Delivery of temozolomide to the tumor bed via biodegradable gel matrices in a novel model of intracranial glioma with resection

Introduction We have completed in vivo safety and efficacy studies of the use of a novel drug delivery system, a gel matrix-temozolomide formulation that is injected intracranially into the post-resection cavity, as a candidate for glioma therapy. Methods A rat intracranial resection model of C6-GFP intracranial glioma was used for safety and toxicity studies. Biodistribution studies were performed using gel matrix-gallocyanine formulations and were evaluated at various time intervals using real-time analysis of dye distribution. Additionally, the resection model was used to determine the efficacy of gel matrix-temozolomide as compared to blank gel matrix. A subcutaneous human xenograft glioma model was used to further assess the efficacy of gel matrix-temozolomide in reducing the overall tumor load. Results Gel matrix-temozolomide exhibited minimal cytotoxicity toward normal brain tissue while displaying high levels of oncolytic activity toward glioma cells. In the intracranial glioma resection and subcutaneous glioma model, administration of gel matrix-temozolomide directly to the tumor bed was well tolerated and effective at reducing the tumor load. A significant reduction of tumor load was observed (P < 0.0001) in the 30% temozolomide group (~95%) as compared to blank control. There was little morbidity and no mortality associated with gel matrix treatment. Conclusions Gel matrix-temozolomide appears to be safe and effective when used in vivo to treat intracranial glioma and warrants further development as a potential adjuvant therapy.     

Optimizing interstitial delivery of BCNU from controlled release polymers for the treatment of brain tumors

 Two approaches for improving the interstitial administration of carmustine (BCNU) using 3.8% loaded poly(carboxyphenoxypropane-sebacic acid), an implantable biodegradable anhydride which significantly prolongs survival in patients with recurrent malignant gliomas, were evaluated. First, increasing the ratio of carboxyphenoxypropane (CPP) to sebacic acid (SA) in the polymer increases its hydrolytic stability, thus prolonging its half-life in vivo, and extending the period of drug release. A second approach is to increase the dose of drug loaded into the polymer. This study evaluated the relative merits of these two approaches by comparing release kinetics, safety, and efficacy of escalating BCNU doses in polymers with 20:80 and 50:50 ratios of CPP to SA. At the highest dose tested, the 50:50 polymer released BCNU 2.5 times as long in vitro as the 20:80 polymer. Both formulations were nontoxic in rat brains for all BCNU doses tested except 32%. The 20:80 and 50:50 polymers were equally effective in the rat intracranial 9L-glioma model. A dose-response relationship for BCNU was observed (hazard ratio 0.8354 for each mg/kg increase, P<0.001). The two highest loading doses of BCNU improved survival 40-fold (P<0.001). The 20% BCNU-loaded 20:80 polymer achieved the best balance of toxicity and antitumor efficacy, yielding a 75% long-term survival rate. Further evaluation of this polymer in monkeys suggests that it might be used with acceptable toxicity. This study establishes that a dose-escalation strategy for improving BCNU controlled-release polymers is more effective than adjusting the ratio of CPP to SA to prolong drug release. Received: 21 February 1996/Accepted: 14 August 1996     

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.     

A pharmacogically guided phase I study of carboplatin in combination with methotrexate and vinblastine in advanced urothelial cancer

 Carboplatin is an alternative for cisplatin in the treatment of urothelial cancers. A pharmacologically guided phase I study of carboplatin in combination with methotrexate (30 mg/m²) and vinblastine (4 mg/m²) was conducted in ten patients by increment of the area under the plasma concentration versus time curve (AUC) for ultrafilterable carboplatin using the Calvert formula. The maximal tolerated AUC was 5 mg ml^-1 min, with neutropenia being the dose-limiting toxicity. There was a significant linear correlation between the percentage of decrease in neutrophil count and the carboplatin AUC. Determination of the glomerular filtration rate by the isotopic method allowed us to adapt the dose of carboplatin given to patients suffering from urothelial cancer, who frequently have impaired renal function. The recommended AUC for phase II study is 4 mg ml^-1 min. Received: 9 May 1994/Accepted: 16 August 1994     

Assessment of a balloon-tipped catheter modified for intracerebral convection-enhanced delivery

Numerous improvements in the understanding of the biology of primary brain tumors have been reported. The resultant application of this information to the therapy of these lesions offers promising alternatives. For any of a number of reasons delivery of these therapies to the target neoplasm can be challenging. Convection enhanced delivery has been established as a modality that has been shown to circumvent some of the impediments to treatment agent delivery. This report described the preliminary preclinical use of a balloon tipped catheter with a channel built in for infusion of therapy directly into the brain. A series of 10 canines were studied using bolus and continuous infusions with the balloon either inflated or deflated. The infusates contained gadolinium to allow imaging of the convection process. The character of the cerebral penetration is described ranging from minimal cerebral penetration with uninflated balloons used with bolus injections to extensive bilateral penetration using inflated balloons and continuous infusions. This data demonstrates the feasibility and potential value of such a system and warrants a more detailed analysis of the device using a wider variety of infusion parameters, assessment of larger infusate molecule sizes likely to be solely dependent on convection and direct comparison to standard catheter convection techniques.     

A single-sample assay for the estimation of the area under the free carboplatin plasma concentration versus time curve

 The aim of this study was to develop and validate a simple and rapid method for the estimation of the area under the free carboplatin plasma concentration versus time curve (AUC). The relationship between the carboplatin AUC and the total plasma platinum (Pt) concentration 24 h after treatment was studied using data from 49 patients treated with 20–1600 mg/m² carboplatin as a 60–100 min infusion (median 60 min). The relationship was confirmed by the in vitro incubation of carboplatin in human plasma and prospectively validated in 13 ovarian cancer patients. Free carboplatin was separated by ultrafiltration (MW cut off 30,000), and free and total Pt measured by atomic absorption spectrophotometry. There was a linear relationship in vivo between the 24 h (median 24.4; range 16.3–27.3 h) total plasma Pt concentration (μM) and free carboplatin AUC (mg/ml.min): AUC=(24 h Pt+0.3)/0.82 (r ²=0.93, AUC median 5.8 (0.13–28)mg/ml.min, 24 h Pt median 4.4 (0.1–23) μM). A similar relationship was observed in vitro [AUC=(24 h Pt +0.1)/0.93 (r ²=0.98, AUC median 7.9 (2.0–17) mg/ml.min, 24 h Pt median 7.1 (1.8–15) μM)]. The relationship derived from the in vivo data gave an unbiased and reasonably accurate estimate of the measured carboplatin AUC in 13 patients (AUC=5.1–8.7 mg/ml.min, GFR=59–129 ml/min, infusion time 30–45 min, 24 h sampling time 22.9–24.5 h), giving a percentage mean error of −4.2% and root mean squared percentage error of 11.5%. These results show that the analysis of a single blood sample taken 24 h after carboplatin administration can be used to produce an unbiased and reasonably accurate measure of the free carboplatin AUC. Unlike published limited sampling strategies, this method is not complicated by the need to accurately control the duration of the carboplatin infusion or the time at which the sample is taken. Received: 13 January 1995/Accepted: 14 May 1995     

L-Amino acid oxidase (LOX) modulation of melphalan activity against intracranial glioma

 These studies evaluated the efficacy of sequential pretreatment with L-amino acid oxidase (LOX) and LOX antiserum in the modulation of melphalan activity against intracranial glioma in athymic nude mice. LOX produced statistically significant (P<0.01) depletion of the large neutral amino acids isoleucine, leucine, methionine, phenylalanine, tyrosine, and valine in murine plasma at doses of 100 and 200 μg administered intravenously. Polyclonal anti-LOX antibody was successfully produced in mice, rabbits, and goats subsequent to immunization with LOX. Staphylococcal protein A-purified rabbit anti-LOX serum inhibited approximately 50% of LOX activity in vitro relative to control samples. This antiserum was used in vivo to inactivate LOX after it had depleted the large neutral amino acids, thereby preventing LOX-mediated catabolism of melphalan. Inoculation of three mice with rabbit anti-LOX serum after the treatment with LOX (100 μg) reduced LOX activity by 100%, 89%, and 100% at 6 h compared with reductions of 80%, 59%, and 52% over the same period in animals receiving LOX alone. In three separate studies using groups of eight to ten mice bearing intracranial human glioma xenografts, pretreatment with LOX followed by anti-LOX serum increased the antitumor activity of melphalan as compared with treatments with melphalan plus LOX, melphalan plus anti-LOX serum, or melphalan alone. Received: 4 December 1995/Accepted: 25 May 1996     

Local delivery of rapamycin: a toxicity and efficacy study in an experimental malignant glioma model in rats

Rapamycin, an anti-proliferative agent, is effective in the treatment of renal cell carcinoma and recurrent breast cancers. We proposed that this potent mammalian target of rapamycin inhibitor may be useful for the treatment of gliomas as well. We examined the cytotoxicity of rapamycin against a rodent glioma cell line, determined the toxicity of rapamycin when delivered intracranially, and investigated the efficacy of local delivery of rapamycin for the treatment of experimental malignant glioma in vivo. We also examined the dose-dependent efficacy of rapamycin and the effect when locally delivered rapamycin was combined with radiation therapy. Rapamycin was cytotoxic to 9L cells, causing 34% growth inhibition at a concentration of 0.01 μg/mL. No in vivo toxicity was observed when rapamycin was incorporated into biodegradable caprolactone-glycolide (35:65) polymer beads at 0.3%, 3%, and 30% loading doses and implanted intracranially. Three separate efficacy studies were performed to test the reproducibility of the effect of the rapamycin beads as well as the validity of this treatment approach. Animals treated with the highest dose of rapamycin beads tested (30%) consistently demonstrated significantly longer survival durations than the control and placebo groups. All dose-escalating rapamycin bead treatment groups (0.3%, 3% and 30%), treated both concurrently with tumor and in a delayed manner after tumor placement, experienced a significant increase in survival, compared with controls. Radiation therapy in addition to the simultaneous treatment with 30% rapamycin beads led to significantly longer survival duration than either therapy alone. These results suggest that the local delivery of rapamycin for the treatment of gliomas should be further investigated.     

The effects of anesthesia on the biodistribution of drugs in rats: a carboplatin study

 Purpose: Anesthetics can alter the biodistribution profile of drugs and, consequently, the regional pharmacokinetics of antineoplastic drugs at the tumor site. The effect of coadministered anesthetics on the biodistribution profile of carboplatin was studied in rats. Methods: Female Wistar rats were used to compare the effects of ketamine/xylazine, thiopental and pentobarbital on the biodistribution of 30 mg/kg radiolabelled ^195mPt-carboplatin administered intravenously, with conscious rats as the control group. Blood and urine samples were collected between 5 and 120 min. Results: The percentage values of the injected dose of platinum per ml (%ID/ml) in plasma at the final time-point were respectively, 0.557%, 0.156%, 0.115% and 0.086%, in pentobarbital-, ketamine/xylazine- and thiopental-injected rats, and in conscious animals. Following the same sequence of groups, the %ID/ml values of platinum in the cumulative urine were 0.001%, 0.619%, 0.184% and 0.118%, respectively. Urine output varied from very little in the pentobarbital group, to several milliliters in the other groups. Conclusions: There was an increase of almost 100-fold in total platinum uptake in the kidneys, cerebrum and cerebellum of rats receiving pentobarbital over the uptake in the control rats, whereas the biodistribution profile of the thiopental group had the least variance. These results demonstrate the importance of anesthetic selection in animal pharmacokinetic studies, as it influences the biodistribution and pharmacokinetic profile of the drug being studied. Received: 12 September 1996 / Accepted: 21 February 1997     

Distribution of estramustine in the BT4C rat glioma model

 Estramustine (EaM), a carbamate ester of 17β-estradiol and nor-nitrogen mustard, is a cytotoxic compound with antitumoral effect in malignant glioma in vitro and in vivo . However, knowledge of the pharmacokinetics of EaM in experimental glioma is limited. The objective of this study was therefore to investigate further the distribution of EaM in the BT4C rat glioma model. Assessment of EaM uptake and distribution was performed by quantitative whole-body autoradiography. In addition, the uptake of EaM and its metabolites estromustine (EoM), estradiol, and estrone were analyzed by gas chromatography. EaM was taken up from the circulation and was found to be the main product in glioma tissue. Whole-body autoradiography after [¹⁴C]-EaM administration revealed a strong ¹⁴C label simultaneously in tumor and normal brain tissue at 0.5 h after drug administration. In tumor tissue, sustained high levels of ¹⁴C label were detected at 12 h after drug administration. In contrast to the tumor, radioactivity in normal brain tissue rapidly leveled off, indicating a retention of radioactivity in the tumor. The tumor/brain radioactivity ratio reached a peak of 4.5 at 12 h after drug administration. High levels of ¹⁴C label were also found in pulmonary tissue. By gas chromatography, EoM was found to be the main metabolite in plasma. However, EaM reached higher levels in tumor tissue, with the mean tumor/plasma ratio being 11.7 as compared with 2.0 for EoM. Only low plasma levels of the estrogen metabolites were detected. In conclusion, EaM is taken up in the BT4C rat glioma tissue and is retained in the tumor as compared with normal brain tissue and plasma. EaM showed a greater selectivity for tumor tissue, exhibiting a high tumor/plasma ratio as compared with EoM. The distribution pattern after administration of EaM, as evaluated by both whole-body autoradiography and gas chromatography, supports the earlier suggestion that the uptake is related to a protein with EaM-binding characteristics. Received: 12 January 1997 / Accepted: 28 August 1997     

The effect of treatment with high dose melphalan,cisplatin or carboplatin on levels of glutathione in plasma,erythrocytes, mononuclear cells and urine

 Glutathione (GSH) has been implicated as an important factor in the detoxification of many electrophilic xenobiotics, including agents used in cytotoxic chemotherapy. Maintenance of high levels of GSH in normal tissues is believed to be important in the prevention of drug-induced toxicity. Previous studies have indicated that exposure of cells to some toxic electrophiles both in vitro and in vivo can cause a temporary decrease in intracellular levels of GSH. In this paper we report that in a series of 22 children and young adults treated with high dose melphalan (ten courses studied, all 200 mg/m²), cisplatin (eight courses, 80–104 mg/m²) or carboplatin (seven courses, 507–750 mg/m²) there was no significant alteration in the level of plasma, erythrocyte or urine GSH in the period immediately following drug administration. Fluctuations in the level of GSH in mononuclear cells were observed in some patients but did not follow any consistent pattern and were similar to those observed in a series of nine normal adult controls over the same time course. These results suggest that for melphalan, cisplatin and carboplatin, drug-GSH adduct formation is insufficient to cause a measurable decrease in intracellular GSH levels in normal peripheral haematopoietic cells during the course of treatment. Received: 6 October 1994/Accepted: 22 May 1995     

Digitonin enhances the efficacy of carboplatin in liver tumour after intra-arterial administration

 Platinum-containing drugs enter the cell slowly and have a poor tissue penetration. Increasing the permeability of the cell membrane might increase the intracellular drug concentration. Digitonin, a detergent that increases cell permeability by binding to cholesterol molecules in the cell membrane, can increase cisplatin accumulation and reduce tumour growth in vitro. The aim of this study was to determine whether digitonin could increase the efficacy of carboplatin (CBDCA) in vivo. In LH rats, a hepatoma was implanted in the liver. At 7 days after implantation, digitonin (or saline in the control group) was infused via the hepatic artery and, 10 min later, CBDCA was injected. Biopsies from the tumour and liver parenchyma were obtained after 1 h. The concentration of platinum measured in the liver tumours was higher in the digitonin group than in the control groups. In the liver parenchyma the concentrations were of the same magnitude. Measured with the ¹³³Xe-clearance technique, digitonin did not alter the tumour blood flow. Digitonin enhanced the tumour-growth-retarding effect of CBDCA given intra-aterially at 5 mg/kg but not at 25 mg/kg. No increase in toxicity was observed for digitonin given together with CBDCA at 5 mg/kg. Systemic administration of CBDCA was not influenced by digitonin. These findings demonstrate that pretreatment with digitonin increases the tumour uptake of CBDCA and potentiates the cytotoxic effect of CBDCA. Received: 21 September 1996 / Accepted: 17 March 1997     

Phase II study of the combination of carboplatin and 5-fluorouracil in metastatic nasopharyngeal carcinoma

 A carboplatin and 5-fluorouracil (CF) chemotherapy protocol was designed to evaluate tumor response and toxicity in patients with metastatic nasopharyngeal carcinoma (NPC). Patients with metastatic NPC were treated with a maximum of eight courses of CF. Carboplatin was given at 300 mg/m² by intravenous bolus on day 1 and 5-fluorouracil at 1 g/m² per day by continuous infusion on days 1 – 3; cycles were repeated once every 3 weeks. A total of 42 patients were evaluable for response and toxicity. They received a median of 6 courses (range 2 – 8) of chemotherapy. The overall response rate was 38% (16/42), comprising 7 complete responses (CR, 17%) and 9 partial responses (PR, 21%). The median survival was 12.1 months (range 6 – 54.2 months). The treatment was well tolerated. Toxicity was mainly bone marrow suppression. There were four episodes of neutropenic fever, but no renal toxicity or treatment-related death was documented. The combination of carboplatin given at a fixed dose of 300 mg/m² for 1 day and 5-fluorouracil given at 1 g/m² per day for 3 days produced an objective response rate of 38% and tolerable side effects. Received: 21 October 1995 / Recepted: 1 March 1996     

In vivo antitumor activity of cis-bis-neodecanoato-trans- R,R-l,2-diaminocyclohexane platinum(II) formulated in long-circulating liposomes

 A lipophilic cisplatin derivative, cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum(II) (NDDP), was formulated in liposomes composed of phosphatidylcholine (PC) and cholesterol (Chol) additionally containing monosialoganglioside (G_M1) or polyethyleneglycol conjugated to phosphatidylethanolamine (PEG-PE). These NDDP-containing long-circulating liposomes were examined for in vivo antitumor activity using the mouse RIF-1 solid tumor as a target residing outside the reticuloendothelial system (RES). Biodistribution studies, using C3H/HeJ mice and ¹¹¹In-labelled DTPA-SA as a lipid marker, showed that the activity of G_M1 and PEG-PE in prolonging the circulation times of liposomes was preserved in the presence of 3.0 mol% of NDDP in the liposome membranes. The high levels of liposomes remaining in the blood for PC/Chol/G_M1 and PC/Chol/PEG3000-PE liposomes were associated with high levels of platinum in the blood as determined by atomic absorption spectrophotometry. These NDDP-containing long-circulating liposomes showed approximately a three-fold increase in tumor accumulation as compared to the conventional PC/Chol liposomes. In vitro cytotoxicity studies using RIF-1 tumor cells showed that the presence of PEG-PE, but not G_M1, significantly enhanced the cytotoxicity of liposomal NDDP. RIF-1 tumor-bearing C3H/HeJ mice were treated twice with 25 mg/kg NDDP in various liposomal formulations on days 12 and 16 after tumor cell inoculation. A significant reduction in the tumor growth rate was observed when NDDP was formulated in PC/Chol/PEG3000-PE liposomes which support both efficient tumor accumulation and enhanced cytotoxicity of liposomal NDDP. On the other hand, NDDP formulated in PC/Chol/G_M1 liposomes, which display only a high tumor accumulation, had no effect on the tumor growth rate. Furthermore, NDDP formulated in dimyristoylphosphatidylglycerol (DMPG) containing liposomes, exhibiting in vitro cytotoxicity comparable to NDDP formulated in PC/Chol/PEG3000PE liposomes, but showing poor tumor accumulation, was also not effective. These results indicate a potential effectiveness of NDDP formulated in PEG-PE-containing liposomes for therapy of tumors in non-RES organs. Received: 13 November 1994/Accepted: 14 May 1995     

Adoptive transfer via immune T-lymphocytes of effective anti-tumor immunity against a malignant rat glioma in the brain

The aim of the present study was to develop an animal model to test the therapeutic potential of purified CD4 and CD8 T-lymphocytes against the intracerebrally implanted rat glioma cell line TZ363. Peripheral immunization of donor rats was performed by subcutaneous injection of viable TZ363 tumor cells while control animals received buffer injection. Donor splenic T-lymphocytes were prepared 14 days later and enriched by immune-bead MACS sorting. FACScan analysis revealed that of the pooled and sorted cells 91% of the tumor immune group were T-lympocytes and from the control animals 96%. The purified immune CD4/CD8 T-lymphocytes (1.2 to 5x10(7) cells) were injected intraperitoneally into 12 adult rats (three groups; each four animals), which were challenged five days later by an intracerebral injection of 5x10(4) TZ363 glioma cells. Four rats received 1.4x10(7) T-cells from control animals. While 3 of 4 animals developed a brain tumor and died in the control group, all animals, which received 5x10(7) immune T-cells survived the intracerebral tumor challenge. In the other groups survival rate depended on the amount of T-cells given. All other rats were sacrificed 32 days after intracerebral grafting. No tumor was found in these animals. Our data demonstrate that an anti-tumor T-cell response can be raised against the malignant rat glioma TZ363 and that purified CD4 and CD8 T-lymphocytes from tumor immunized donors can transfer protective immunity across the blood-brain barrier into recipient rats which are tumor challenged intracerebrally.     

Intracerebral delivery of Carboplatin in combination with either 6 MV Photons or monoenergetic synchrotron X-rays are equally efficacious for treatment of the F98 rat glioma

Background

The purpose of the present study was to compare side-by-side the therapeutic efficacy of a 6-day infusion of carboplatin, followed by X-irradiation with either 6 MV photons or synchrotron X-rays, tuned above the K-edge of Pt, for treatment of F98 glioma bearing rats.

Methods

Carboplatin was administered intracerebrally (i.c.) to F98 glioma bearing rats over 6 days using Alzet^TM osmotic pumps starting 7 days after tumor implantation. Radiotherapy was delivered in a single 15 Gy fraction on day 14 using a conventional 6 MV linear accelerator (LINAC) or 78.8 keV synchrotron X-rays.

Results

Untreated control animals had a median survival time (MeST) of 33 days. Animals that received either carboplatin alone or irradiation alone with either 78.8 keV or 6 MV had a MeSTs 38 and 33 days, respectively. Animals that received carboplatin in combination with X-irradiation had a MeST of > 180 days with a 55% cure rate, irrespective of whether they were irradiated with either 78.8 KeV synchrotron X-rays or 6MV photons.

Conclusions

These studies have conclusively demonstrated the equivalency of i.c. delivery of carboplatin in combination with X-irradiation with either 6 MV photons or synchrotron X-rays.     

Intraarterial O6-benzylguanine enables the specific therapy of nitrosourea-resistant intracranial human glioma xenografts in athymic rats with 1,3-bis(2-chloroethyl)-1-nitrosourea

 The prognosis for patients with malignant gliomas continues to be dismal. The high degree of resistance of gliomas to nitrosourea-based chemotherapy is one major factor in poor treatment outcome. The identification of O ⁶-alkylguanine-DNA alkyltransferase (AGAT) as a major determinant of nitrosourea resistance has resulted in the development of several agents to inactivate this repair protein and counteract tumor cell resistance. However, a major problem in preclinical trials has been the marked nitrosourea dose limitations imposed by the prior administration of AGAT-depleting agents. We investigated the AGAT depletion and selective enhancement of BCNU activity of intraarterial (i.a.) O ⁶-benzylguanine (O ⁶BG) in the human malignant glioma xenograft D-456 MG growing intracranially (i.c.) in athymic rats. Whereas i.a. O ⁶BG at 2.5 mg/kg produced 100% inhibition of D-456 MG AGAT i.c. activity 8 h after administration, intraperitoneal (i.p.) O ⁶BG at this dose produced only 40% inhibition, requiring dose escalation to 10 mg/kg to produce 100% AGAT depletion. Prior administration of i.p. O ⁶BG (10 mg/kg) and i.a. O ⁶BG (2.5 mg/kg) limited maximum tolerated intravenous (i.v.) BCNU doses (37.5 mg/kg when given alone) to 6.25 and 25 mg/kg, respectively. Higher doses of BCNU alone or in combination with O ⁶BG produced histopathologic evidence of cerebral and hepatic toxicity. Therapy experiments revealed a significantly improved median survival for rats treated with O ⁶BG i.a. (2.5 mg/kg) plus BCNU i.v. (25 mg/kg, days 61 and 59 in duplicate experiments) compared with saline (day 21, P=0.001), O ⁶BG i.a. or i.p. (days 22 and 23, P=0.001), BCNU i.v. (37.5 mg/kg, day 29, P=0.001), and O ⁶BG i.p. (10 mg/kg) plus BCNU i.v. (6.25 mg/kg, day 37, P<0.001). Therefore, O ⁶BG i.a., by virtue of rapid AGAT depletion and selective uptake into i.c. tumors, offers significant potential for regional chemomodulation of AGAT-mediated nitrosourea resistance in malignant human gliomas with concomitant reduction of systemic toxicity. Received: 30 January 1996/Accepted: 16 June 1996     

Investigations on the mechanisms of methotrexate resistance in a cisplatin-resistant L1210 murine leukemia cell subline

 We report a murine leukemia cell variant (L1210/DDP), selected for cisplatin (DDP) resistance, to be cross-resistant to methotrexate (MTX). Cross-resistance of L1210 cells to DDP and MTX has been observed by others, and has also been recorded in P388 murine leukemia and SSC-25 human squamous carcinoma cells. We demonstrated that MTX resistance is not due to dihydrofolate reductase (DHFR) gene amplification, increased DHFR enzyme activity or decreased MTX binding to the target enzyme. Of the mechanisms commonly proposed for MTX resistance, only differences in transport were observed when comparing sensitive (L1210/0) and resistant (L1210/DDP) cells. Our results suggest that MTX resistance in L1210/DDP cells is due to altered methotrexate uptake. Received: 3 August 1994/Accepted: 14 May 1995     

On the mechanism of action of doxorubicin encapsulation in nanospheres for the reversal of multidrug resistance

 We had previously shown that doxorubicin encapsulation in polyisohexylcyanocrylate nanospheres could circumvent the P-glycoprotein-mediated multidrug resistance (MDR) exhibited by C6 rat glioblastoma in culture. We then investigated what could be the mechanism of such a circumvention. The cytotoxicity of free and encapsulated doxorubicin was evaluated in two MDR variants of the C6 cell line in a device allowing the separation of cells from drugs by a polycarbonate membrane of 0.2-μm pore size. We observed that the progressive disruption of the nanospheres allowed their doxorubicin content to reach the cell monolayer and exert its cytotoxicity in a fashion similar to that exhibited by free doxorubicin. However, no circumvention of MDR is obtained by doxorubicin encapsulation when drug-containing nanospheres are separated from the cells by the polycarbonate membrane. In addition, no effect on azidopine binding to P-glycoprotein-enriched membranes is exerted by unloaded nanospheres, even after their spontaneous degradation in cell-culture medium. Taken together, these results suggest that a physical contact between doxorubicin-containing nanospheres and the cells is required for the circumvention of MDR. The role of degradation products from the nanospheres as modulators of P-glycoprotein activity can be ruled out. Received: 8 March 1995/Accepted: 6 June 1995     

Interferon augments the cytotoxicity of hydroxyurea without enhancing its activity against the M2 subunit of ribonucleotide reductase: effects in wild-type and resistant human colon cancer cells

 The effects of prolonged exposure to the ribonucleotide reductase (RR) inhibitor, hydroxyurea (HU), were assessed in the presence or absence of recombinant interferon alfa-2a (IFN) in wild-type human colon cancer cells (HT-29) and variants expressing low-level resistance to HU (R200). IFN at nontoxic concentrations decreased the IC₅₀ of HU from 368 μM to 215 μM (P<0.01) in wild-type cells, but not in the resistant variants. Potential cellular targets for the HU/IFN interaction were examined. In wild-type, but not resistant cells, treatment with HU at clinically achievable concentrations (1000 μM) resulted in rapid early inhibition of RR activity between 4 and 24 h after treatment with a maximal decrease of 65% at 12 h, decreases in cellular levels of dATP, dCTP and dGTP by 50–90% over the same time course, and a two- to fourfold increase in the level of mRNA for both the M1 and M2 subunits of RR, at 24, but not between 1 and 4 h, which probably represents a response to the earlier decrease in RR activity. IFN at a clinically achievable concentration (500 U/ml) failed to augment the effects of HU on RR protein, RR mRNA levels or RR enzyme activity in either the wild-type or resistant cells, suggesting that the mechanism by which IFN augments the effects of HU in the wild-type cells is independent of the effects of HU on M2. Received: 22 August 1995/Accepted: 18 December 1995