Polymers is effective against experimental glioma in the rat

 Purpose: Carboplatin has shown promise experimentally as an antineoplastic agent against both primary central nervous system (CNS) tumors and several solid tumors that frequently metastasize to the brain. Unfortunately, carboplatin is limited in its clinical use for tumors in the CNS by systemic toxicity and poor penetration through the blood–brain barrier. Recent advances in polymer technology have made feasible the intracranial implantation of a biodegradable polymer capable of local sustained delivery of chemotherapy for brain neoplasms. This study assessed the toxicity and efficacy of carboplatin delivered from intracranial sustained release polymers in the treatment of experimental gliomas in rodents. Methods: Two biodegradable anhydride polymer systems were tested: a copolymer of 1,3-bis-(p-carboxyphenoxy propane) and sebacic acid, and a copolymer of fatty acid dimer and sebacic acid. The polymers were loaded with carboplatin and dose escalation studies evaluating toxicity were performed by implanting carboplatin-loaded polymers into the brains of rats. Next, efficacy was tested. F-98 glioma cells were injected intracranially into rats, and 5 days later polymers containing the highest tolerated doses were implanted at the site of tumor growth. The survival of animals receiving carboplatin-loaded polymer was compared with that of animals receiving intraperitoneal doses of the same agent. Results: Carboplatin-polymer was well tolerated at doses up to 5% loading in both polymer systems. Locally delivered carboplatin effectively prolonged survival of rats with F98 gliomas. Maximal treatment effect was seen with 5% loading of either polymer, with median survival increased threefold over control (P<0.004). Systemic carboplatin also significantly prolonged survival, but the best intracranial polymer dose was significantly more effective than the best systemic dose tested. Conclusions: Carboplatin can be safely delivered intracranially by biodegradable sustained-release polymers. This treatment improves survival in rodents with experimental gliomas, with locally delivered carboplatin being more effective than systemic carboplatin. Received: 25 August 1995/Accepted: 21 February 1996     

Interstitial Docetaxel (Taxotere), Carmustine and Combined Interstitial Therapy: a Novel Treatment for Experimental Malignant Glioma

Docetaxel (Taxotere) is a hemisynthetic, anti-cancer compound with good preclinical and clinical activity in a variety of systemic neoplasms. We tested its activity against malignant gliomas using local delivery methods. Antitumor activity was assessed in vitro against human (U87 and U80 glioma) and rat brain-tumor (9L gliosarcoma and F98 glioma) cell lines. For in vivo evaluation, we incorporated docetaxel into a biodegradable polymer matrix, determined associated toxicity in the rat brain, and measured efficacy at extending survival in a rat model of malignant glioma. Also, we examined the combined local delivery of docetaxel with carmustine (BCNU) against the experimental intracranial glioma. Rats bearing intracranial 9L gliosarcomas were treated 5 days after tumor implantation with various polymers (placebo, 5% docetaxel, 3.8% BCNU, or 5% docetaxel and 3.8% BCNU combination). Animals receiving docetaxel polymers (n = 15, median survival 39.1 days) had significantly improved survival over control animals (n = 12, median survival 22.5 days, P = 0.01). Similarly, animals receiving BCNU polymers (n = 15, median survival 39.3 days, 13.3% long-term survivors) demonstrated an increase in survival compared to the controls (P = 0.04). Animals receiving the combination polymers demonstrated a modest increase in survival compared to either chemotherapeutic agent alone (n = 14, median survival 54.9 days, 28.6% long-term survivors) with markedly improved survival over controls (P = 0.003). We conclude that locally delivered docetaxel shows promise as a novel anti-glioma therapy and that the combination of drug regimens via biodegradable polymers may be a great therapeutic benefit to patients with malignant glioma.     

Implantable polymers for tirapazamine treatments of experimental intracranial malignant glioma.

Malignant gliomas remain refractory to intensive radiotherapy and cellular hypoxia enhances clinical radioresistance. Under hypoxic conditions, the benzotriazine di-N-oxide (3-amino-1,2,4-benzotriazine 1,4-dioxide) (tirapazamine) is reduced to yield a free-radical intermediate that results in DNA damage and cellular death. For extracranial xenografts, tirapazamine treatments have shown promise. We therefore incorporated tirapazamine into the synthetic, biodegradable polymer, measured the release, and tested the efficacy both alone and in combination with external beam radiotherapy in the treatment of experimental intracranial human malignant glioma xenografts. The [(poly(bis(p-carboxyphenoxy)-propane) (PCPP):sebacic acid (SA) (PCPP:SA ratio 20:80)] polymer was synthesized. The PCPP:SA polymer and solid tirapazamine were combined to yield proportions of 20% or 30% (wt/wt). Polymer discs (3 x 2 mm) (10 mg) were incubated (PBS, 37 degrees C), and the proportion of the drug released vs. time was recorded. Male nu/nu nude mice were anesthetized and received intracranial injections of 2 x 10(5) U251 human malignant glioma cells. For single intraperitoneal (i.p.) drug and/or external radiation treatments, groups of mice had i.p. 0.3 mmol/kg tirapazamine, 5 Gy cranial irradiation, or combined treatments on day 8 after inoculation. For fractionated drug and radiation treatments, mice had i.p. 0.15 mmol/kg tirapazamine, 5 Gy radiation, or combined treatments on days 8 and 9 after inoculation. For intracranial (i.c.) polymer treatments, mice had craniectomies and intracranial placement of polymer discs at the site of cellular inoculation. The maximally tolerated percentage loading of tirapazamine in the polymer.disc was determined. On day 7 after inoculation, groups of mice had i.c. empty or 3% tirapazamine alone or combined with radiation (5 Gy x 2 doses) or combined with i.p. drug (0.15 mmol/kg x 2 doses on days 8 and 9). Survival was recorded. Polymers showed controlled, protracted in vitro release for over 100 days. The 5 Gy x 1 treatment resulted in improved survival; 28.5 +/- 3.7 days (P = 0.01 vs. controls), while the single i.p. 0.3 mmol/kg tirapazamine treatment, 17.5 +/- 1.9 days (P = NS) and combined treatments; 21.5 +/- 5.0 days (P = NS) were not different. The fractionated treatments: 5 Gy x 2, i.p. 0.15 mmol/kg tirapazamine x 2 and the combined treatments resulted in improved survival: 44.5 +/- 3.9 (P < 0.001), 24.5 +/- 2.3 (P = 0.05) and 50.0 +/- 6.0 (P < 0.001), respectively. Survival after intracranial empty polymer was 16.5 +/- 3.0 days and increased to 31.0 +/- 3.0 (P = 0.003) days when combined with the 5 Gy x 2 treatment. The survival after the polymer bearing 3% tirapazamine alone vs. combined with radiation was not different. The combined 3% tirapazamine polymer, i.p. tirapazamine, and radiation treatments resulted in both early deaths and the highest long-term survivorship. The basis for potential toxicity is discussed. We conclude that implantable biodegradable polymers provide controlled intracranial release for treatment of experimental glioma. For treatment of malignant gliomas, the combination of continuous polymer-mediated delivery and fractionated systemic delivery of tirapazamine with external beam radiotherapy warrants further exploration.     

Polymer Delivery of Camptothecin against 9L Gliosarcoma: Release,Distribution, and Efficacy

Camptothecin is a potent antineoplastic agent that has shown efficacy against multiple tumor lines in vitro; unfortunately, systemic toxicity has limited its in vivo efficacy. This is the first study to investigate the release, biodistribution, and efficacy of camptothecin from a biodegradable polyanhydride polymer. Tritiated camptothecin was incorporated into biodegradable polymers that were implanted intracranially in 16 male Fischer 344 rats and the animals were followed up to 21 days post-implant. A concentration of 11–45g of camptothecin-sodium/mg brain tissue was within a 3mm radius of the polymer disc, with levels of 0.1g at the outermost margin of the rat brain, 7mm from the site of implantation. These tissue concentrations are within the therapeutic ranges for human and rat glioma lines tested against camptothecin-sodium in vitro. The in vivo efficacy of camptothecin-sodium was evaluated with male Fischer 344 rats implanted intracranially with 9L gliosarcoma and compared with the efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The animals were divided into four groups. Group 1 (control) had a median survival of 17 days. Group 2 (3.8% BCNU polymer) had a median survival of 23 days (P=0.006). Group 3 (20% camptothecin polymer) had a median survival of 25 days (P=0.023). Group 4 (50% camptothecin polymer) had a median survival of 69 days (P<0.001). Drug loadings of 20% and 50% camptothecin released intact camptothecin for up to 1000h in vitro. We conclude that the biodegradable polymer p(CPP:SA) releases camptothecin-sodium, produces tumoricidal tissue levels, results in little or no systemic toxicity, and prolongs survival in a rat glioma model.     

O6-benzylguanine potentiates the antitumor effect of locally delivered carmustine against an intracranial rat glioma

Local delivery of carmustine (BCNU) via biodegradable polymers prolongs survival against experimental brain tumors and in human clinical trials. O6-benzylguanine (O6-BG), a potent inhibitor of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), has been shown to reduce nitrosourea resistance and, thus, enhance the efficacy of systemic BCNU therapy in a variety of tumor models. In this report, we demonstrate that O6-BG can potentiate the activity of BCNU delivered intracranially via polymers in rats challenged with a lethal brain tumor. Fischer 344 rats received a lethal intracranial challenge of 100,000 F98 glioma cells (F98 cells have significant AGT activity, 328 fmol/mg protein). Five days later, animals receiving an i.p. injection of O6-BG (50 mg/kg) 2 h prior to BCNU polymer (3.8% BCNU by weight) implantation had significantly improved survival (n = 7; median survival, 34 days) over animals receiving either O6-BG alone (n = 7; median survival, 22 days; P = 0.0002) or BCNU polymer alone (n = 8; median survival, 25 days; P = 0.0001). Median survival for the control group (n = 8) was 23.5 days. Moreover, there was no physical, behavioral, or pathological evidence of treatment-related toxicity. These findings suggest that O6-BG can potentiate the effects of interstitially delivered BCNU and, for tumors expressing significant AGT, may be necessary for the BCNU to provide a meaningful therapeutic benefit. Given the clinical use of BCNU polymers against malignant gliomas, concurrent treatment with O6-BG may provide an important addition to our therapeutic armamentarium.     

Lactacystin Exhibits Potent Anti-tumor Activity in an Animal Model of Malignant Glioma when Administered via Controlled-release Polymers

Summary Lactacystin, a proteasome-inhibitor, has been shown to induce apoptosis of experimental gliomas in vitro. However, its systemic toxicity prevents further clinical use. To circumvent this problem, lactacystin can be delivered intratumorally. We tested the efficacy of lactacystin incorporated into controlled-release polymers for treating experimental gliomas. 9L-gliosarcoma and F98-glioma cell lines were treated with lactacystin (10–100 μg/ml) for 72 h in vitro. Cell-viability was measured with MTT-assays. Toxicity of lactacystin/polycarboxyphenoxypropane-sebacic-acid (pCPP : SA) polymers was tested in vivo using Fischer-344 rats intracranially implanted with lactacystin polymers loaded from 0.1 to 2% lactacystin by weight. The efficacy of 1, 1.3, 1.5 and 1.7% lactacystin/pCPP : SA polymers was determined in Fischer-344 rats intracranially challenged with 9L and treated either simultaneously or 5 days after tumor implantation. Lactacystin was cytotoxic in 9L cells, causing a 16 ± 8% growth inhibition at 10-μg/ml that increased to 78 ± 4% at 100-μg/ml. Similarly, lactacystin inhibited growth of F98 by 18 ± 8% at 10-μg/ml and 74 ± 2% at 100-μg/ml in vitro. Polymers released lactacystin for 21 days and intracranial implantation in rats neither generate local nor systemic toxicity at doses lower than 2%. Treatment with lactacystin/pCPP : SA polymers with loading concentrations of 1.0, 1.3, and 1.5% prolonged survival of animals intracranially challenged with 9L when polymers where inserted in the day of tumor implantation. In conclusion, lactacystin exhibits potent cytotoxic-activity against 9L and F98 in vitro, it can be efficiently incorporated and delivered using controlled-release polymers, and at the proposed concentrations lactacystin polymers are safe for CNS delivery and prolong survival in the 9L model. These authors equally contributed in all aspects of this study.     

Synthetic,implantable polymers for local delivery of IUdR to experimental human malignant glioma

Purpose: Recently, polymeric controlled delivery of chemotherapy has been shown to improve survival of patients with malignant glioma. We evaluated whether we could similarly deliver halogenated pyrimidines to experimental intracranial human malignant glioma. To address this issue we studied the in vitro release from polymers and the in vivo drug delivery of IUdR to experimental human U251 glioblastoma xenografts.Methods and Materials: In vitro: To measure release, increasing (10%, 30%, 50%) proportions of IUdR in synthetic [(poly(bis(p-carboxyphenoxy)-propane) (PCPP):sebacic acid (SA) polymer discs were serially incubated in buffered saline and the supernatant fractions were assayed. In vivo: To compare local versus systemic delivery, mice bearing flank xenografts had intratumoral or contralateral flank IUdR polymer (50% loading) treatments. Mice bearing intracranial (i.c.) xenografts had i.c. versus flank IUdR polymer treatments. Four or 8 days after implantation of polymers, mice were sacrificed and the percentage tumor cells that were labeled with IUdR was measured using quantitative microscopic immunohistochemistry.Results: In vitro: Increasing percentage loadings of IUdR resulted in higher percentages of release: 43.7 + 0.1, 70.0 + 0.2, and 90.2 + 0.2 (p < 0.001 ANOVA) for the 10%, 30%, and 50% loadings, respectively. In vivo: For the flank tumors, both the ipsilateral and contralateral IUdR polymers resulted in similarly high percentages labeling of the tumors versus time. For the ipsilateral IUdR polymers, the percentage of tumor cellular labeling after 4 days versus 8 days was 45.8 ± 7.0 versus 40.6 ± 3.9 (p = NS). For the contralateral polymer implants, the percentage of tumor cellular labeling were 43.9 ± 10.1 versus 35.9 ± 5.2 (p = NS) measured 4 days versus 8 days after implantation. For the i.c. tumors treated with extracranial IUdR polymers, the percentage of tumor cellular labeling was low: 13.9 ± 8.8 and 11.2 ± 5.7 measured 4 and 8 days after implantation. For the i.c. tumors having the i.c. IUdR polymers, however, the percentage labeling was comparatively much higher: 34.3 ± 4.9 and 35.3 ± 4.0 on days 4 and 8, respectively. For the i.c. tumors, examination of the percentage cellular labeling versus distance from the implanted IUdR polymer showed that labeling was highest closest to the polymer disc.Conclusion: Synthetic, implantable biodegradable polymers provide the local, controlled release of IUdR and result in the high, local delivery of IUdR to experimental intracranial human malignant glioma. This technique holds promise for the local delivery of IUdR for radiosensitization of human brain tumors.     

Cure of advanced L1210 leukemia after correction of abnormal red blood cell deformability

Summary Chemotherapeutic efficacy is inversely related to pretreatment tumor burden. A possible contributory factor in chemotherapy resistance is the occurrence of decreased red blood cell deformability in mice with advanced tumors. Poorly deformable red blood cells may prevent adequate drug delivery to tumor cells. Two methods for improving red cell deformability were found in this study. The first involved treatment of L1210 leukemia-bearing mice with red cell metabolic substrates, including inosine, adenosine, glucose, sodium pyruvate, and ascorbic acid. The combination of inosine plus sodium pyruvate (3 mg of each drug in 0.5 cm³ phosphate-buffered saline) was most effective in restoring deformability to normal. Administration of an active chemotherapeutic agent (BCNU or cyclophosphamide) also improved red cell deformability, with maximal restoration occurring 4–5 days after drug treatment. Standard and 50% of standard drug doses were equally effective in restoring deformability. The optimal therapy program for day 7 L1210 leukemia utilized inosine plus sodium pyruvate given 10–15 min before BCNU 15 mg/kg on day 7 and before BCNU 30 mg/kg on day 12. This treatment yielded 44% cures, whereas BCNU alone, in identical dose and schedule, gave no cures. Median survival was 50 days for the inosine-pyruvate-treated mice, as against 30 days for BCNU alone. Therefore, treatment with non-toxic doses of red blood cell metabolic substrates plus optimal timing of chemotherapy, two maneuvers that significantly increased red blood cell deformability, resulted in significant therapeutic benefit.     

Early chemotherapy and concurrent radio-chemotherapy in high grade glioma

Summary Purpose: The poor results from treatment of high grade glioma prompted us to explore new protocols involving concurrent radio-chemotherapy. Our primary objective was to evaluate the feasibility of very early postoperative chemotherapy with BCNU, concurrent radio-chemotherapy with carboplatin and teniposide, and post-radiotherapy BCNU. Our secondary objectives were to evaluate time to progression, and overall survival. Patients and methods: We treated 24 newly diagnosed patients (pts) with BCNU 150 mg/m² seven days after surgery. Thirty days later, we started radiotherapy, 1.8 to 2 Gy/day for 5 days a week on limited fields up to 60 Gy, and concurrent chemotherapy with carboplatin 250 mg/m² on days 1, 22, and 43, and teniposide 50 mg/m² on days 1, 2, 3, 22, 23, 24, 43, 44 and 45. Two cycles of 150 mg/m² BCNU were then given at 30 and 70 days, respectively, after the end of the radio-chemotherapy course. Therapy was then suspended, but if disease progression was evident, treatment was resumed with drugs that had not been previously employed. Surgical reintervention was not routinely considered. Results: Following radio-chemotherapy treatment in the 24 pts evauble for response, we observed partial remissions in 8 cases (33%) and stable disease in 12 (50%). Actuarial estimates of progression free survival (PFS) were 33 weeks, with 56 wks for anaplastic astrocytoma and 31 weeks for glioblastoma. Median survival time (MST) of all pts was 58 weeks; 51 weeks for glioblastoma and was not reached for anaplastic astrocytoma. This regimen was feasible. Of 144 planned cycles, 139 were delivered, and among these only in 13 and 9 cycles the doses were reduced by 75 and 50%, respectively. We did not observe any gastrointestinal toxicity. Grade 2 hematological toxicity occurred in 25% of pts, grade 3 in 4% and neurological toxicity in 3% of the pts during BCNU delivery, probably due to a sharp increase in intracranial pressure. Conclusion: Early chemotherapy, concurrent chemo-radiotherapy and brief post-radio-therapy chemotherapy are feasible and well tolerated. The objective response and disease stabilization rates appear similar to previous experiences.     

IUdR polymers for combined continuous low-dose rate and high-dose rate sensitization of experimental human malignant gliomas

Local polymeric delivery enhances IUdR radiosensitization of human malignant gliomas (MG). The combined low-dose rate (LDR) (0.03 Gy/h) and fractionated high-dose rate (HDR) treatments result in cures of experimental MGs. To enhance efficacy, we combined polymeric IUdR delivery, LDR, and HDR for treatments of both subcutaneous and intracranial MGs. In vitro: Cells (U251 MG) were trypsinized and replated in triplicate 1 day prior to LDR irradiation in media either without (control) or with 10 microM IUdR. After 72 hr, LDR irradiation cells were acutely irradiated (1.1 Gy/min) with increasing (0, 1.25, 2.5, 5.0, or 10 Gy) single doses. Implantable IUdR polymers [(poly(bis(p-carboxyphenoxy)-propane) (PCPP): sebaic acid (PCPP:SA), 20:80] (50% loading; 10 mg) were synthesized. In vivo: For flank vs. intracranial tumors, mice had 6 x 10(6) subcutaneous vs. 2 x 10(5) intracranial cells. For intracranial or subcutaneous MGs, mice had intratumoral blank (empty) vs. IUdR polymer treatments. One day after implantation, mice had immediate external LDR (3 cGy/h x 3 days total body irradiation) or HDR (2 Gy BID x 4 days to tumor site) or concurrent treatments. For the in vitro IUdR treatments, LDR resulted in a striking increase in cell-killing when combined with HDR. For the in vivo LDR treatments of flank tumors, the growth delay was greater for the IUdR vs. blank polymer treatments. For the combined LDR and HDR, the IUdR treatments resulted in a dramatic decrease in tumor volumes. On day 60 the log V/V0 were -1.7 +/- 0.22 for combined LDR + HDR + IUdR polymer (P < 0.05 vs. combined LDR + HDR + blank polymer). Survival for the intracranial controls was 22.9 +/- 1.2 days. For the blank polymer + LDR vs. blank polymer + LDR + HDR treatments, survival was 25.3 +/- 1.7 (P = NS) vs. 48.1 +/- 3.5 days (P < 0.05). For IUdR polymer + LDR treatment survival was 27.3 +/- 2.3 days (P = NS). The most striking improvement in survival followed the IUdR polymer + LDR + HDR treatment: 66.0 + 6.4 days (P < 0.05 vs. blank polymer + LDR + HDR). The polymeric IUdR delivery plus combined continuous LDR and HDR treatments results in growth delay and improved survival in animals bearing the MG xenografts. This treatment may hold promise for the treatment of human MGs.     

Hyperthermic potentiation of BCNU toxicity in BCNU-resistant human glioma cells

Summary Experimental evidence indicating potentiation of the cytotoxic effect of drugs at high temperatures suggests that the utilization of drug-heat combinations for gliomas of the brain might be therapeutically useful. Hyperthermia may increase the cytotoxicity of a particular drug in areas of low drug concentration/time and in cell populations resistant to the drug. We report in vitro experiments with a BCNU resistant, U-373MG, and a BCNU sensitive, U-87MG, human derived glioma cell lines under hyperthermic conditions. Temperatures equal or above 42°C potentiate BCNU cell kill in both lines. The thermo-sensitizer lidocaine increases thermal cell kill but only minimally with concentrations corresponding to therapeutic plasma lidocaine levels. Within our experimental conditions, the best strategy to overcome BCNU resistance involved a combination of heat, BCNU and cis-DDP. BCNU resistant cells have no cross resistance to cis-DDP and the combination of BCNU and cis-DDP is synergystic. At modest hyperthermic conditions (42°C) 99.4% BCNU resistant cells are killed by a combination of BCNU and cis-DDP at drug concentrations identical to plasma concentrations after standard IV doses. Clinical protocols using heat and drug may need to incorporate two or more drugs for optimal effects.Abbreviations BCNU 1,3-bis (2-Chloroethyl)-1-nitrosourea - cis-DDP Cis-Diamminedichloroplatinum (II) - AZO Aziridinylbenzoquinone - TER Thermal Enhancement Ratio     

Enhancement of the antitumor effect of 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) by phenylethylbiguanide (phenformin).

Phenlethylbiguanide (phenformin) a commonly used antidiabetic medication has been found to enhance the antitumor effect of 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) in advanced subcutaneously implanted murine L1210 leukemia. Enhancement required two doses of phenformin given twelve to 18 hours apart, the treatment starting either before or after BCNU administration. With BCNU alone median survival (MS) was 18 days with 5% cures. BCNU plus phenformin, in optimal dose and schedule, gave a MS of 25 days with 29% cures. Th mechanism of action of phenformin is unknown but may involve several established metabolic effects of this drug.     

Glutathione monoethyl ester can selectively protect liver from high dose BCNU or cyclophosphamide

Normal Swiss Webster mice were treated with monocrotaline or high doses of three antitumor alkylating agents (BCNU, cyclophosphamide, or mitomycin C), all of which have been connected with hepatic veno-occlusive disease at our clinic. Prior administration of WR-2721 did not improve the survival of monocrotaline-treated animals. Glutathione (GSH) improved the survival of these animals to a small degree. Glutathione monoethyl ester (GSHet) almost completely protected animals from the toxicity of monocrotaline. Pretreatment with WR-2721 produced moderate increases in survival at the highest doses of BCNU, and at the lower BCNU doses none of the animals pretreated with WR-2721 died before they were killed on day 150. Pretreatment with GSHet gave good protection from BCNU toxicity at the highest dose of the drug, and there were no deaths in the groups of animals treated with GSHet 1 hour before BCNU. On a multiple dose schedule, GSH provided some protection from cyclophosphamide toxicity; GSHet gave a very good level of protection from cyclophosphamide. In none of these treatment groups were lesions suggestive of hepatic or pulmonary venoocclusive disease identified. In all three experimental protocols (monocrotaline, BCNU, and cyclophosphamide), there was a consistent decrease in hepatic toxicity after GSHet pretreatment; this was not observed in GSH- or WR-2721-pretreated animals. There was no evidence of protection of the FSaIIC fibrosarcoma growing in C3H mice as assayed by tumor growth delay or tumor cell survival in groups treated with two different doses of GSHet 1 hour before each drug injection compared to those treated with the BCNU or cyclophosphamide alone, or BCNU with cyclophosphamide. Pretreatment with GSHet did not alter the toxicity of these drugs to bone marrow. GSHet appears to be an effective protector of critical normal tissue and does not appear to protect tumor.     

Toxicity of intracranial and intraperitoneal O6-benzyl guanine in combination with BCNU delivered locally in a mouse model

PURPOSE: The DNA-repair protein, O6-alkylguanine-DNA alkyl transferase, may account for resistance of CNS tumors to DNA-alkylating drugs, such as bis-(2-chloroethyl)-1-nitrosourea (BCNU). The therapeutic effects of BCNU can be potentiated by inhibiting the repair protein with an alkylated guanine analog, O6-benzyl guanine (O6BG). To investigate potential toxicity of this inhibition, we examined the effects of O6BG in mice treated with intracranial (i.c.) BCNU given via a biodegradable polymer. METHODS: Mice were treated with escalating doses of BCNU chronically delivered i.c., and with chronically delivered O6BG. The O6BG was delivered via a 7-day intraperitoneal (i.p.) or i.c. osmotic minipump. Toxicity of the combination therapies was measured from survival data. Bone marrow response was estimated from white blood cell counts. RESULTS: Combining systemic (i.p.) O6BG with locally (i.c.) delivered BCNU resulted in a decrease in the maximum tolerated dose (MTD) of local BCNU. With local delivery of O6BG, the MTD of BCNU in combination with O6BG was increased. CONCLUSIONS: Based on the results of this study, a dose escalation study will be necessary when combining systemic O6BG with the higher doses of i.c. BCNU.     

Influence of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice

The effects of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice have been investigated using a versatile procedure for encapsulating doxorubicin inside liposomes. In this procedure, vesicles exhibiting transmembrane pH gradients (acidic inside) were employed to achieve drug trapping efficiencies in excess of 98%. Drug-to-lipid ratios as high as 0.3:1 (wt:wt) could be obtained in a manner that is relatively independent of lipid composition and vesicle size. Egg phosphatidylcholine (EPC)/cholesterol (55:45; mol/mol) vesicles sized through filters with a 200-nm pore size and loaded employing transmembrane pH gradients to achieve a doxorubicin-to-lipid ratio of 0.3:1 (wt/wt) increased the LD50 of free drug by approximately twofold. Removing cholesterol or decreasing the drug-to-lipid ratio in EPC/cholesterol preparations led to significant decreases in the LD50 of liposomal doxorubicin whereas, the LD50 increased 4- to 6-fold when distearoylphosphatidylcholine was substituted for EPC. The results suggest that the stability of liposomally entrapped doxorubicin in the circulation is an important factor in the toxicity of this drug in liposomal form. In contrast, the antitumor activity of liposomal doxorubicin is not influenced dramatically by alterations in lipid composition. Liposomal doxorubicin preparations of EPC, EPC/cholesterol (55:45; mol:mol), EPC/egg phosphatidylglycerol (EPG)/cholesterol (27.5:27.5:45; mol:mol), and distearoylphosphatidylcholine/cholesterol (55:45; mol:mol) all demonstrated similar efficacy to that of free drug when given at doses of 20 mg/kg and below. Higher dose levels of the less toxic formulations could be administered, leading to enhanced increases in life span (ILS) values. Variations in vesicle size, however, strongly influenced the antitumor activity of liposomal doxorubicin. At a dose of 20 mg/kg, large EPC/cholesterol systems are significantly less effective than free drug (with ILS values of 65% and 145%, respectively). In contrast, small systems sized through filters with a 100-nm pore size are more effective than free drug, resulting in an ILS of 375% and a 30% long term (greater than 60 days) survival rate when administered at a dose of 20 mg/kg. Similar size-dependent effects are observed for distearoylphosphatidylcholine/cholesterol systems.