Reductive activation of the prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively occurs in oxygen-deficient cells and overcomes O-alkylguanine-DNA alkyltransferase mediated KS119 tumor cell resistance

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) is a prodrug of the 1,2-bis(sulfonyl)hydrazine class of antineoplastic agents designed to exploit the oxygen-deficient regions of cancerous tissue. Thus, under reductive conditions in hypoxic cells this agent decomposes to produce the reactive intermediate 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which in turn generates products that alkylate the O⁶-position of guanine in DNA. Comparison of the cytotoxicity of KS119 in cultured cells lacking O⁶-alkylguanine-DNA alkyltransferase (AGT) to an agent such as Onrigin™, which through base catalyzed activation produces the same critical DNA G-C cross-link lesions by the generation of 90CE, indicates that KS119 is substantially more potent than Onrigin™ under conditions of oxygen deficiency, despite being incompletely activated. In cell lines expressing relatively large amounts of AGT, the design of the prodrug KS119, which requires intracellular activation by reductase enzymes to produce a cytotoxic effect, results in an ability to overcome resistance derived from the expression of AGT. This appears to derive from the ability of a small portion of the chloroethylating species produced by the activation of KS119 to slip through the cellular protection afforded by AGT to generate the few DNA G-C cross-links that are required for tumor cell lethality. The findings also demonstrate that activation of KS119 under oxygen-deficient conditions is ubiquitous, occurring in all of the cell lines tested thus far, suggesting that the enzymes required for reductive activation of this agent are widely distributed in many different tumor types.     

The polymorphic human glutathione transferase T1-1, the most efficient glutathione transferase in the denitrosation and inactivation of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea

A member of the Theta class of human glutathione transferases (GST T1-1) was found to display the greatest catalytic activity towards the cytostatic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of the GSTs studied. In this investigation (the most extensive to date), enzymes from four classes of the soluble human GSTs were heterologously expressed, purified, and kinetically characterized. From the 12 enzymes examined, only GST M2-2, GST M3-3 and GST T1-1 had significant activities with BCNU. This establishes that the activity is not a characteristic of a particular class of GSTs. Although GST M3-3 was previously reported to have the greatest activity with BCNU, the current investigation demonstrates that GST M2-2 is equally active and that GST T1-1 has an approximately 20-fold higher specific activity than either of the Mu class enzymes. A more rigorous kinetic analysis of GST T1-1 gave the following parameters with BCNU: a k(cat) of 0.035 +/-0.003s(-1) and a K(M) of 1.0 +/- 0.1mM. The finding that GST T1-1 has the highest activity towards BCNU is significant since GST T1-1 is expressed in the brain, a common target for BCNU treatment. Furthermore, the existence of a GST T1-1 null allele in up to 60% in some populations, may influence both the sensitivity of tumors to chemotherapy and the severity of adverse side-effects in patients treated with this agent.     

KS900: A hypoxia-directed, reductively activated methylating antitumor prodrug that selectively ablates O-alkylguanine-DNA alkyltransferase in neoplastic cells

To most effectively treat cancer it may be necessary to preferentially destroy tumor tissue while sparing normal tissues. One strategy to accomplish this is to selectively cripple the involved tumor resistance mechanisms, thereby allowing the affected anticancer drugs to gain therapeutic efficacy. Such an approach is exemplified by our design and synthesis of the intracellular hypoxic cell activated methylating agent, 1,2-bis(methylsulfonyl)-1-methyl-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS900) that targets the O-6 position of guanine in DNA. KS900 is markedly more cytotoxic in clonogenic experiments under conditions of oxygen deficiency than the non-intracellularly activated agents KS90, and 90M, when tested in O⁶-alkylguanine-DNA alkyltransferase (AGT) non-expressing cells (EMT6 mouse mammary carcinoma, CHO/AA8 hamster ovary, and U251 human glioma), and than temozolomide when tested in AGT expressing cells (DU145 human prostate carcinoma). Furthermore, KS900 more efficiently ablates AGT in HL-60 human leukemia and DU145 cells than the spontaneous globally activated methylating agent KS90, with an IC₅₀ value over 9-fold lower than KS90. Finally, KS900 under oxygen-deficient conditions selectively sensitizes DU145 cells to the chloroethylating agent, onrigin, through the ablation of the resistance protein AGT. Thus, under hypoxia, KS900 is more cytotoxic at substantially lower concentrations than methylating agents such as temozolomide that are not preferentially activated in neoplastic cells by intracellular reductase catalysts. The necessity for intracellular activation of KS900 permits substantially greater cytotoxic activity against cells containing the resistance protein O⁶-alkylguanine-DNA alkyltransferase (AGT) than agents such as temozolomide. Furthermore, the hypoxia-directed intracellular activation of KS900 allows it to preferentially ablate AGT pools under the oxygen-deficient conditions that are present in malignant tissue.     

The effect of alkylating agents and other drugs on the accumulation of melphalan by murine L1210 leukaemia cells in vitro

The effect of cytotoxic and other drugs on the accumulation of melphalan by L1210 murine leukaemia cells was studied. We have confirmed that uptake is an active process competitively inhibited by l-leucine. In 36 experiments in amino acid-free medium the mean concentration of melphalan taken up was 225 pmoles/10⁶ cells. High pressure liquid Chromatographie analysis showed that the majority of the drug is present as free native melphalan. 1, 3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) was the only drug that stimulated accumulation, but without significant effect on influx or efflux rates. Busulphan, chlorambucil, cyclophosphamide, interferon, methotrexate and prednisolone had no effect on accumulation after 30 min melphalan transport. Adriamycin, CCNU, methyl CCNU, mustine and vincristine all impaired melphalan accumulation as did the non-cytotoxic drugs aminophylline, chlorpromazine and ouabain. Adriamycin, aminophylline, chloropromazine, indomethacin and ouabain all reduced melphalan influx.     

Mechanism of action of 2-haloethylnitrosoureas on deoxyribonucleic acid. Nature of the intermediates from nitrosourea decomposition.

Direct current (DC) and differential pulse polarographic analyses were used to measure the rates of decomposition of a series of 2-haloethylnitrosoureas in aqueous solution. Measured by these methods, the rates of the first and rate-determining step which show a marked pH and solvent dependence agree with the overall rate of decomposition measured by gas evolution. In the 1,3-bis(haloethyl)-1-nitrosourea series, changing the nature of the halogen X has a small effect on the rate of decomposition. In the 3-cyclohexyl-1-(2-haloethyl)-1-nitrosourea series, changing X for OH or OCH₃ results in the rate of hydrolysis being reduced considerably. A free—NH₂ group in the nitrosourea structure as in CNU, MNU, ENU, CPNU, 4-CBNU and 5-CPNU accelerates considerably the rate of decomposition relative to the BCNU and CCNU series. Arrhenius parameters for the decomposition in aqueous pH 7.1 solution in the temperature range 28–47° were obtained for BFNU, BCNU and BBNU: log A, ?20.1± 1.4,?21.6± 0.7 and ?22.3±1.6; E_a, 24.4 ± 2.0, 26.5± 1.0 and 27.2 m 2.3 kcal/mole. The corresponding values for BINU were estimated as log A,?23.3± 3.0; E_a, 28.0± 3.0 kcal/mole. Examination of the decomposition products of 1,3-bis(2-chloropropyl)-1-nitrosourea (BCNU-β-Me) and 1,3-bisl 1-(chloromethyl)ethyl]-1-nitrosourea (BCNU-α-Me) favors decomposition pathway B via the diazohydroxide and cyclic chloronium ion for BCNU-β-Me and via the diazohydroxide and/or 2-chloro-1-methylethyl carbonium ion for BCNU-α-Me. While there is no evidence for the contribution of pathway A via a 2-imino-N-nitrosooxazolidinone for these compounds, consideration of product type and yields implicates a third decomposition pathway, via a 1,2,3-oxadiazoline intermediate. Additional evidence for an oxadiazoline intermediate is obtained by the isolation of 2-bromoethanol when BCNU is decomposed in the presence of a high concentration of sodium bromide.     

In vitro pharmacokinetics and pharmacodynamics of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

The relationship between treatment efficacy and the pharmacokinetics (PK) and pharmacodynamics (PD) of anticancer drugs is poorly defined. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an alkylating agent used in the treatment of brain and other forms of cancer. It is postulated that BCNU kills cells by forming DNA interstrand cross-links. The present study was undertaken to characterize the PK and PD of BCNU in mouse L1210 cells. L1210 cells were exposed to BCNU (0-160 microM) and analyzed for intracellular BCNU concentrations, DNA interstrand cross-links, cell cycle phase, and cytotoxicity. The half-life of BCNU in cells was approximately 40 min. The maximum reduction of mitochondrial enzyme activity (maximum cell death) achieved within 24 hr after exposure to BCNU was concentration-dependent and could be described by a Hill equation. At lower concentrations, the area under the DNA interstrand cross-link-time curve linearly correlated with the maximum cell death and the area under the BCNU concentration-time curve. BCNU induced cell accumulation in the G(2)/M phase of the cell cycle, which continued even after apparent completion of cross-link repair. Loss of membrane permeability was minimal (approximately 2%) during the first 24 hr. Thereafter, cells died exponentially over the next 9 days, primarily by necrosis. In conclusion, while cytotoxicity was concentration-dependent, an indirect relationship was found among the time-course of BCNU concentrations, DNA interstrand cross-links, and cell death. Because of the disparity between the time-scale of PK and PD, focusing only on the early events may provide limited information about the process of anticancer drug-induced cell death.     

Antitumour imidazotetrazines--IV. An investigation into the mechanism of antitumour activity of a novel and potent antitumour agent, mitozolomide (CCRG 81010, M & B 39565; NSC 353451)

8-Carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4-(3H )-one- mitozolomide (CCRG 81010, M & B 39565, NSC 353451) is a potent inhibitor of the growth of a number of experimental tumours and can potentially decompose to give either an isocyanate or the monochloroethyltriazene (MCTIC). In vitro CCRG 81010 is not cross-resistant with the bifunctional alkylating agents against the Walker carcinoma. To investigate the mechanism of the antitumour activity of CCRG 81010 a comparison has been made with BCNU and MCTIC on precursor incorporation into macromolecules in TLX5 mouse lymphoma cells. Whereas BCNU produces a rapid and extensive inhibition of both (methyl 3H) thymidine and [5-3H]uridine incorporation into acid-insoluble material, neither CCRG 81010 or MCTIC have an early effect on precursor incorporation. Inhibition of precursor uptake is also not produced by concentrations of 2-chloroethylisocyanate that inhibit intracellular glutathione reductase activity. The potential carbamoylating activity of CCRG 81010 has also been assessed by comparing its effect with that of BCNU and 2-chloroethyl isocyanate on enzymes known to be inhibited by carbamoylation. Such enzymes, glutathione reductase, chymotrypsin and gamma-glutamyltranspepidase are not inhibited by CCRG 81010 under conditions where BCNU and 2-chloroethyl isocyanate show complete inhibition of enzyme activity, suggesting an absence of carbamoylating species. The results suggest that the most likely antitumour metabonate produced from CCRG 81010 is the triazene MCTIC.     

Quantitative relationship between guanine O-alkyl lesions produced by Onrigin™ and tumor resistance by O-alkylguanine-DNA alkyltransferase

O⁶-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O⁶-chloroethyl (Onrigin™ and carmustine) and O⁶-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O⁶-methylguanines/cell and ∼300 O⁶-chloroethylguanines/cell, respectively. AGT repaired O⁶-methylguanines until the AGT pool was exhausted, while its repair of O⁶-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O⁶-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ∼10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O⁶-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.     

Protective role of the glutathione redox cycle against adriamycin-mediated toxicity in isolated hepatocytes

Incubation of isolated rat hepatocytes with 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) resulted in the selective and extensive (> 90 per cent) inactivation of glutathione reductase. BCNU also depleted intracellular glutathione by 70 per cent but had no significant effect on Cell viability or lipid peroxidation. Incubation of BCNU-treated hepatocytes with adriamycin (ADR) resulted in a decrease in Cell viability concurrent with an increase in lipid peroxidation. These effects were not observed with untreated hepatocytes incubated with ADR. Glutathione depletion with diethylmaleate and incubation with ADR did not result in a significant decrease in Cell viability or increase in lipid peroxidation. Incubation of BCNU-treated hepatocytes with ADR in the presence of exogenous α-tocopherol resulted in a significant amount of protection from ADR-mediated damage.     

Differential inactivation of polymorphic variants of human O6-alkylguanine-DNA alkyltransferase

The human DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) is an important source of resistance to some therapeutic alkylating agents and attempts to circumvent this resistance by the use of hAGT inhibitors have reached clinical trials. Several human polymorphisms in the MGMT gene that encodes hAGT have been described including L84F and the linked double alteration I143V/K178R. We have investigated the inactivation of these variants and the much rarer variant W65C by O(6)-benzylguanine, which is currently in clinical trials, and a number of other second generation hAGT inhibitors that contain folate derivatives (O(4)-benzylfolic acid, the 3' and 5' folate esters of O(6)-benzyl-2'-deoxyguanosine and the folic acid gamma ester of O(6)-(p-hydroxymethyl)benzylguanine). The I143V/K178R variant was resistant to all of these compounds. The resistance was due solely to the I143V change. These results suggest that the frequency of the I143V/K178R variant among patients in the clinical trials with hAGT inhibitors and the correlation with response should be considered.     

Depletion in vitro of mitochondrial glutathione in rat hepatocytes and enhancement of lipid peroxidation by adriamycin and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

Treatment of isolated rat hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and adriamycin (ADR) produced a complete depletion of cellular glutathione accompanied by a significant increase in lactate dehydrogenase (LDH) leakage. Separation of the mitochondrial and cytoplasmic pools of glutathione by digitonin disruption showed that, although BCNU, a specific inhibitor of glutathione, completely depleted the cytoplasmic pool of glutathione, the mitochondrial supply was not entirely expended and LDH leakage was only moderately stimulated. Only after depletion of the mitochondrial supply of glutathione by ADR and BCNU did LDH leakage increase markedly. Measurement of lipid peroxidation, by monitoring malondialdehyde through the thiobarbituric acid procedure, showed that malondialdehyde accumulated more extensively and at a rate mirroring release of LDH from ADR/BCNU treated cells. The time of increase in LDH leakage and malondialdehyde production corresponded to the time of depletion of mitochondrial glutathione to less than 10% of the initial pool size. No such increase in LDH leakage was observed with BCNU or ADU treatment alone or when aminopyrine, an inhibitor of lipid peroxidation, was included. Aminopyrine was found to prevent, in a dose-dependent manner, both LDH leakage and malondialdehyde production stimulated by ADR/BCNU treatment. The protective effect peaked at 5 mM aminopyrine, and higher concentrations produced significant LDH leakage exhibiting LDH release kinetics different than those observed with ADR/BCNU. Although aminopyrine had no effect on the rate or extent of cytoplasmic glutathione depletion by ADR/BCNU treatment, the mitochondrial pool was conserved significantly in those cells protected by aminopyrine. These data suggest that enhanced hepatocyte damage observed after treatment with a combination of ADR and BCNU versus BCNU or ADR alone is due to the extensive depletion of mitochondrial glutathione supported by ADR after glutathione reductase inhibition. Further, enhancement of lipid peroxidation is strongly implicated in the mechanism of adriamycin toxicity.     

Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity

The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex-GS, 0.22 microm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax and Potter-Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10 microM) or HI 6 (30 microM) of sarin-inhibited human muscle acetylcholinesterase (0.142+/-0.004 min(-1) and 0.166+/-0.008 min(-1), respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker.     

Interaction of mammalian O(6)-alkylguanine-DNA alkyltransferases with O(6)-benzylguanine

Human O(6)-alkylguanine-DNA alkyltransferase (hAGT) activity is a major factor in providing resistance to cancer chemotherapeutic alkylating agents. Inactivation of hAGT by O(6)-benzylguanine (BG) is a promising strategy for overcoming this resistance. Previous studies, which have focused on the region encompassed by residues Pro138 to Gly173, have identified more than 100 individual mutations located at 23 discrete sites at which alterations can render AGT less sensitive to BG. We have now extended the examination of possible sites in hAGT at which alterations might lead to BG resistance to include the residues from Val130 to Asn137, which also make up part of the binding pocket into which BG is postulated to fit. A further 21 mutations located at positions Gly132, Met134, Arg135, and Gly136 were found to lower sensitivity to BG. Mutants R135L, R135Y, and G136P were the most strikingly resistant, with a 50-fold increase in the amount of BG needed to obtain 50% inactivation. These results therefore increase the number of sites at which BG resistance can occur in response to a single amino acid change to 27. Although mammalian AGTs are very similar in amino acid sequence, mouse AGT (mAGT) is significantly less sensitive to BG than rat AGT (rAGT) or hAGT. Construction of chimeric proteins in which portions came from the rAGT and the mAGT indicated that the difference in inactivation resided solely in the amino acids located in the sequence from residues 150 to 188. Individual mutations of the three residues where rAGT and mAGT differ in this region showed that the principal reason for the reduced ability of the mAGT to react with BG was the presence of a histidine residue at position 161, which is occupied by asparagine in rAGT and hAGT. These experiments indicate that many minor changes in amino acids forming all parts of the nucleoside binding pocket of AGT can alter its ability to react with BG and that the possibility that polymorphisms or variants may occur reducing the effectiveness of combination therapy with BG and alkylating agents must be considered.     

Kinetic analysis of the protection afforded by reversible inhibitors against irreversible inhibition of acetylcholinesterase by highly toxic organophosphorus compounds

In organophosphate poisoning, the underlying mechanism of the therapeutic efficacy of carbamate prophylaxis, which was successfully tested in animal experiments, still awaits complete understanding. In particular, it is unclear whether survival is improved by increased acetylcholinesterase activity during the acute phase, when both carbamate and organophosphate are present. This question should be solved experimentally by means of a dynamically working in vitro model. Immobilized human erythrocytes were continuously perfused while acetylcholinesterase activity was monitored in real-time by a modified Ellman method. The concentrations of reversible inhibitors and of paraoxon were varied to assess the influence of both components on the enzyme activity under steady-state conditions. Physostigmine, pyridostigmine and huperzine A were tested for their prophylactic potential. Upon pretreatment with these reversible inhibitors the enzyme was inhibited by 20-90%. Additional perfusion with 1 microM paraoxon for 30 min resulted in a residual activity of 1-4%, at low and high pre-inhibition, respectively. The residual activity was significantly higher than in the absence of reversibly blocking agents (0.3%). After discontinuing paraoxon, the activity increased even in the presence of the reversible blockers. Stopping the reversibly blocking agents resulted in 10-35% recovery of the enzyme activity, depending on the degree of pre-inhibition. The experimental results agreed with computer simulations upon feeding with the essential reaction rate constants, showing that physostigmine was somewhat superior to pyridostigmine in enhancing residual activity in the presence of 1 microM paraoxon for 30 min. The model predicts that inhibitors with a faster dissociation rate, e.g. huperzine A, may be superior in case of a 'hit-and-run' poison such as soman.     

Comparative study of oxime-induced reactivation of erythrocyte and muscle AChE from different animal species following inhibition by sarin or paraoxon

Standard treatment of acute poisoning by organophosphorus compounds (OP) includes administration of an antimuscarinic (e.g. atropine) and of an oxime-based reactivator of OP-inhibited acetylcholinesterase (AChE). A recently introduced dynamically working in vitro model with real-time determination of membrane-bound AChE activity was shown to be a very versatile and promising model to investigate oxime-induced reactivation kinetics of OP-inhibited enzyme. In this assay, human AChE from erythrocytes or muscle tissue was immobilized on a particle filter. This bioreactor was continuously perfused with substrate and chromogen and AChE activity was analyzed on-line in a flow-through detector. The model has been successfully adopted to Rhesus monkey, swine and guinea pig erythrocytes and intercostal muscle AChE. In addition, the basic kinetic constants of inhibition, aging, spontaneous- and oxime-induced-reactivation of erythrocyte AChE from these species were determined with a standard static model. The major findings were, in part substantial species differences in the inhibition (sarin, paraoxon) and reactivation kinetics (obidoxime, HI 6) of erythrocyte AChE, but comparable kinetics of inhibition and reactivation between erythrocyte and muscle AChE. Hence, these data provide further support of the assumption that erythrocyte AChE is an adequate surrogate of muscle (synaptic) AChE and admonish that major species differences have to be considered for the design and evaluation of therapeutic animal models.     

Astaxanthin can alter CYP1A-dependent activities via two different mechanisms: Induction of protein expression and inhibition of NADPH P450 reductase dependent electron transfer

Astaxanthin (Ax), a xanthophyll carotenoid, is reported to induce cytochrome P450 (CYP) 1A-dependent activity. CYP1A is one of the most important enzymes participating in phase I metabolism for chemicals, and it can activate various mutagens. To investigate the effect of Ax on the metabolic activation of a typical promutagen, benzo[a]pyrene by CYP1A, we orally administrated Ax-containing oil (100 mg Ax/kg body weight/day for 3 days) to male Wistar rats. In the treated rat liver, expression of CYP1A1 mRNA, protein, and its activity were significantly increased (5.5-, 8.5-, and 2.5-fold, respectively). In contrast, the activities of phase II enzymes (glutathione S-transferase and glucuronosyl-transferase) were not modulated by Ax-containing oil. As a consequence, the mutagenicity of benzo[a]pyrene was more enhanced in Ax-treated rats, compared with controls in the Ames assay. On the other hand, NADPH P450 reductase activity was decreased in liver microsomes from the treated group. This result suggests the possibility that Ax inhibits the electron supply necessary for CYP catalytic activities and decreases CYP1A activity indirectly. In conclusion, Ax-containing oil intake can alter CYP1A-dependent activities through two different mechanisms: (1) induction of CYP1A1 mRNA, protein expression, and activity; and (2) inhibition of the electron supply for the enzyme.     

On the formation of 4-[N,N-bis(2-chloroethyl)amino]phenyl acetic acid esters of hecogenin and aza-homo-hecogenin and their antileukemic activity

The p-[N,N-bis(2-chloroethyl)amino]phenylacetic acid esters of hecogenin and aza-homo-hecogenin have been prepared and their antineoplastic activity was evaluated against two basic drug screening systems in rodents, P388 lymphocytic and L1210 lymphoid murine leukemias. Among the compounds tested, the p-[N,N-bis(2-chloroethyl)amino]phenylacetic acid ester of aza-homo-hecogenin was appeared to possess a significant higher antileukemic effect. These results support that the alkylating esters of hecogenin produce important antitumor activity as well as, indicate that the aza-homo-hecogenin ester exhibits significantly higher activity due to lactam group (-NHCO-) modification.     

Biochemical changes associated with a multidrug-resistant phenotype of a human glioma cell line with temozolomide-acquired resistance

Temozolomide (TMZ) is a newly approved alkylating agent for the treatment of malignant gliomas. To investigate resistance mechanisms in a multidrug therapeutic approach, a TMZ-resistant human glioma cell line, SF188/TR, was established by stepwise exposure of human SF188 parental cells to TMZ for approximately 6 months. SF188/TR showed 6-fold resistance to TMZ and cross-resistance to a broad spectrum of other anticancer agents that included 3-5-fold resistance to melphalan (MEL), gemcitabine (GEM), paclitaxel (PAC), methotrexate (MTX), and doxorubicin (DOX), and 1.6-2-fold resistance to cisplatin (CDDP) and topotecan (TPT). Alkylguanine alkyltransferase (AGT) activity was increased significantly in the resistant cell line compared with the parental cell line (P<0.05), whereas no significant differences occurred in the cellular uptake of TMZ and PAC between resistant and parental cells. Depletion of AGT by O(6)-benzylguanine significantly increased the cytotoxicity of TMZ in both the sensitive and resistant cell lines, but did not influence the cytotoxicity of the other drugs tested. Treatment with TMZ caused SF188 cells to accumulate in S phase, whereas SF188/TR cells were unaffected. Expression of Bcl-2 family members in SF188/TR cells compared with SF188 cells indicated that the pro-apoptotic proteins (i.e. Bad, Bax, Bcl-X(S)) were reduced 2-4-fold in the resistant cell line, whereas the anti-apoptotic proteins Bcl-2 and Bcl-X(L) were expressed at similar levels in both cell lines. In conclusion, the mechanism of resistance of SF188/TR cells to TMZ involved increased activity of AGT, a primary resistance mechanism, whereas the broad cross-resistance pattern to other anticancer drugs was due to a common secondary resistance mechanism related to alterations in the relative expression of the pro-apoptotic and anti-apoptotic proteins.     

The role of adenosine A2A and A2B receptors in the regulation of TNF-alpha production by human monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes through the activation of its four receptors: A(1), A(2A), A(2B) and A(3). Previous studies have identified the involvement of A(2) receptors in the inhibitory activity of adenosine analogues on tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS) activated monocytes, but the relative contributions of A(2A) versus A(2B) receptors have not been determined in human primary monocytes. Nor has the role of A(1) and A(3) been clearly identified in the system. The lack of such information impacts on the selection of adenosine receptor agonists for disease intervention. Using LPS-stimulated human primary monocytes, we found that the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) or the A(2A) receptor agonist, 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680) produced a concentration-dependent inhibition of TNF-alpha production, with IC(50)s of 58.4nM (32.7-104.5nM, 95% confidence interval) and 49.2nM (22.7-105.9nM, 95% confidence interval), respectively. The selective A(2A) receptor blocker, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylaminso]ethyl)phenol (ZM241385, 30nM), antagonized the effects of NECA and CGS21680 (pK(B) estimates were 8.7+/-0.1 and 8.9+/-0.1, respectively), while the selective A(2B) antagonist, N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754, 100nM), failed to antagonize the effects of either agonist. Furthermore, neither the A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) nor the A(3) receptor agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-d-ribofuranuronamide (2-Cl-IB-MECA) showed significant inhibitory activity at concentrations that effectively bind to their respective receptors. We conclude that A(2A) receptor activation is predominantly responsible for the inhibitory effects of adenosine receptor agonists on TNF-alpha production from LPS-stimulated monocytes.     

Modulation of agonist responses at the A(1) adenosine receptor by an irreversible antagonist,receptor-G protein uncoupling and by the G protein activation state

Potency and intrinsic activity of agonists depend on ligand structure, but are also regulated by receptor-G protein stoichiometry. A potential functional reserve in adenosine A(1) receptor-mediated G protein activation was investigated by stimulation of guanosine-5'-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding by the full agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) and the partial agonist 5'-deoxy-5'-methylthioadenosine (MeSA). Pretreatment of rat brain membranes with the irreversible antagonist 1-propyl-3-[3-[[4-(fluorosulfonyl)benzoyl]oxy]-propyl]-8-cyclopentylxanthine revealed no classical receptor reserve for either agonist. The functional significance of the G protein coupling state of the receptor and occupancy of G proteins by guanine nucleotides was assessed after partial uncoupling of receptor-G protein complexes with N-ethylmaleimide and in the presence of increasing GDP concentrations. Agonist EC(50) values in G protein activation were increased after NEM pretreatment and at higher GDP concentrations, and a decrease in the relative intrinsic activity of MeSA was observed. The shift of agonist concentration-response curves to the right, the decrease in maximal effects and the decrease in relative intrinsic activity of the partial agonist point to a functional reserve which has to be attributed to GDP-free receptor-G protein complexes. The mechanisms of action of FSCPX, NEM and GDP were fully consistent with the two-state model of receptor activation. The apparent reserve revealed by GDP reflects a shift from spontaneously active GDP-free receptor-G protein complexes (RG)(*), which can bind [35S]GTPgammaS, to (RG) occupied by GDP. The abundance of (RG)(*) is favored by agonists and by the absence of GDP.