Controlled delivery of 1,3-bis(2-chloroethyl)-1-nitrosourea from ethylene-vinyl acetate copolymer.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) has been found to be an effective chemotherapeutic agent against brain tumors. However, because it has a very short half-life in plasma, the exposure of neoplastic cells to BCNU is very brief. The delivery of BCNU may be enhanced by using controlled release polymers. We measured the release of BCNU from ethylene-vinyl acetate copolymer (EVAc) into blood, phosphate buffer, and brain tissue. BCNU-EVAc cylinders that weighed 60 mg were implanted in the peritoneum of rats, and BCNU was detected in blood for 6 days. Studies carried out in vitro showed that BCNU was released from EVAc at a decreasing rate for 195 h. BCNU-EVAc cylinders that weighed 15 mg were implanted either intracranially (i.c.) or i.p. in Fischer 344 rats. Controlled release of BCNU from the i.c. BCNU-EVAc implants was observed over 9 days, with peak drug levels of 49.6 micrograms/g of brain tissue in the implanted hemisphere. The BCNU levels in the contralateral hemisphere and the peripheral circulation were much lower and were detectable for only 1 day. By contrast, peak BCNU levels in the brain from the i.p. BCNU-EVAc implants were 2.7-3.0 micrograms/g for only 12 h, accompanied by peak BCNU levels in blood of 1.0 micrograms/ml tapering over 1 day. These results demonstrate the controlled release of intact BCNU from EVAc in vitro and in vivo. Furthermore, the i.c. implants resulted in localized, prolonged, high levels of the drug in the implanted hemisphere. Hence, the i.c. controlled delivery of BCNU may be more efficacious for the treatment of localized brain tumors.

Comparative pharmacokinetics of 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)-1-nitrosourea in rats and patients and extrapolation to clinical trials.

The plasma pharmacokinetics of 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)1-nitrosourea (PCNU) were determined in ambulatory rats and in patients receiving PCNU chemotherapy in Phase 1 and II studies. After derivativization to the methyl carbamate, both rat and human PCNU plasma levels were measured by gas chromatography-mass spectrometry. Comparison of the tolerated dose levels and pharmacokinetics of PCNU to the values determined for 1,3-bis(2-chloroethyl)-1-nitrosourea in humans indicated that PCNU has a lower plasma drug area under the curve at equitoxic doses. We conclude that PCNU may show less clinical efficacy than 1,3-bis(2-chloroethyl)-1-nitrosourea in the treatment of solid tumors.

Sensitivity of human and murine hematopoietic precursor cells to 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose and 1,3-bis(2-chloroethyl)-1-nitrosourea.

The sensitivity of mouse and human bone marrow hematopoietic precursor cells [colony-forming units committed to granulocyte-macrophage differentiatin (CFU-C)] was determined after in vitro incubation with chlorozotocin (2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose), or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with the use of drug concentrations achieved during clinical administration. Chlorozotocin, at a concentration of 1 X 10(-4) M, did not decrease mouse CFU-C below the control of 44 colonies/10(5) nucleated cells; 5 X 10(-4) M produced a 70% reduction in CFU-C, and 1 X 10(-3) M chlorozotocin eliminated all colony formation. In contrast, BCNU at 1 X 10(-4) M resulted in a 55% reduction in CFU-C, and at 5 X 10(-4) M it eliminated all colony formation. For human marrow the threshold concentration for chlorozotocin toxicity was 1 X 10(4) M, which resulted in 25% reduction in CFU-C as compared to the control of 32 colonies/10(5) nucleated cells. In contrast, BCNU at 5 X 10(-5) M decreased human CFU-C to 47% of control, and at 1 X 10(-4) M it eliminated all colony formation. Twenty-four hr after in vitro exposure to 1 X 10(-4) M chlorozotocin, there was no reduction in human bone marrow DNA synthesis in contrast to a 42% reduction with 1 X 10(-4) M BCNU. The plasma concentration of drugs following a therapeutic dose in patients was measured and was found to correspond to the concentration range used in the in vitro studies of marrow toxicity.

Effect of administration schedules on the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) by misonidazole in subcutaneous 9L tumors.

Our previous studies demonstrated that metabolism of misonidazole (MISO) by hypoxic cells is required to potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in sc 9L tumors. To determine the influence of administration schedules on this chemosensitization, tumors were either clamped to produce a reversible hypoxia or left unclamped. MISO (2.5 mmoles kg-1) was administered to rats with unclamped tumors simultaneously with BCNU (9 or 12 mg kg-1), 20 min before BCNU, or 2.5 hr before BCNU, and the drug pharmacokinetics and BCNU cytotoxicity were measured. MISO administered 20 min or 2.5 hr before BCNU increased the plasma elimination half-time (t1/2) of BCNU, but MISO administered simultaneously with BCNU did not change the plasma elimination t1/2 of BCNU. In unclamped sc 9L tumors, all administration schedules decreased the peak BCNU concentration and increased the initial BCNU elimination t1/2; however, the BCNU exposure dose (AUC0-infinity) calculated from these data did not change significantly. In agreement with the AUC calculations, none of the administration schedules altered the BCNU cytotoxicity in unclamped tumors. If the tumors were clamped for 5-120 min after the peak MISO concentration was reached, BCNU-induced cell kill was increased by a constant factor of 3 over the first hour of the clamping period and by an additional factor of 7 over the second hour of the clamping period. If the tumors were clamped for 2 hr after the peak MISO concentration was reached and then BCNU administered 0-60 min after the clamp was released, this chemosensitization remained at a constant factor of approximately 20 for the first 10 min, and then decreased rapidly to a factor of approximately 3 by 20 min after the clamp was released. These data indicate that in sc 9L tumors, (1) at least two biochemical mechanisms are involved in this MISO-BCNU interaction, one of which depends on the duration and extent of the metabolism of MISO by hypoxic cells, and (2) reoxygenation does not immediately eliminate the potentiation of BCNU by MISO. These data also suggest that MISO should be given 2-4 hr before BCNU to achieve the maximum chemosensitization in clinical trials.

Nitroimidazoles as modifiers of nitrosourea pharmacokinetics.

We have studied the effect of a number of nitroimidazole sensitizers of varying lipophilicity on the pharmacokinetics of CCNU in mice. It was found that the effectiveness of these compounds in producing pharmacokinetic effects correlated directly with their lipophilicity, viz. in the order: benznidazole (Benzo) greater than Ro07-1902 misonidazole greater than (MISO) greater than Ro05-9963. The effects of MISO on the pharmacokinetics of 4 nitrosoureas of differing lipophilicity were also investigated. The plasma clearances of CCNU, BCNU and MeCCNU (high lipophilicity) were slowed by MISO whereas that of chlorozotocin (Chlz) (low lipophilicity) was unaffected. Thus, it seems that for a pharmacokinetic interaction to occur between a nitroimidazole and a nitrosourea, both the modifier and the cytotoxic agent must have a requisite degree of lipophilicity. As the same requirement appears to hold for enhancement of tumor response, these data provide further evidence that pharmacokinetic modification plays a major role in chemosensitization.

Pharmacokinetics of superselective intra-arterial and intravenous [11C]BCNU evaluated by PET.

The pharmacokinetics of i.v. and superselective intra-arterial carbon-11 1,3-bis-(2-chloroethyl)-1-nitrosourea ([11C]BCNU) were directly compared for the first time in ten patients with recurrent gliomas using positron emission tomography (PET). Intra-arterial administration of [11C]BCNU achieved concentrations of the drug in the tumor that averaged 50 times higher than with a comparable i.v. dose. These preliminary results suggest that the degree of early metabolic trapping of BCNU in tumor correlates with the clinical response to this chemotherapy.

Relationship of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) pharmacokinetics of uptake, distribution, and tissue/plasma partitioning in rat organs and intracerebral tumors.

To obtain a clearer definition of the relationship between the structure of BCNU and CCNU and their antitumor activity, we determined the uptake, distribution, and tissue/plasma partition ratios of both compounds in normal organs and intracerebral (ic) 9L tumors in rats. Greater uptake, distribution, and tissue/plasma partition ratios were obtained for parent CCNU in fat, liver, and brain, and for parent BCNU in kidney. CCNU distributes more rapidly and extensively than BCNU only in fatty tissues. BCNU distributed more extensively in kidney and liver. Rats received bolus IV injections of 14C-labeled BCNU or CCNU in increasing doses; measurements taken 30 min after injection showed that three- to fourfold more BCNU than CCNU was bound to nucleic acids in brain and ic 9L tumor tissue. Because the chloroethyl group is the alkylating moiety for both drugs, these findings implied that BCNU biotransformed to its reactive intermediate more rapidly than did CCNU. These observations, together with previous findings, indicate that one reason for the greater effectiveness of BCNU than CCNU against ic 9L tumors is its superior ability to form an intermediate that can bind to ic 9L tumor cell nucleic acids.

A randomised phase II study of carmustine alone or in combination with tumour necrosis factor in patients with advanced melanoma.

Laboratory data suggest a synergistic interaction between carmustine (BCNU) and tumour necrosis factor (TNF) in melanoma. We therefore studied the activity of 200 mg/m2 BCNU given alone or in combination with 88 micrograms/m2 recombinant human TNF-alpha (rhTNF alpha) as a daily i.v. infusion for 5 days at 48-day intervals to patients with metastatic melanoma. In this randomised phase II trial, the rate of response to BCNU alone was 20% [95% confidence interval (CI), 2%-38%], and this was not improved by the addition of TNF (response rate, 10.5%; 95% CI, 1.3%-33%). Toxicity was higher in the combination arm, and there was no difference in survival.

Metabolism of a cholesterol-rich microemulsion (LDE) in patients with multiple myeloma and a preliminary clinical study of LDE as a drug vehicle for the treatment of the disease.

PURPOSE: Previously we have shown that cholesterol-rich microemulsions that bind to LDL receptors have the ability to concentrate in acute myeloid leukemia cells and in ovarian and breast carcinomas. Thus, LDE may be used as a vehicle for drugs directed against neoplastic cells. Indeed, we subsequently showed that when carmustine is associated with LDE the toxicity of the drug is significantly reduced in patients with advanced cancers. The aim of the present study was to verify whether LDE may be taken up by multiple myeloma cells and whether patients with multiple myeloma respond to treatment with LDE associated with carmustine. METHODS: A total of 131 consecutive volunteer patients with recently diagnosed multiple myeloma classified as clinical stage IIIA had their plasma lipid profile determined. LDE plasma kinetics were performed in 14 of them. Cell uptake of LDE and the cytotoxicity of carmustine associated with the emulsion were evaluated in a multiple myeloma cell line. A pharmacokinetic study of LDE-carmustine was performed in three patients. Finally, an exploratory clinical study of LDE-carmustine (carmustine dose 180 mg/m(2) body surface every 4 weeks) was performed in seven untreated multiple myeloma patients. RESULTS: LDL cholesterol was lower in the 131 multiple myeloma patients than in healthy controls and the fractional clearance rate (FCR, in units per minute) in the 14 multiple myeloma patients was twice that in 14 paired healthy control subjects. Moreover, entry of LDE into multiple myeloma cells was shown to be mediated by LDL receptors. Taken together, these findings indicate that LDE may target multiple myeloma. The exploratory clinical study showed that gammaglobulin decreased by 10-70% (mean 36%) after three cycles and by 25-75% (mean 44%) after six cycles. Furthermore, there was amelioration of symptoms in all patients. Cholesterol concentrations increased after treatment, suggesting that the treatment resulted in at least partial destruction of neoplastic cells with receptor upregulation. Side effects of the treatment were negligible. CONCLUSIONS: Because it targets multiple myeloma and, when associated with an antineoplastic agent, produces therapeutic responses in patients with fewer side effects, LDE has the potential for use as a drug vehicle in the treatment of the disease.

Differential plus polarographic determination of submicrogram quantities of carmustine and related compounds in biological samples.

A polarographic method was developed to determine the antineoplastic agent carmustine and other nitrosoureas, such as N-methyl-N-nitrosourea and N-cyclohexyl-N-nitrosourea, in biological fluids at levels well below 1 microgram/ml or g. The stability of carmustine in different media was investigated to prevent losses during administration or assay. Examples of nitrosourea determination in biological samples are given.

Generation and functional characterization of mice with a disrupted glutathione S-transferase, theta 1 gene.

Glutathione S-transferase (GST) theta 1 (GSTT1) has been regarded as one of the key enzymes involved in phase II reactions because of its unique substrate specificity. In this study, we generated mice with the disrupted Gstt1 gene (Gstt1-null mice) by gene targeting and analyzed the metabolic properties in cytosolic and in vivo studies. The resulting Gstt1-null mice failed to express the Gstt1 mRNA and GSTT1 protein by reverse transcriptase-polymerase chain reaction analysis and two-dimensional fluorescence difference gel electrophoresis/mass spectrometry analysis, respectively. However, the Gstt1-null mice appeared to be normal and were fertile. In an enzymatic study using cytosolic samples from the liver and kidney, GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), dichloromethane (DCM), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was markedly lower in Gstt1-null mice than in the wild-type controls, despite there being no difference in GST activity toward 1-choloro-2,4-dinitrobenzene between Gstt1-null mice and the wild-type controls. Gstt1-null mice had GST activity of only 8.7 to 42.1% of the wild-type controls to EPNP, less than 2.2% of the wild-type controls to DCM, and 13.2 to 23.9% of the wild-type controls to BCNU. Plasma BCNU concentrations after a single i.p. administration of BCNU to Gstt1-null mice were significantly higher, and there was a larger area under the curve(5-60) min (male, 2.30 times; female, 2.28 times, versus the wild-type controls) based on the results. In conclusion, Gstt1-null mice would be useful as an animal model of humans with the GSTT1-null genotype.

Intratumoral injection of BCNU in ethanol (DTI-015) results in enhanced delivery to tumor--a pharmacokinetic study.

Solvent facilitated perfusion (SFP) has been proposed as a technique to increase the delivery of chemotherapeutic agents to tumors. SFP entails direct injection of the agent into the tumor in a water-miscible organic solvent, and because the solvent moves easily through both aqueous solutions and cellular membranes it drives the penetration of the solubilized anticancer agent throughout the tumor. To test this hypothesis, we compared the pharmacokinetics (PK) of 14C-labeled 1,3-bis-chlorethyl-1-nitrosourea (BCNU) in intra-cerebral 9L rat gliomas after intravenous (IV) infusion in 90% saline--10% ethanol or direct intratumoral (IT) injection of 14C-BCNU in 100% ethanol (DTI-015). Treatment with DTI-015 yielded a peak radioactive count (Cmax) for the 14C label that was 100-1000 fold higher in the tumor than in all other tissues in addition to a concentration in the tumor that was 100-fold higher than that achieved following IV infusion of 14C-BCNU. Pathologic and auto-radiographic analysis of tissue sections following IT injection of 14C-BCNU in ethanol into either tumor or normal rat brain revealed both an enhanced local volume of distribution and an increased concentration of BCNU delivered to tumor compared to non-tumor bearing brain. To investigate the mechanism behind the SFP of BCNU to the tumor both dynamic contrast and perfusion MRI were performed on 9L tumors before and after treatment and demonstrated a decrease in tumor perfusion following IT injection of DTI-015. Finally, initial PK of patient blood samples following administration of DTI-015 into relapsed high-grade glioma indicated a 20-fold decrease in systemic exposure to BCNU compared to IV infusion of BCNU providing further evidence for the enhanced therapeutic ratio observed for DTI-015.

Lipophilic drugs and lipoproteins: partitioning effects on chloroethylnitrosourea reaction rates in serum.

Chloroethylnitrosoureas are anticancer agents that undergo chemical reactions in vitro and in vivo to form active alkylating agents. The rate at which lipophilic chloroethylnitrosourease decompose in serum is faster than in aqueous solution and comparable to in vivo clearance rates. The increase in reaction rate is known to be caused by a reaction catalyzed by protein. Addition of lipoproteins to serum stabilizes chloroethylnitrosoureas to degradation. A model is presented that correlates serum decomposition rates to lipoprotein concentrations. This model involves partitioning of lipophilic chloroethylnitrosoureas into the hydrophobic core region of the lipoproteins where the chloroethylnitrosourea is chemically stable and is not free to undergo protein binding nor aqueous chemical degradation reactions. Normal interindividual variations of serum lipoprotein concentrations and hence partitioning may be a significant factor affecting the tissue distribution and pharmacokinetics of lipophilic drugs.

Hepatic arterial BCNU: a pilot clinical-pharmacologic study in patients with liver tumors.

BCNU at a dose of 200 mg/m2 was administered in a 60-minute hepatic arterial (HA) infusion to three patients with liver-predominant neoplastic disease. Nitrosourea blood levels were measured during the 60-minute infusion and for 30 minutes after completion of the infusion in samples obtained simultaneously from a hepatic venous (HV) catheter and a peripheral venous (PV) site. Direct extraction of blood with diethyl ether was used in order to remove quantitatively the nitrosourea from blood and thereby stabilize it against in vitro breakdown in blood prior to colorimetric analysis. As determined by blood levels, steady-state was achieved within 50 minutes of infusion. Mean HV levels at steady-state were 2.5-fold higher in the HV compared to PV samples. The higher HV compared to PV levels must reflect higher tumor exposure to nitrosourea with HA compared to the usual iv route of administration. No drug-related hepatic toxicity was noted. Myelosuppression was noted in only one patient in whom reversible leukopenia (granulocyte nadir, 500/mm3) and thrombocytopenia (platelet nadir, 20,000/mm3) developed.

Pharmacokinetics of BCNU in man: a preliminary study of 20 patients.

Using the technique of direct sample insertion selected ion monitoring chemical ionization mass spectroscopy, BCNU levels were measured in vivo in patients and in vitro in serum, sera ultrafiltrates, and buffered Ringer's solution. The disappearance of BCNU in vitro was found to be first-order with a half-time of 11.6 minutes (+/- 0.5 SD) in volunteers and 15.6 minutes (+/- 2.3 SD) in patients. The disappearance from serum was catalyzed by a macromolecular component of the serum and was slowed by serum lipids. Analysis of the pharmacokinetics of BCNU in 20 patients using a two-compartment open model demonstrated a volume of distribution of 3.25 liters/kg (+/- 1.69 SD), a clearance of 56 ml/minute/kg (+/- 56 SD), and a transfer constant from the central compartment to the outside (K10) OF 0.0324 MINUTE-1 (+/- 41% SD), which was close to the decomposition rate observed for BCNU in serum in vitro. The pharmacokinetics of BCNU in patients may be affected by the percent of body fat and the lipid content of the serum.

Analysis for 1,3-bis(2-chloroethyl)-1-nitrosourea by chemical ionization mass spectrometry.

We used chemical ionization mass spectrometry to quantify the cancer chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea in biological fluids. A stable isotope-labeled internal standard was added to the fluid being analyzed. Protonated molecular ion intensity ratios were determined by selected ion monitoring of sample extracts inserted directly into the chemical ionization source. Peak ratios were calculated for chlorine isotope ratios in their natural abundance, to verify the integrity of the data acquired, and to compute the drug concentration. The assay was applied to measure the compound in rat blood and plasma and in human plasma, and to monitor in vitro metabolism in rat-liver microsomal preparations. For the analyses we used a quadrupole mass spectrometer interfaced to a commercial computer system. Data-system characteristics are described.

Differential pulse polarographic determination of BCNU pharmacokinetics in patients with lung cancer.

BCNU levels were determined by differential pulse polarography in 12 patients with lung cancer. A simple assay method for submicrogram quantities of nitrosoureas is described. The half-life of BCNU added to plasma in vitro was 1413 mins at 0 degrees C and 12 mins at 37 degrees C during incubation. BCNU kinetics in blood of treated patients was calculated using the two-compartment open model; the half-life of the drug was 1.4 mins in the first phase and 17.8 mins in the second. Mean distribution volume was 2.59 liters/kg and plasma clearance was 16.7 ml/min/kg.

Specific high-performance liquid chromatographic assay with ultraviolet detection for the determination of 1-(2-chloroethyl)-3-sarcosinamide-1-nitrosourea in plasma.

A facile, sensitive and highly specific HPLC method for assaying 1-(2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) in plasma has been developed. The drug was efficiently isolated from plasma by extraction with tert.-butyl methyl ether. A structurally related compound with similar physicochemical properties served as the internal standard (I.S.). Following evaporation of the organic solvent, the extract was reconstituted with 0.05 M ammonium acetate buffer, pH 5.0, and loaded onto a 4 micron Nova-Pak C18 column (15 cm x 3.9 mm), which was preceded by a 7 micron Brownlee RP-18 precolumn (1.5 cm x 3.2 mm). Chromatography was performed at ambient temperature using a mobile phase of methanol-0.1 M ammonium formate buffer, pH 3.7 (25:75, v/v). UV absorbance of the effluent was monitored at 240 nm. A flow-rate of 1.0 ml/ min was used for analyzing mouse and dog plasma extracts. Under these conditions, the drug eluted at 4.0 min and was followed by the I.S. at 6.1 min. An automatic switching valve was employed to allow the precolumn to be flushed 1.5 min into the run, without interrupting the flow of the mobile phase to the analytical column, thereby preventing the apparent build-up of extractable, strongly retained, UV-absorbing components present in mouse and dog plasma. Operating in this manner, more than 100 samples could be analyzed during a day using a refrigerated autosampler for overnight injection. The method was readily adapted to the determination of SarCNU in human plasma by simply decreasing the eluent flow-rate to 0.6 ml/min, whereby SarCNU and the I.S. eluted at approximately 5.8 and 9.1 min, respectively. Furthermore, the switching valve was not necessary for the analysis of human plasma samples. With a 50-microliter sample volume, the lowest concentration of SarCNU included in the plasma standard curves, 0.10 micrograms/ml, was quantified with a 7.8% R.S.D. (n = 27) over a 2 month period. Plasma standards, with concentrations of 0.26 to 5.1 micrograms/ml, exhibited R.S.D. values ranging from 1.3 to 4.7%. Thermospray-ionization MS detection was used to definitively establish the specificity of the method. The sensitivity of the assay was shown by application to be more than adequate for characterizing the plasma pharmacokinetics of SarCNU in mice.

Pharmacokinetic profile of fotemustine in rat plasma by electrochemical detection.

1. A differential pulse polarographic (DDP) assay of diethyl-1-[3-(2-chloroethyl)-3-nitrosoureido] ethylphosphonate (fotemustine) was developed to determine the kinetics of this nitrosourea in plasma, brain, liver, lung and kidney. The optimized polarographic determination, previously applied to BCNU and CCNU, attained a limit of detection of 0.3 micrograms fotemustine/ml plasma and 1 microgram/g in other tissues; the calibration curve in electrolyte or in plasma was linear between 0.5 and 100 micrograms/ml. 2. The choice of electrolyte, the effects of pH, temperature, light, and the stability of fotemustine in samples were investigated. Recovery of fotemustine was 76-90% from lung greater than kidney greater than plasma greater than brain greater than liver; the variability coefficients were low (4.0-7.3%). Tissue samples could be stored for 20 days at -20 degrees C without loss of the compound. 3. Plasma kinetics of fotemustine and BCNU given to male rats at therapeutic doses (20 mg/kg i.v.) fitted a bi-exponential equation. Two minutes after injection plasma, levels of unchanged nitrosoureas were 15 and 11 micrograms/ml respectively. Fotemustine could be measured (0.92 microgram/ml) for 3 h, while BCNU could not be detected after 60 min. Unchanged fotemustine was cleared from the blood stream 3-5 times more slowly than BCNU.