Acquired temozolomide resistance in human glioblastoma cell line U251 is caused by mismatch repair deficiency and can be overcome by lomustine

Purpose. Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. While the alkylating agent temozolomide (TMZ) has prolonged overall survival, resistance evolution represents an important clinical problem. Therefore, we studied the effectiveness of radiotherapy and CCNU in an in vitro model of acquired TMZ resistance. Methods. We studied the MGMT-methylated GBM cell line U251 and its in vitro derived TMZ-resistant subline, U251/TMZ-R. Cytotoxicity of TMZ, CCNU, and radiation was tested. Both cell lines were analyzed for MGMT promotor status and expression of mismatch repair genes (MMR). The influence of MMR inhibition by cadmium chloride (CdCl2) on the effects of both drugs was evaluated. Results. During the resistance evolution process in vitro, U251/TMZ-R developed MMR deficiency, but MGMT status did not change. U251/TMZ-R cells were more resistant to TMZ than parental U251 cells (cell viability: 92.0% in U251/TMZ-R/69.2% in U251; p = 0.032) yet more sensitive to CCNU (56.4%/80.8%; p = 0.023). The effectiveness of radiotherapy was not reduced in the TMZ-resistant cell line. Combination of CCNU and TMZ showed promising results for both cell lines and overcame resistance. CdCl2-induced MMR deficiency increased cytotoxicity of CCNU. Conclusion. Our results confirm MMR deficiency as a crucial process for resistance evolution to TMZ. MMR-deficient TMZ-resistant GBM cells were particularly sensitive to CCNU and to combined CCNU/TMZ. Effectiveness of radiotherapy was preserved in TMZ-resistant cells. Consequently, CCNU might be preferentially considered as a treatment option for recurrent MGMT-methylated GBM and may even be suitable for prevention of resistance evolution in primary treatment (AU)

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A Phase II randomized study of galunisertib monotherapy or galunisertib plus lomustine compared with lomustine monotherapy in patients with recurrent glioblastoma.

BACKGROUND: The combination of galunisertib, a transforming growth factor (TGF)-ß receptor (R)1 kinase inhibitor, and lomustine was found to have antitumor activity in murine models of glioblastoma. METHODS: Galunisertib (300 mg/day) was given orally 14 days on/14 days off (intermittent dosing). Lomustine was given as approved. Patients were randomized in a 2:1:1 ratio to galunisertib + lomustine, galunisertib monotherapy, or placebo + lomustine. The primary objective was overall survival (OS); secondary objectives were safety, pharmacokinetics (PKs), and antitumor activity. RESULTS: One hundred fifty-eight patients were randomized: galunisertib + lomustine (N = 79), galunisertib (N = 39), and placebo + lomustine (N = 40). Baseline characteristics were: male (64.6%), white (75.3%), median age 58 years, ECOG performance status (PS) 1 (63.3%), and primary glioblastoma (93.7%). The PKs of galunisertib were not altered with lomustine, and galunisertib had a median half-life of ∼8 hours. Median OS in months (95% credible interval [CrI]) for galunisertib + lomustine was 6.7 (range: 5.3-8.5), 8.0 (range: 5.7-11.7) for galunisertib alone, and 7.5 (range: 5.6-10.3) for placebo + lomustine. There was no difference in OS for patients treated with galunisertib + lomustine compared with placebo + lomustine [P (HR < 1) = 26%]. Median progression-free survival of ∼2 months was observed in all 3 arms. Among 8 patients with IDH1 mutation, 7 patients were treated with galunisertib (monotherapy or with lomustine); OS ranged from 4 to 17 months. Patients treated with galunisertib alone had fewer drug-related grade 3/4 adverse events (n = 34) compared with lomustine-treated patients (10% vs 26%). Baseline PS, post-discontinuation of bevacizumab, tumor size, and baseline levels of MDC/CCL22 were correlated with OS. CONCLUSIONS: Galunisertib + lomustine failed to demonstrate improved OS relative to placebo + lomustine. Efficacy outcomes were similar in all 3 arms. CLINICAL TRIAL REGISTRATION: NCT01582269, ClinicalTrials.gov.

Lomustine Nanoparticles Enable Both Bone Marrow Sparing and High Brain Drug Levels - A Strategy for Brain Cancer Treatments.

PURPOSE: The blood brain barrier compromises glioblastoma chemotherapy. However high blood concentrations of lipophilic, alkylating drugs result in brain uptake, but cause myelosuppression. We hypothesised that nanoparticles could achieve therapeutic brain concentrations without dose-limiting myelosuppression. METHODS: Mice were dosed with either intravenous lomustine Molecular Envelope Technology (MET) nanoparticles (13 mg kg(-1)) or ethanolic lomustine (6.5 mg kg(-1)) and tissues analysed. Efficacy was assessed in an orthotopic U-87 MG glioblastoma model, following intravenous MET lomustine (daily 13 mg kg(-1)) or ethanolic lomustine (daily 1.2 mg kg(-1) - the highest repeated dose possible). Myelosuppression and MET particle macrophage uptake were also investigated. RESULTS: The MET formulation resulted in modest brain targeting (brain/ bone AUC0-4h ratios for MET and ethanolic lomustine = 0.90 and 0.53 respectively and brain/ liver AUC0-4h ratios for MET and ethanolic lomustine = 0.24 and 0.15 respectively). The MET formulation significantly increased mice (U-87 MG tumours) survival times; with MET lomustine, ethanolic lomustine and untreated mean survival times of 33.2, 22.5 and 21.3 days respectively and there were no material treatment-related differences in blood and femoral cell counts. Macrophage uptake is slower for MET nanoparticles than for liposomes. CONCLUSIONS: Particulate drug formulations improved brain tumour therapy without major bone marrow toxicity.

EORTC 26083 phase I/II trial of dasatinib in combination with CCNU in patients with recurrent glioblastoma.

The treatment of patients with recurrent glioblastoma remains a major oncologic problem, with median survival after progression of 7-9 months. To determine the maximum tolerated dose and dose-limiting toxicity (DLT), the combination of dasatinib and cyclonexyl-chloroethyl-nitrosourea (CCNU) was investigated in this setting. The study was designed as multicenter, randomized phase II trial, preceded by a lead-in safety phase. The safety component reported here, which also investigated pharmacokinetics and preliminary clinical activity, required expansion and is therefore considered a phase I part to establish a recommended dosing regimen of the combination of CCNU (90-110 mg/m(2)) and dasatinib (100-200 mg daily). Overall, 28 patients were screened, and 26 patients were enrolled. Five dose levels were explored. DLTs, mainly myelosuppression, occurred in 10 patients. Grade 3 or 4 neutropenia was recorded in 7 patients (26.9%) and thrombocytopenia in 11 patients (42.3%). No significant effect of CCNU coadministration on dasatinib pharmacokinetics was found. Median progression-free survival (PFS) was 1.35 months (95% confidence interval: 1.2-1.4) and 6-month PFS was 7.7%. In this phase I study of recurrent glioblastoma patients, the combination of CCNU and dasatinib showed significant hematological toxicities and led to suboptimal exposure to both agents.

Role of glutathione-S-transferase (GST) polymorphisms in patients with advanced Hodgkin lymphoma: results from the HD2000 GISL trial.

Polymorphisms of the Glutathione-S Transferase (GST) family may influence the prognosis in lymphoma patients. We aimed to validate the impact of GSTT1 and GSTM1 deletions and of the GSTP1Ile105Val polymorphism on outcome and toxicity in 140 patients with advanced Hodgkin's lymphoma enrolled in the prospective multicenter HD2000-GISL trial, comparing ABVD, BEACOPP and CEC regimens. Carriers of the GSTP1Ile105Val polymorphism had a higher rate of grade 3-4 anemia following treatment. Overall, our study failed to validate GST genotyping as prognostic factor for progression-free survival (PFS). Only the small cohort of patients with an international prognostic score (IPS) >3 and undeleted GSTT1 and/or GSTM1, treated with ABVD had worse progression-free survival (PFS) (GSTT1 + vs GSTT1-: HR 5.02, 95% C.I., 1.16-21.8, p = 0.031, GSTM1 + /GSTT1 + vs GSTM1-and/or GSTT1-: HR 4.61, 95% C.I. 1.28- 16.6, p = 0.019, respectively). No differences were observed for patients treated with intensified regimens, as BEACOPP and CEC. In conclusion, the prognostic role of GST polymorphism, if at all, is limited to a small subgroup of patients treated with standard ABVD regimen.

HPLC method validation for the quantification of lomustine to study pharmacokinetics of thermosensitive liposome-encapsulated lomustine containing iohexol for CT imaging in C6 glioma rats.

To study the pharmacokinetics of the novel lomustine liposome, a specific, simple, and reliable high-performance liquid chromatography with ultraviolet detection (HPLC/UV) method for quantification of lomustine in rat plasma was established and validated. The calibration curve was linear over the concentration range of 0.05-5 µg/mL with the linearity (r (2)) being ≥0.9994. The intra- and inter-day precision was less than 5.87 and 10.47%, respectively, and the accuracy was within ±7.95%. The validated method has been successfully applied to a pharmacokinetic study of thermosensitive liposome-encapsulated lomustine containing iohexol and lomustine solution after intravenous administration to C6 glioma rats. Population pharmacokinetic (PPK) analysis was performed using NONMEM program for the plasma concentration versus time data. A one-compartment model with first-order elimination was established and proved to be the best description of the lomustine profile. Bootstrap analysis (n = 1,000) was executed to evaluate the stability and robustness of the model. The pharmacokinetic parameters of lomustine liposome containing iohexol and lomustine solution in rats were simulated using the final PPK model: t (1/2), AUC(0-∞), C (max) were 0.28 ± 0.12, 0.19 ± 0.08, and 0.30 ± 0.13 h; 2.37 ± 0.76, 1.32 ± 0.42, and 0.90 ± 0.29 mg h/L; 5.15 ± 2.22, 3.91 ± 1.90, and 1.87 ± 0.35 µg/mL for heated, non-heated, and control group, respectively. The result indicated that thermosensitive liposome-encapsulated lomustine containing iohexol for CT imaging had different pharmacokinetic characteristics than lomustine solution. Compared with non-heated group, the bioavailability of lomustine was obviously increased in C6 glioma rat plasma.

Synthesis of trans- and cis-4'-hydroxylomustine and development of validated analytical method for lomustine and trans- and cis-4'-hydroxylomustine in canine plasma.

In veterinary medicine, lomustine has been successfull used primarily for the treatment of resistant lymphoma and also for the treatment of mast cell tumors, intracranial meningioma, epitheliotropic lymphoma, and histiocytic sarcoma in dogs either alone or in combination with other chemotherapeutic agents. Even though lomustine is commonly used in dogs primarily for the treatment of resistant lymphoma, there is no pharmacokinetics information available regarding this compound in dogs. In the present study, we developed and validated a simple high-performance liquid chromatography (HPLC) method with a one-step liquid-liquid extraction procedure to detect and quantify lomustine and its two monohydroxylated metabolites (trans- and cis-4'-hydroxylomustine) in canine plasma for future pharmacokinetic studies. The HPLC-diode-array detection method reported here readily detects lomustine, cis-4'-hydroxylomustine, and trans-4'-hydroxylomustine in canine plasma with a limit of detection of lomustine, cis-4'-hydroxylomustine, and trans-4'-hydroxylomustine in plasma of about 10 ng/120 microL, 5 ng/120 microL, and 5 ng/120 microL, respectively. The mean extraction efficiency values for lomustine, cis-4'-hydroxylomustine, and trans-4'-hydroxylomustine were 73%, 90%, and 89%, respectively, from canine plasma samples on HPLC. The present study also provides stability information about lomustine and its two monohydroxylated metabolites in canine plasma and methanol solution stored at various conditions.

Nitroxyl radicals for labeling of conventional therapeutics and noninvasive magnetic resonance imaging of their permeability for blood-brain barrier: relationship between structure, blood clearance, and MRI signal dynamic in the brain.

The present study describes a novel nonradioactive methodology for in vivo noninvasive, real-time imaging of blood-brain barrier (BBB) permeability for conventional drugs, using nitroxyl radicals as spin-labels and magnetic resonance imaging (MRI). Two TEMPO-labeled analogues (SLENU and SLCNUgly) of the anticancer drug lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] were synthesized, using a substitution of the cyclohexyl part with nitroxyl radical. Nonmodified nitroxyl radical TEMPOL was used for comparison. The nitroxyl derivatives were injected intravenously in healthy mice via the tail vein, and MR imaging of the brain was performed on a 7.0 T MRI. The MRI signal dynamic of SLENU and SLCNUgly followed the same kinetics as nonmodified TEMPO radical. SLENU and SLCNUgly were rapidly transported and randomly distributed in the brain tissue, which indicated that the exchange of cyclohexyl part of lomustine with TEMPO radical did not suppress the permeability of the anticancer drug for BBB. The selected nitroxyl derivatives possessed different hydrophobicity, cell permeabilization ability, and blood clearance. Based on these differences, we investigated the relationship betweenthe structure of nitroxyl derivatives, their half-life in the circulation, and their MRI signal dynamic in the brain. This information was important for estimation of the merits and demerits of the described methodology and finding pathways for overcoming the restrictions.

Improved efficacy of chemotherapy for glioblastoma by radiation-induced opening of blood-brain barrier: clinical results.

PURPOSE: To improve the efficacy of chemotherapy for glioblastoma through the radiation-induced opening of the blood-brain barrier (BBB). METHODS AND MATERIALS: In two previous articles, we have described the results of brain scanning using technetium 99m-labeled somatostatin and the measurement of methotrexate (MTX) concentrations in blood and cerebrospinal fluid (CSF) after i.v. injection. We discovered that the BBB and blood-cerebrospinal fluid barrier opened to a certain extent after 20- to 40-Gy irradiation, thus increasing the degree to which MTX permeated the brain tissue. On the basis of these findings, we retrospectively analyzed the outcome in 56 patients with glioblastoma given either chemotherapy (CCNU) after 20- to 40-Gy irradiation (28 patients) or radiation therapy alone (28 patients). RESULTS: The 1-, 3-, and 5-year survival rates were 57.14%, 22.50%, and 15.00% in the combined-therapy group and 17.86%, 7.14%, and 3.57% in the radiotherapy alone group, respectively. The respective median survival times were 29.11 +/- 6.99 and 9.86 +/- 3.45 months (p < 0.001), which represented a statistically significant difference. CONCLUSION: Our study further confirms that opening of the BBB induced by irradiation with 20-40 Gy may optimize the effects of intracranial chemotherapy.

Lipid association improves the therapeutic index of lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] to suppress 36B-10 tumor growth in rats.

The therapeutic efficacy and tumor accumulation of a liposome formulation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), an effective agent used in the treatment of malignant brain tumors, was examined in an animal tumor model. Pharmacokinetic studies in normal and tumor-bearing rats indicated that a 2-fold greater plasma exposure was achieved with liposome-formulated CCNU compared with the free drug. In Fisher rats bearing s.c. tumors 36B-10, tumor growth was delayed substantially when liposomal CCNU was delivered compared with free-drug treatment. In single-dose treatments of 20, 35, and 50 mg/kg, tumor progression after each dose was reduced approximately 2-fold with liposomal compared with free CCNU (four animals in each treatment group). Multiple-dose treatments (given as three weekly doses with eight animals in each treatment group) with cumulative doses of 80 and 100 mg/kg of free and liposomal CCNU also resulted in a 2-fold reduction in tumor progression when compared with free-drug treatment. When drug levels in tumors relative to plasma were examined, it was observed that tumor drug concentrations did not exceed those found in plasma after administration of free CCNU; after administration of liposomal CCNU, however, tumor concentrations exceeded those in plasma by nearly 10-fold. These results suggest that the increased efficacy of liposome-formulated CCNU may be attributable to enhanced drug accumulation in tumor tissues.

Improved controlled release study of lomustine.

Lomustine (CCNU) microcapsules was prepared by improved recoacervation method, then mixed microcapsules with 0.7% collagen swelling solution to prepare the emulsion, spreaded the emulsion on the plate to form membrane and cross-linked it, the membrane would be planted into body and was expected to release at steady speed. The concentration of CCNU and the CCNU content of microcapsules were measured by ultraviolet spectrophotometry to observe the release of CCNU be slow and constant, approach to 0-class release approximately.

Cytochrome P450-inducing antiepileptics increase the clearance of vincristine in patients with brain tumors.

BACKGROUND: Vincristine is at least partly metabolized by CYP3A4. We have studied the possible effect of the CYP3A4-inducing antiepileptic agents carbamazepine and phenytoin on the pharmacokinetics of vincristine. METHODS: Fifteen adult patients with brain tumors receiving combination chemotherapy with procarbazine, lomustine, and vincristine volunteered for this open parallel-group study. Nine of the patients used either carbamazepine or phenytoin and six of the patients used no obvious CYP3A4-inducing medication. After intravenous infusion of 2 mg vincristine, timed blood samples were collected up to 24 hours. Plasma vincristine concentrations were measured by liquid chromatography-tandem mass spectrometry. The pharmacokinetics of vincristine were compared between the two patient groups. RESULTS: The systemic clearance of vincristine was 63% higher (925 +/- 61 versus 569 +/- 76 mL/min [mean +/- SEM]; P = .004), the elimination half-life was 35% shorter (12.7 +/- 0.6 versus 19.4 +/- 3.6 hours; P = .13), and the total area under the plasma concentration-time curve was 43% smaller (37.3 +/- 2.4 versus 65.1 +/- 10.1 ng x h/mL; P = .04) in patients who were receiving carbamazepine or phenytoin than in the control group. CONCLUSIONS: Drugs that induce CYP3A4 can increase the elimination of vincristine. Further studies are needed to determine whether the increased clearance of vincristine by carbamazepine or phenytoin decreases the efficacy of vincristine.

Pharmacokinetics of high-dose oral CCNU in bone marrow transplant patients.

PURPOSE: CCNU (lomustine) and other nitrosourea compounds are rapidly metabolized to alkylating moieties, which are active against various malignancies. In humans, CCNU undergoes biotransformation to the geometric isomers of 4'-hydroxyCCNU. The pharmacokinetics of trans-and cis-4'-hydroxyCCNU were determined in five patients with Hodgkin's or non-Hodgkin's lymphoma receiving sequential doses of CCNU (15 mg/kg), etoposide (60 mg/kg) and cyclophosphamide (100 mg/kg) prior to autologous bone marrow transplantation. METHODS: Plasma concentrations of the isomeric forms of the metabolites were determined by high-performance liquid chromatography. Data were analysed using noncompartmental pharmacokinetic methods. RESULTS: Formation of the trans-isomer predominated over that of the cis-isomer, with an average exposure ratio of 1.4 (range 1.0-2.1). Peak plasma concentrations occurred between 1 and 4.1 h postdosing and averaged 1.56 mg/l for the trans-isomer and 1.10 mg/l for cis isomer. Peak plasma concentrations and systemic exposures varied approximately six- and ninefold, respectively, reflecting significant interindividual variability in alkylating activity after high doses of CCNU. Plasma half-lives of the metabolites were 3.1 h (range 1.1-4.5 h) for the trans-isomer and 3.5 h (range 1.3-6.4 h) for the cis- isomer, and varied linearly with increasing patient body weight. There was no significant difference in plasma half-lives after high-dose CCNU administration observed in this study and those reported previously after the administration of substantially lower doses of CCNU. CONCLUSION: Despite linearity in the pharmacokinetics of the isomeric forms of 4'-hydroxyCCNU at high doses, large interindividual variability in exposure to the CCNU metabolites was observed. Potential saturation of metabolic pathways to the isomers at these doses may manifest as toxic symptoms since alkylating moieties formed directly from the parent, CCNU, may be associated with greater toxicity than those formed from the isomeric forms of the 4'-hydroxylated metabolite.

Identification of carbamoylated thiol conjugates as metabolites of the antineoplastic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, in rats and humans.

The metabolic fate of 1-(2-chloroethyl)-3-cyclohexyl)-nitrosourea (CCNU) in rats and humans was investigated with a view to characterizing the nature of the carbamoylating species released upon in vivo transformation of the drug. CCNU undergoes oxidation in vivo to afford 4-hydroxy and 3-hydroxy CCNU which, along with the parent drug, decomposes to the corresponding isocyanates. Although the highly reactive nature of the isocyanate species precludes their identification in vivo, their existence as electrophilic intermediates was detected in the likeness of trapped glutathione (GSH) and N-acetylcysteine (NAC) conjugates. Conjugated thiol metabolites were purified by HPLC from the bile and urine of CCNU-dosed rats, and the urine of a patient on CCNU therapy. The metabolites were identified by atmospheric pressure chemical ionization LC/MS and LC/MS/MS. In addition, LC/MS and LC/MS/MS spectra of synthesized authentic standards corroborated the identity of the metabolites. In rats, 4-hydroxycyclohexyl, 3-hydroxycyclohexyl, and cyclohexyl isocyanate were identified as their GSH conjugates in bile and NAC conjugates in urine. In the case of the patient, the NAC conjugates of 4-hydroxycyclohexyl and 3-hydroxycyclohexyl isocyanate were identified as urinary metabolites. The identification of GSH and NAC conjugates reported herein marks a significant advance in the assessment of the in vivo carbamoylating activity of CCNU and its phase I metabolites.

Pharmacokinetics of alkylating agents.

Alkylating agents have been used for over 30 years in the treatment of malignant disease. Because of their very reactive nature, studies of their intermediate metabolism have been difficult. However, this is now possible with modern analytical techniques. Further understanding of their metabolism and pharmacokinetics should lead to a more rational use in the clinic.

Improved delivery through biological membranes. XLV. Synthesis, physical-chemical evaluation, and brain uptake studies of 2-chloroethyl nitrosourea delivery systems.

The dihydropyridine in equilibrium with pyridinium redox chemical delivery system (CDS) was supplied to two 2-chloroethylnitrosoureas, i.e., HECNU and CCNUOH, and the physicochemical properties of the delivery systems were studied to assess their potential as improved delivery forms to the CNS. Detailed physicochemical evaluation and brain uptake studies were performed on one of the delivery systems (CCNUOH-CDS) derived from trans-4-hydroxy-CCNU, an active metabolite of CCNU. Two aqueous-based formulations derived from hydroxypropyl-beta-cyclodextrin (HP beta CD) and Tween 80:ethanol:water system were developed for CCNUOH-CDS to overcome the poor aqueous solubility conferred upon it by its high lipophilicity. The formulations enabled a 200- to 400-fold improvement in the water solubility of CCNUOH-CDS. Dose- and vehicle-dependent comparative tissue distribution studies in rats indicated improved brain-to-organ ratios of the delivery system at lower doses.

Chemosensitization of the nitrosoureas by 2-nitroimidazoles in the subcutaneous 9L tumor model: pharmacokinetic and structure-activity considerations.

Alterations of the pharmacokinetics and cytotoxic effects of the nitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) by the 2-nitroimidazoles, misonidazole (MISO) and SR-2508 were investigated using the subcutaneous (sc) 9L tumor model in male Fisher 344 rats. When 50 mg/kg of CCNU was given i.p., the peak plasma concentration of CCNU was about 3 micrograms/ml. CCNU was eliminated with biphasic kinetics that had a terminal half-time (T1/2) of approximately 47 min. When 2.5 mmole/kg of MISO was given i.p. 150 min before CCNU, the peak plasma concentration of CCNU was increased by approximately 63% with no change in the elimination kinetics. Clamping did not change the pharmacokinetics of CCNU in either plasma or tumors. MISO pretreatment increased the peak CCNU concentration in unclamped tumors by 3-fold, but had no effect on the CCNU pharmacokinetics in clamped tumors. With the exception of a decrease in the peak BCNU concentration in tumors similar to that observed with MISO, SR-2508 (3.75 mmole/kg, i.p.) did not change the pharmacokinetics of BCNU or CCNU in plasma and tumors. CCNU had no effect on the MISO concentration in plasma and unclamped tumors. However, in the clamped tumors, CCNU delayed the return of the MISO concentration to the unclamped tumor level by about an additional 60 min after the clamp was released. SR-2508 was eliminated from the plasma with biphasic kinetics having an initial and terminal T1/2 of approximately 11 and approximately 76 min, respectively. SR-2508 reached a peak tumor concentration of about 500 micrograms/ml in 30 min. The elimination T1/2 for SR-2508 in unclamped and clamped tumors was approximately 81 and approximately 42 min, respectively. When the clamp was released, the SR-2508 concentration returned to the level found in the unclamped tumors approximately 90 min after it reached its nadir; BCNU and CCNU had no effect on the kinetics of this process. MISO significantly potentiated the cytotoxicity of BCNU in clamped tumors at surviving fractions less than or equal to 0.5. MISO did not potentiate the cytotoxicity of CCNU until the surviving fraction reached 0.05. SR-2508 did not potentiate the cytotoxicity of either BCNU or CCNU.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution and activity of antineoplastic drugs in a tumor model.

Antineoplastic drugs can be effective in solid tumors only if they can penetrate several cell layers and retain their activity in the tumor microenvironment. The capacity of several common chemotherapeutic agents to meet these requirements was evaluated in an in vitro tumor model, V79 Chinese hamster cells grown as spheroids. The delivery and toxicity of radioactively labeled 5-fluorouracil, lomustine, tetraplatin, and chlorambucil were determined by use of cell-sorting techniques to select cells as a function of their position (depth) within these spheroids, and the delivery and toxicity of doxorubicin (DOX) were evaluated on the basis of fluorescence intensity. Simultaneous measurement of drug level and toxicity in cells at the time of recovery from different depths within the spheroids led to the conclusion that drug delivery was a problem only for DOX. In contrast, several of the other agents showed a dissociation between cellular drug levels and activity, implicating a major role of the cellular microenvironment in modulating drug toxicity.

Capillary gas chromatography and thermionic N-P-specific detection of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in stability and pharmacokinetic studies.

An expedient, rapid, and sensitive capillary gas chromatographic method for the analysis of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in plasma is described. Separation of the underivatized nitrosourea compounds was performed on a 0.33-mm-i.d., 25-m fused-silica, SE-30 capillary column, and detection was carried out using a thermionic N-P-specific detector. The compounds were extracted from plasma with benzene with a yield of greater than 87%. The assay was linear in the ranges of 0.001 to 0.5 and 0.5 to 25 micrograms/ml for CCNU or 0.003 to 0.50 and 0.5 to 25 micrograms/ml for BCNU, with correlation coefficients from 0.9914 to 0.9999 and coefficients of variation (CV) of less than 3.3%. Other antineoplastic agents did not interfere in the assay. The method was employed to study the pharmacokinetics of BCNU in rabbits. The plasma concentration-time curves were fit to a two-compartment model with a mean (SE) alpha, beta, and total-body clearance of 2.898 (0.913) hr-1, 0.1228 (0.0179) hr-1, and 7.211 (2.862) liters/hr.kg, respectively. Further, the stability of BCNU and CCNU in solution was examined at different temperatures. Both compounds were stable in benzene or acetone (4 to 37 degrees C) but labile in plasma even if refrigerated. The apparent rate constants for degradation of BCNU and CCNU were 0.09921 and 0.02853 hr-1 at 4 degrees C and 5.998 and 2.553 hr-1 at 37 degrees C, respectively.

Improved delivery through biological membranes. XXX. Synthesis and biological aspects of a 1,4-dihydropyridine based chemical delivery system for brain-sustained delivery of hydroxy CCNU.

A redox chemical delivery system based on the NADH in equilibrium NAD+ model was applied to an active metabolite (D) of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), i.e. CCNU-OH. The 1,4-dihydrotrigonelline ester of CCNU-OH, N-(2-chloro ethyl)-N'-[trans-4-(1,4-dihydro-1-methyl-3-pyridinecarbonyloxy)cyc lohexyl]- N-nitrosourea (D-CDS) was prepared by a direct hydride transfer reaction of the corresponding pyridinium precursor (D-Q+) with a highly reactive 1-benzyl-1,2-dihydroisonicotinamide. The in vitro kinetics in biological fluids indicated facile oxidative conversion of D-CDS to D-Q+. An in vivo study showed that one intravenous injection to rats of D-CDS resulted in rapid brain accumulation of D-Q+, followed by a sustained release of CCNU-OH, while D-Q+ was rapidly eliminated from systemic circulation. The ratio of brain/blood concentration of D-Q+ was found to increase progressively with time. At an equimolar dose of CCNU-OH, the ratio of brain/blood concentration for CCNU-OH was found to be close to unity.