Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs.

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

Mathematical Modelling of Convection Enhanced Delivery of Carmustine and Paclitaxel for Brain Tumour Therapy.

PURPOSE: Convection enhanced delivery (CED) is a promising method of anticancer treatment to bypass the blood-brain barrier. This paper is aimed to study drug transport under different CED operating conditions. METHODS: The convection enhanced delivery of chemotherapeutics to an intact and a remnant brain tumour after resection is investigated by means of mathematical modelling of the key physical and physiological processes of drug transport. Realistic models of brain tumour and its holding tissue are reconstructed from magnetic resonance images. Mathematical modelling is performed for the delivery of carmustine and paclitaxel with different infusion rates, solution concentrations and locations of infusion site. RESULTS: Modelling predications show that drug penetration can be improved by raising the infusion rate and the infusion solution concentration. The delivery of carmustine with CED is highly localised. High drug concentration only can be achieved around the infusion site. The transport of paclitaxel is more sensitive to CED-enhanced interstitial fluid as compared to carmustine, with deeper penetration into tumour interior. Infusing paclitaxel in the upstream of interstitial fluid flow leads to high spatial averaged concentration and relatively uniform distribution. CONCLUSION: Results obtained in this study can be used to guide the design and optimisation of CED treatment regimens.

Microclaw Array Fabricated by Single Exposure of Femtosecond Airy Beam and Self-Assembly for Regulating Cell Migratory Plasticity.

The highly aligned extracellular matrix of metastatic breast cancer cells is considered to be the "highway" of cancer invasion, which strongly promotes the directional migration of cancer cells to break through the basement membrane. However, how the reorganized extracellular matrix regulates cancer cell migration remains unknown. Here, a single exposure of a femtosecond Airy beam followed by a capillary-assisted self-assembly process was used to fabricate a microclaw-array, which was used to mimic the highly oriented extracellular matrix of tumor cells and the pores in the matrix or basement membrane during cell invasion. Through the experiment, we found that metastatic breast cancer MDA-MB-231 cells and normal breast epithelial MCF-10A cells exhibit three major migration phenotypes on microclaw-array assembled with different lateral spacings: guidance, impasse, and penetration, whereas guided and penetrating migration are almost completely arrested in noninvasive MCF-7 cells. In addition, different mammary breast epithelial cells differ in their ability to spontaneously perceive and respond to the topology of the extracellular matrix at the subcellular and molecular levels, which ultimately affects the cell migratory phenotype and pathfinding. Altogether, we fabricated a microclaw-array as a flexible and high-throughput tool to mimic the extracellular matrix during invasion to study the migratory plasticity of cancer cells.

Brain targeted delivery of carmustine using chitosan coated nanoparticles via nasal route for glioblastoma treatment.

This study aims to develop chitosan-coated PLGA nanoparticles intended for nose-to-brain delivery of carmustine. Formulations were prepared by the double emulsion solvent evaporation method and optimized by using Box-Behnken Design. The optimized nanoparticles were obtained to satisfactory levels in terms of particle size, PDI, entrapment efficiency, and drug loading. In vitro drug release and ex-vivo permeation showed sustained release and enhanced permeability (approx. 2 fold) of carmustine compared to drug suspension. The AUC0-t of brain obtained with carmustine-loaded nanoparticles via nasal administration in Albino Wistar rats was 2.8 and 14.7 times that of intranasal carmustine suspension and intravenous carmustine, respectively. The MTT assay on U87 MG cell line showed a significant decrease (P < 0.05) in the IC50 value of the formulation (71.23 µg ml-1) as compared to drug suspension (90.02 µg ml-1).These findings suggest chitosan coated nanoparticles could be used to deliver carmustine via intranasal administration to treat Glioblastoma multiforme.

Protective role of the dried white mulberry extract on the reproductive damage and fertility in rats treated with carmustine.

The present study investigated the protective effect of dried white Mulberry extract (DWME) against carmustine (Crm) induced biochemical alterations and spermatological, histopathological, and fertility damage in Wistar albino rats. Male rats were divided into four groups (control, Crm, Crm + DWME, and DWME group). It was found that Crm decreased the motility. Crm decreased the concentration (not different from control group) compared to DWME groups. Total blood MDA levels were reduced during the recovery period. Also, the recovery period reduced the MDA levels in the Crm group/testicular tissue. The GSH levels in the Crm + DWME group were the highest among all groups in the testicular tissue/experiment period. In the immunohistochemical evaluation of the testicular tissue, a high level of caspase-3 was observed in the cells that underwent meiosis in the Crm group. The most pronounced DNA damage was also detected in the Crm group. The Crm + DWME group showed the highest number of offspring born during recovery period. In conclusion, dried white mulberry extract protects against the spermatological damages caused by carmustine. Moreover, recovery period played a positive effect on spermatological parameters and fertility.

Targeting Inhibition of Notch1 Signaling Pathway on the Study of Human Gastric Cancer Stem Cells with Chemosensitization.

Background: Gastric cancer is the second most frequent cause of cancer death worldwide, although much geographical variation in incidence exists. Prevention and personalized treatment are regarded as the best options to reduce gastric cancer mortality rates (Hartgrink et al., 2009). Numerous studies have suggested that Notch1 and its ligands are overexpressed in gastric cancer, and its knockdown can inhibit the proliferation and survival of gastric cancer cells. Objective: To investigate the effect of Notch1 on the stemness and drug sensitivity of human gastric cancer SGC-7901 cells. Methods: Highly expressed Notch1 intracellular domain (NICD1) and Notch1-shRNA lentiviral expression vector were used to infect human gastric cancer SGC-7901 cells cultured in vitro, and western blot and immunofluorescence staining were used to identify highly expressed NICD and Notch1 silenced cells. The percentage of CD133+ cells was analyzed by flow cytometry, the expression of nestin and CFAP by immunofluorescence staining, the formation rate of tumor cell spheres and the tumorigenicity of SCID mice in vivo, and the regulation of cell stemness by Notch1. The sensitivity of each group of cells to the chemotherapeutic drugs teniposide (VM-26) and carmustine (BCNU) was also detected by the MTT method. Results: The stemness phenotype of tumor cells with the increased NICD expression was enhanced, such as an increased proportion of CD133+ cells, enhanced nestin expression, decreased GFAP expression, increased tumor cell sphere formation rate and tumorigenic rate of SCID mice implantation, and decreased sensitivity to VM-26 and BCNU. In contrast, the stemness phenotype of tumor cells with downregulated Notch1 gene expression was significantly suppressed, while the sensitivity to VM-26 and BCNU was increased. Conclusion: High Notch1 expression increased the stemness of SGC-7901 cells and decreased the sensitivity of SGC-7901 cells to chemotherapeutic drugs.

Effect of tissue permeability and drug diffusion anisotropy on convection-enhanced delivery.

Although convection-enhanced delivery (CED) can successfully facilitate a bypass of the blood brain barrier, its treatment efficacy remains highly limited in clinic. This can be partially attributed to the brain anisotropic characteristics that lead to the difficulties in controlling the drug spatial distribution. Here, the responses of six different drugs to the tissue anisotropy are examined through a parametric study performed using a multiphysics model, which considers interstitial fluid flow, tissue deformation and interlinked drug transport processes in CED. The delivery outcomes are evaluated in terms of the penetration depth and delivery volume for effective therapy. Simulation results demonstrate that the effective penetration depth in a given direction can be improved with the increase of the corresponding component of anisotropic characteristics. The anisotropic tissue permeability could only reshape the drug distribution in space but has limited contribution to the total effective delivery volume. On the other hand, drugs respond in different ways to the anisotropic diffusivity. The large delivery volumes of fluorouracil, carmustine, cisplatin and doxorubicin could be achieved in relatively isotropic tissue, while paclitaxel and methotrexate are able to cover enlarged regions into anisotropic tissues. Results obtained from this study serve as a guide for the design of CED treatments.

Methylguanine methyltransferase-mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model.

Clinical application of gene therapy for genetic and malignant diseases has been limited by inefficient stem cell gene transfer. Here we studied in a clinically relevant canine model whether genetic chemoprotection mediated by a mutant of the DNA-repair enzyme methylguanine methyltransferase could circumvent this limitation. We hypothesized that genetic chemoprotection might also be used to enhance allogeneic stem cell transplantation, and thus we evaluated methylguanine methyltransferase-mediated chemoprotection in an allogeneic setting. We demonstrate that gene-modified allogeneic canine CD34+ cells can engraft even after low-dose total body irradiation conditioning. We also show that cytotoxic drug treatment produced a significant and sustained multilineage increase in gene-modified repopulating cells. Marking in granulocytes rose to levels of up to 98%, the highest in vivo marking reported to date to our knowledge in any large-animal or human study. Increases in transgene-expressing cells after in vivo selection provided protection from chemotherapy-induced myelosuppression, and proviral integration site analysis demonstrated the protection of multiple repopulating clones. Drug treatment also resulted in an increase in donor chimerism. These data demonstrate that durable, therapeutically relevant in vivo selection and chemoprotection of gene-modified cells can be achieved in a large-animal model and suggest that chemoprotection can also be used to enhance allogeneic stem cell transplantation.

In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning.

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment.

Levels and distribution of BCNU in GBM tumors following intratumoral injection of DTI-015 (BCNU-ethanol).

The alkylation products formed by in vitro treatment of DNA with tritium-labeled 1,3-bis(2-chloroethyl)-1-nitrosourea ((3)H-BCNU) were identified and quantified. Twelve adducts were resolved by high-performance liquid chromatography (HPLC). The principal DNA adducts formed by BCNU treatment corresponded to N-7-(2-hydroxyethyl)guanine (N7-HOEtG) (26%), N-7-(2-chloroethyl)guanine (15%), and phosphotriesters (19%). In addition, several minor products were identified as 1,2-(diguan-7-yl)ethane, N-1-(2-hydroxyethyl)-2-deoxyguanosine, 1-(N-1-2-deoxyguanosinyl), 2-(N-3-2-deoxycytidyl)ethane cross-link, and O-6-(2-hydroxyethyl)-2-deoxyguanosine, and individually they represented 1% to 5% of the total alkylation. An HPLC-electrochemical method was applied to quantify the levels of N7-HOEtG in samples treated with BCNU. Treatment of either purified DNA or U87MG cells with various amounts of BCNU produced a linear increase in the amount of N7-HOEtG. These results demonstrated that the levels of N7-HOEtG formed by BCNU treatment could be used as a molecular dosimeter of BCNU treatment dose. We measured the levels of N7-HOEtG in DNA isolated from tumor samples taken from four patients with GBM tumors following stereotactic intratumoral injection with DTI-015 (BCNU-ethanol). The level of N7-HOEtG in these samples ranged from 14.7 to 121.9 micromol N7-HOETG/mol DNA within 1 cm of the site of injection. As the distance from the site of injection increased, the levels of N7-HOEtG in tumor DNA decreased. In two of the samples, the levels of N7-HOEtG were 0.2 to 0.3 micromol N7-HOETG/mol DNA at 3.5 to 3.9 cm from the site of injection, demonstrating significant distribution of BCNU in the tumor. The levels of N7-HOEtG in these tumor samples corresponded to BCNU treatment concentrations of 0.02 to 43.0 mM. These studies demonstrate that stereotactic intratumoral injection of DTI-015 into human GBM tumors produces high concentrations of BCNU up to 2.5 cm from the site of injection in some of the tumors. These observations suggest that intratumoral injection of DTI-015 may be of benefit in the treatment of primary and recurrent GBM tumors.

Direct demonstration in synthetic oligonucleotides that N,N'-bis(2-chloroethyl)-nitrosourea cross links N1 of deoxyguanosine to N3 of deoxycytidine on opposite strands of duplex DNA.

The sequence specificity and covalent structure of the lesion caused by the DNA interstrand cross-linking reaction of N,N'-bis(2-chloroethyl)-nitrosourea (BCNU) were investigated using synthetic oligonucleotides. The efficiency of interstrand cross-linking was found to parallel the efficiency of monoadduct formation, preferring deoxyguanosine-deoxycytidine-rich duplexes and, particularly, runs of deoxyguanosine. No explicit sequence specificity was observed. Enzymatic digestion of purified, interstrand cross-linked DNA returned primarily the unmodified deoxynucleosides, along with 1-[N3-deoxycytidyl]-2-[N1-deoxyguanosyl]ethane. This substance was characterized by comparison of its mass spectrum, high-pressure liquid chromatography retention time, and UV spectrum to an authentic standard prepared by chemical synthesis. These studies provide the first direct evidence that BCNU has no strong sequence preference for interstrand cross-linking and that substance 4, which has been previously isolated from BCNU-treated DNA, derives from alkylation on opposite strands of DNA. The lack of sequence preference and lesion structure together suggest that one source of BCNU interstrand cross-links is linkage of deoxyguanosine and deoxycytidine partners from a single bp.

Quenching of DNA cross-link precursors of chloroethylnitrosoureas and attenuation of DNA interstrand cross-linking by glutathione.

Interstrand DNA cross-linking is essential for the antitumor activity of chloroethylnitrosoureas (CENUs). The critical cross-links have been proposed to involve a rapid O6-guanine chloroethylation on one DNA strand, followed by a rearrangement of the O6-(2-chloroethyl)guanine and slow alkylation of the second DNA strand. In view of the relative intracellular abundance of glutathione (GSH) and nucleophilicity of its thiolate ion, the ability of GSH to react with and to inactivate 2-chlorethylated DNA and the possibility that this interaction decreases net DNA cross-linking by CENUs were investigated. Chloroethylated calf thymus DNA was reacted with GSH, the DNA was precipitated and redissolved, and subsequent DNA interstrand cross-linking was determined. The DNA cross-link index was compared for both GSH-treated and 2-chloroethylated untreated DNA. Simultaneously, Col E1 plasmid DNA was chloroethylated and reacted with GSH, and the extent of DNA interstrand cross-linking was determined by agarose gel electrophoresis and compared with controls. The results show both a time- and GSH concentration-dependent quenching of chloroethylated DNA, with a corresponding decrease in the DNA cross-link index. Using [methyl-3H]GSH, it was also demonstrated that 56% of the total GSH was bound to quenched 2-chloroethylated Col E1 DNA and 25% to quenched 2-chloroethylated calf thymus DNA. GSH binding to cross-linked DNA and native DNA was insignificant. It is concluded that, in addition to direct inactivation of reactive cytotoxic CENU species, GSH may also modulate cellular response to CENUs by quenching chloroethylated DNA, thereby decreasing the formation of potentially lethal DNA cross-links.

Formation of DNA interstrand cross-links by the novel chloroethylating agent 2-chloroethyl(methylsulfonyl)methanesulfonate: suppression by O6-alkylguanine-DNA alkyltransferase purified from human leukemic lymphoblasts.

The formation of DNA interstrand cross-links was compared in DNA treated with either 1,3-bis(2-chloroethyl)-1-nitrosourea or 2-chloroethyl(methylsulfonyl)methanesulfonate. DNA that was pulse treated briefly with either of these drugs continued to form cross-links at 37 degrees C for over 8 h after drug removal, indicating that such DNA contained stable precursors of cross-links. When human O6-alkylguanine-DNA alkyltransferase was added to the drug-treated DNA further cross-link formation was prevented at all points during this protracted time course, indicating that these stable cross-link precursors also remained substrates for this repair enzyme. Although the pattern of 2-chloroethyl(methylsulfonyl)methanesulfonate-induced cross-link formation and susceptibility to suppression by O6-alkylguanine-DNA alkyltransferase resembled that for 1,3-bis(2-chloroethyl)-1-nitrosourea, quantitative differences in the rates of cross-link formation and in the amounts of O6-alkylguanine-DNA alkyltransferase required to suppress cross-link formation suggest that critical differences exist between these agents.

Transport of amino acid amide sarcosinamide and sarcosinamide chloroethylnitrosourea in human glioma SK-MG-1 cells.

The transport of the amino acid amide N-[3H]sarcosinamide (methyl glycinamide) was investigated in human glioma SK-MG-1 cells. Sarcosinamide uptake was found to be temperature dependent, sodium independent, and linear up to 1 min at 22 degrees C. Equilibrium was reached after 10 min at 22 degrees C with accumulation slightly above unity. Sarcosinamide was not metabolized in the cells as shown by thin layer chromatography. The uptake of sarcosinamide was significantly decreased when the extracellular pH was lowered from 7.5 to 6.0 and significantly enhanced at pH values above 7.5. The latter effect may be due mainly to increased cell permeability at high pH. The uptake of the labeled sarcosinamide was trans-stimulated by excess cold sarcosinamide. Sarcosinamide uptake over a 200-fold range of concentrations followed Michaelis-Menten kinetics with a Km of 0.284 +/- 0.041 mM and a Vmax of 0.154 +/- 0.024 nmol/10(6) cells/min. The uptake of sarcosinamide was significantly reduced by iodoacetate but not by the metabolic poisons NaF, ouabain, or dinitrophenyl, suggesting that the uptake is not dependent on energy, rather it proceeds by facilitated diffusion. Several naturally occurring substrates were unable to inhibit the uptake of sarcosinamide. Leucine significantly reduced the uptake of sarcosinamide, while sarcosinamide was a weak inhibitor of leucine transport. 2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid a specific substrate for the sodium-independent, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid-sensitive amino acid system L failed to inhibit the uptake of sarcosinamide. Epinephrine reduced the uptake of sarcosinamide and sarcosinamide was equally potent as an inhibitor of epinephrine transport. Dixon plot analysis demonstrated that epinephrine (Km = 0.270 mM) inhibits the uptake of sarcosinamide competitively (Ki = 0.260 mM). These results indicate that sarcosinamide is a substrate for the catecholamine transporter. The alkylating agent, sarcosinamide chloroethylnitrosourea, was tested for its ability to inhibit the uptake of sarcosinamide. The results of Dixon plot analysis were consistent with competitive inhibition of sarcosinamide uptake and the inhibition constant Ki for SarCNU was found to be 3.26 +/- 0.57 mM. The steady-state intracellular concentration of SarCNU was found to be significantly higher (cell:medium ratio of 1.03 +/- 0.01) than that of BCNU cell:medium ratio of 0.52 +/- 0.12). These findings indicate that SarCNU and sarcosinamide share the same carrier for uptake in SK-MG-1 cells. This transport mechanism may be responsible for the increased accumulation of SarCNU as compared to BCNU, a nitrosourea which enters cells by passive diffusion.

Mechanism of uptake of nitrosoureas by L5178Y lymphoblasts in vitro.

The mechanism of uptake of nitrosoureas by L5178Y cells in vitro was investigated. A time course of the uptake of radioactivity on incubation of L5178Y lymphoblast with [14C]-1,3-bis(2-chloroethyl)-1-nitrosourea was linear for 30 min and then entered a plateau phase; it was markedly temperature dependent. A similar time course for cells incubated with [14C]ethylene-labeled 1-(2-chlorethyl)-3-cyclohexyl-1-nitrosourea reached equilibrium rapidly, was temperature independent, and resulted in a relatively low level of uptake of radioactivity. However, cells treated with 3-[cyclohexyl-14C]-1-(2-chlorethyl)-1-nitrosourea had a time course that was linear for 30 min, resulted in much higher levels of uptake of radioactivity, and was strongly temperature dependent. These findings, at least for 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, suggest that some drug decomposition precedes uptake. The percentage of radioactivity found in the cell sap fraction was at least 85% of total cell activity when cells were incubated with any of the three 14C-labeled nitrosoureas. Furthermore, thin-layer chromatography of the cell sap fraction revealed the presence of free intact drug. These findings indicate that intracellular uptake of intact nitrosoureas occurred. A time course of uptake of intact 1,3-bis(2-chloroethyl)-1-nitrosourea reached equilibrium rapidly with cell/medium distribution ratios of 0.2 to 0.6 and was temperature independent. The addition of excess unlabeled 1,3-bis(2-chlorethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea had no effect on uptake of [14C]-1,3-bis(2-chloroethyl)-1-nitrosourea, These findings suggest that uptake of intact 1,3-bis(2-chloroethyl)-1-nitrosourea was by passive diffusion. A time course of the uptake of intact 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with either [14C]ethylene- or ring-labeled drug rapidly reached equilibrium, was temperature independent, and attained a cell/medium ratio greater than unity. Uptake of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was sodium independent and was unaffected by the metabolic inhibitors (sodium fluoride, sodium cyanide, or 2,4-dinitrophenol) or by urea, a potential physiological competitor. Furthermore, addition of unlabeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea or 1,3-bis(2-chlorethyl)-1-nitrosourea had no effect on uptake of labeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. These findings suggest that uptake of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea also occurs by passive diffusion.

Pharmacokinetics of positron-labeled 1,3-bis(2-chloroethyl)nitrosourea in human brain tumors using positron emission tomography.

The nitrosoureas are widely used in the chemotherapy of brain tumors, two of the most common being 1,3-bis(2-chloroethyl)nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. However, we do not understand how these compounds work, nor do we know which part of the molecule has antitumor activity. In six patients with brain tumor, we measured the kinetic behavior of positron-labeled 1,3-bis(2-chloroethyl)nitrosourea in both the tumor and the normal brain with the aid of positron emission tomography; we also analyzed the distribution of radioactivity in plasma. We found the clearance of total radioactivity from the tumor to be significantly slower than from the contralateral brain and plasma, indicating a different rate of 1,3-bis(2-chloroethyl)nitrosourea decomposition in the tumor than in normal brain.

Synergistic antileukemic effect of theophylline and 1,3-bis(2-chloroethyl)-1-nitrosourea.

A series of experiments evaluated the antileukemic effect of an agent which elevates cellular cyclic adenosine 3':5'-monophosphate levels by phosphate levels by inhibiting phosphodiesterase. When administered alone, theophylline had only modest antileukemic effects, but it had synergistic effects when administered with 1,3-bis(2-chloroethyl)-1-nitrosourea. This synergism produced an improved therapeutic index in a dose-response study and in a comparison between antileukemic effects and effects on white blood cell nadirs. Uptake of 1,3-bis(2-chloroethyl)-1-nitrosourea and studies of timing of treatment support the hypothesis that elevation of cyclic adenosine 3':5'-monophosphate levels is the mechanism of the observed synergistic effect.

Microsomal metabolism of nitrosoureas.

N, N-Bis (2-chloroethyl)-N-nitrosourea (BCNU) is a substrate for a microsomal enzyme of mouse liver. The reaction requires NADPH, and the product is 1, 3-bis (2-chloroethyl) urea. This activity is also found in mouse lungs but not in several other tissues. With reaction conditions under which BCNU is not chemically degraded, the Km for BCNU with liver microsomes is 1.7 mM; nicotine is a competitive inhibitor with a Ki of 0.6 mM. N-Methyl-N-nitrosourea is denitrosated in a similar reaction. N- (2-Chloroetyhy)- N-cyclohexyl-N-nitrosourea and N-(2-chloroethyl)-N-(trans-4-methylcyclohexyl)-N-nitrosourea are also substrates for microsomal enzymes, but the products of these reactions are ring-hydroxylated derivatives. The Km value for N-(2-CHLOROETHYL)-N-cyclohexyl-N-nitrosourea is 3.0 mM and that for N-(2-chloroethyl)-N-(trans-4-methylcyclohexyl)-N-nitrosourea is 1.0 mM. The hydroxylase activity is also present in lungs, but not in the other mouse tissues. The rates of microsomal metabolism of BCNU, N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea, and N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea, and N-(2-chloroethyl-N-(trans-4-methylcyclohexyl)-N-nitrosourea are fast enough to allow metabolism of large portions of administered doses before chemical decomposition of the drugs occurs.

Effect of thymidine on uptake, DNA alkylation, and DNA repair in L1210 cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea or 3'-[3-(2-chloroethyl)-3-nitrosoureido]-3'-deoxythymidine.

In order to define the mechanism for the enhancement by thymidine (dThd) of the antitumor activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 3'-[3-(2-chloroethyl)-3-nitrosoureido]-3'-deoxythymidine (3'-CTNU) in mice, we have investigated the effect(s) of dThd on the uptake of nitrosourea by L1210 cells in culture, DNA alkylation, and repair of the alkyl lesion. Using a rapid centrifugation technique through silicone:paraffin oil, we observe a 1.3- and 1.5-fold increase in the uptake of radioactivity from 0.1 mM [chloroethyl-14C]BCNU in the presence of a 5- and 25-fold excess of dThd, respectively. Similarly, an enhancement of DNA alkylation was observed upon treatment of L1210 cells for up to 3 h with 0.1 mM [chloroethyl-14C]BCNU from 70 pmol 14C/mg DNA in control to 85, 95, and 120 pmol 14C/mg DNA with equimolar 5- and 25-fold excess dThd, respectively. No effect of dThd on the uptake of 0.1 mM [chloroethyl-14C]-3'-CTNU was observed, although a small increase in DNA alkylation at 3 h was evident. DNA repair, as measured by the amount of radioactivity remaining associated with the DNA after an initial 2-h treatment with labeled BCNU was largely unaffected by dThd. Although dThd appears to enhance the cellular uptake of BCNU and the alkylation of DNA by both BCNU and 3'-CTNU, dealkylative repair proceeds unhindered in the presence of dThd.

Denitrosation of 1,3-bis(2-chloroethyl)-1-nitrosourea by class mu glutathione transferases and its role in cellular resistance in rat brain tumor cells.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is known to be detoxified by a denitrosation reaction catalyzed by glutathione-dependent enzymes in rat liver cytosol (R. E. Talcott and V. A. Levin, Drug Metab. Dispos., 11:175-176, 1983). Using a modification of their procedure, we have measured the ability of different purified rat glutathione transferase isoenzymes to denitrosate BCNU. The catalytic efficiencies of the isoenzymes for the denitrosation reaction expressed as the ratio of Vmax to Km were as follows (isoenzyme, Vmax/Km): 1-2, 2.3; 3-3, 12.2; 3-4, 29.2; and 4-4, 26.1. Thus, the class mu isoenzymes containing subunit 4 are by far the best catalysts of the BCNU denitrosation reaction. The class pi transferase 7-7 and class alpha transferases 1-1 and 1-2 demonstrated very weak catalytic activity with BCNU. Determination of the glutathione transferase isoenzyme profiles of 9L rat brain tumor cells and the BCNU-resistant 9L-2 subline by immunoblotting revealed that although the resistant 9L-2 cells contain lower total glutathione transferase activity than 9L cells, they have elevated levels of the class mu transferases. Also, the class pi transferases were found to be down-regulated in 9L-2 as compared with 9L cells. Thus, the increased resistance of 9L-2 cells to BCNU may, in part, be explained by up-regulation of class mu transferase expression with consequent increased capacity for BCNU detoxication. Further support for this hypothesis comes from the fact that pretreatment of 9L-2 cells with the glutathione transferase inhibitors ethacrynic acid or triphenyltin chloride enhanced the cytotoxic effects of BCNU. These results suggest that the class mu transferases play a role in the resistance of brain tumor cells to BCNU.