Cytotoxicity of combinations of prostaglandin D2 (PGD2) and antitumor drugs for B16 melanoma cells in culture

Prostaglandin D₂ (PGD₂) is lethal to murine and human melanoma cells at high doses, but synchronizes cells at G₁ at non-toxic doses (2.5 or 5 g/ml). We tested the lethality to B16 mouse melanoma cells of combinations of PGD₂ with anticancer drugs. The drugs selected were mostly those used in treating human melanoma: actinomycin D, Bleomycin, BCNU, cis-platin, melphalan, 5-fluorouracil, and 1--D-arabinofuranosylcytosine (ara-C). PGD₂ was combined with the drugs according to 3 different protocols:Protocol 1 An asynchronous culture was given a long term (24 hr) exposure simultaneously to PGD₂ + drug. Combinations with Bleomycin, ara-C or melphalan were additive or slightly antagonistic whereas PGD₂ plus actinomycin D was significantly antagonistic.Protocol 2 Cells synchronized in G₁ by 24 hr PGD₂ exposure were then given a short-term (2 hr) treatment with PGD₂ + drug. Combinations with cis-platin, Bleomycin, BCNU or 5-fluorouracil were additive or slightly antagonistic, whereas melphalan and actinomycin D combinations were significantly antagonistic.Protocol 3 Cells were released from a PGD₂-induced G₁ block and were exposed to drug at different times during cell progression. Actinomycin D was antagonistic when added immediately after release from the G₁ block, but was significantly synergistic when added 10 to 12 hr later.The effect of the combinations cannot be explained by available cell cycle or biochemical information. The antagonism between PGD₂ and several of the drugs resembles the cytoprotective effect of PGD₂ towards various noxious agents.     

Sequence-dependent cytotoxicity of combination chemotherapy using paclitaxel, carboplatin and bleomycin in human lung and ovarian cancer

Combination chemotherapy for non-small cell lung cancer (NSCLC) and ovarian cancer typically consists of a regimen of a taxane such as paclitaxel and a platinum-containing agent. Bleomycin, which halts cell cycle progression at G2 phase, is an agent which might thereby increase taxane cytotoxicity. The goal of this study was to evaluate the effect of different paclitaxel-platinum or paclitaxel-bleomycin schedules on cytotoxicity in human NSCLC and ovarian cancer cells. The simultaneous combination of paclitaxel and carboplatin exhibited simple additivity in vitro, while sequential exposure studies indicated that carboplatin followed by paclitaxel produced greater than additive cytotoxicity using the isobologram analysis of combinatorial effects. In contrast, the simultaneous combination of paclitaxel and bleomycin consistently exhibited greater than additive effects indicating a potentially synergistic combination. Sequential exposure studies of bleomycin followed by paclitaxel produced similar synergistic findings. Experiments in SCID mice evaluating the combinations of paclitaxel and bleomycin supported the in vitro results, as significantly enhanced A549 lung tumor growth inhibition was observed when paclitaxel was administered 1 h after bleomycin. The synergistic activity shown by the combination of bleomycin and paclitaxel indicates a potentially beneficial novel combination for treatment of NSCLC and ovarian cancer.     

Effects of perturbations of pools of deoxyribonucleoside triphosphates on expression of ribonucleotide reductase, a G1/S transition state enzyme, in p53-mutated cells

Effects of drug treatment with antimetabolites on a human colon cancer cell line, SW480, were studied. Cells were treated with 10 microM of 5-fluorouracil (5FU), an inhibitor of pyrimidine synthesis, or 1000 microM of hydroxyurea (HU), an inhibitor of both purine and pyrimidine syntheses, or the combination. Recombinant alpha-2a-interferon (IFN), 500 U/mL, also was employed, as this augments the effects of both antimetabolites in vitro and in vivo. The predominant effect of this combination was to block cells in early S phase as measured by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation. By 24 hr, 86% of the cells had accumulated in S phase, but failed to progress to G2/M. This was accompanied by an early, rapid decline in all four deoxyribonucleoside triphosphates (dNTPs) by 38-86% at 4-24 hr. Despite these effects, expression of the G1/S transition state enzyme, ribonucleotide reductase (RR), increased at 24 hr as measured by a 3 to 5-fold increase in mRNA levels for the M2 subunit, in the absence of a measurable effect on protein levels. The rise in levels of RR mRNA and the continued progression of cells into S phase were associated with a synergistic inhibition of cell cycle proliferation resulting from treatment with the three-drug combination. This suggests that in the presence of antimetabolite-induced depletion of dNTPs, SW480 cells, which lack a normal p53 gene, will proceed into S phase, and that this is associated with a rise in expression of the G1/S transition state enzyme, RR. Cells arrested in S phase by a p53-independent mechanism will undergo a synergistic enhancement of cell death.     

DUP 785 (NSC 368390): schedule-dependency of growth-inhibitory and antipyrimidine effects

DUP 785 (NSC 368390; Brequinar sodium) is a new inhibitor of pyrimidine de novo biosynthesis with antitumor activity against several experimental tumors. DUP 785 inhibits the mitochondrial enzyme dihydroorotate dehydrogenase, blocking the conversion of dihydroorotate to orotate. We examined the influence of exposure time to DUP 785 on its growth-inhibitory effects in L1210 murine leukemia and WiDR human adenocarcinoma cells and the effects of pyrimidine (deoxy) nucleosides on reversal of growth-inhibition. The results were correlated with changes in intracellular pyrimidine nucleotide pools and cell cycle distribution. In L1210 cells, a continuous exposure to 25 microM DUP 785 up to 96 hr caused complete growth inhibition. A 2 hr exposure of cells to the drug did not affect growth. In WiDR cells, exposure to the drug for 1-24 hr, followed by cultivation in drug-free medium resulted in recovery of growth. However, cells exposed to the drug for 48 hr or longer were not able to resume growth when recultured in drug-free medium. Reversal studies were performed to know whether selective depletion of one of the pyrimidine (deoxy) nucleotides might be related to the growth-inhibitory effects of DUP 785. Neither thymidine, deoxycytidine alone, deoxycytidine plus tetrahydrouridine; nor cytidine plus tetrahydrouridine added after 24 hr were able to reverse cell growth inhibition induced by 25 microM DUP 785. However, uridine and cytidine alone reversed growth inhibition. UTP and CTP pools in L1210 cells decreased to about 30-40% of control levels after 4 hr of drug exposure, while dTTP and dCTP pools decreased to about 30% of control levels. There were no significant changes in purine nucleotide pools. In WiDR cells, UTP and CTP pools decreased rapidly after drug exposure and were substantially depleted after 24 hr. Reculture of cells in drug-free medium resulted in a significant recovery of UTP and CTP levels only for cells exposed to DUP 785 for 1-24 hr. For cells exposed to the drug for 48 and 72 hr recovery of nucleotide pools was minimal. In L1210 cells, a 12-hr exposure to the drug caused an accumulation of cells in the early S-phase. In WiDR cells, there was a clear accumulation of cells in the S-phase of the cell cycle after 24 hr drug exposure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synergistic interaction of nitrogen dioxide and ozone on rat lungs: acute responses.

Rats were exposed for 6 hr per day to either ozone alone (0.2-0.8 ppm), nitrogen dioxide (NO2) alone (3.6-14.4 ppm), or to combinations of these two oxidant air pollutants. Their response was quantified by changes in the total protein content of lung lavage supernatants or by changes in the content of specific cell types in the lung lavage pellets. A concentration-dependent synergistic response was observed when rats were exposed to the combination of ozone and NO2. Apparent threshold concentrations for the observation of synergistic interaction between ozone and NO2 were assay specific, with epithelial cell content of lung lavage fluid being the most sensitive parameter evaluated, showing positive interaction (greater than additive response) at the lowest concentrations tested. Concurrent exposure to ozone and NO2 was necessary to elicit greater than additive responses; no such interactions were seen upon sequential exposure to ozone or NO2 in either order of presentation. Based upon apparent disappearance rates of ozone in the chambers during exposure of rats to ozone and NO2, we modelled the predicted outcomes based upon the assumption that the two oxidant gases were reacting to form nitrogen pentoxide (N2O5) in the chambers. Agreement between predicted concentrations of ozone and NO2 and those actually observed was excellent. Based upon such modelling estimates and our acute toxicological data, we conclude that synergistic toxicologic interactions between ozone and NO2 are found only at concentrations very much higher than would be encountered in environmental or occupational settings. It remains to be determined whether there are any chronic toxicological responses to exposure to combinations of ozone and NO2 at concentrations below the thresholds for observing acute responses.     

Enhanced antitumor activity of combinations of free and HPMA copolymer-bound drugs

The synergism in anticancer effect toward human renal carcinoma A498 cells by binary combinations of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound anticancer drugs, SOS thiophene (SOS), doxorubicin (DOX), and mesochlorin e6 monoethylenediamine (Mce6), was evaluated. The combination index (CI) analysis was used to quantify the synergism, antagonism, and additive effects. Both free drugs and HPMA copolymer conjugates, when used as single agents or in combination, exhibited cytotoxic activities against A498 cells, as determined using a modified MTT assay. As single agents, SOS and P-GFLG-SOS (HPMA copolymer conjugates containing SOS bound via glycylphenylalanylleucylglycine [GFLG] spacer) were significantly more effective than the other agents evaluated. The synergistic effects ranked in the order SOS+DOX>P-GFLG-DOX+P-GFLG-Mce6 approximately DOX+Mce6>P-GFLG-SOS+P-GFLG-DOX approximately SOS+Mce6>P-GFLG-SOS+P-GFLG-Mce6. The combination of SOS+DOX proved to be synergistic over all cell growth inhibition levels. All other combinations exhibited synergism in a wide range of drug effect levels. The SOS+Mce6 and P-GFLG-SOS+P-GFLG-Mce6 combinations displayed synergism up to drug affected fraction (fa) values of about 0.8 and reached slight antagonism and nearly additivity at fa=0.95, respectively. However, all other combinations were synergistic up to fa<0.9 and were additive at higher fa values. The observations that most combinations produced synergistic effects will be important for clinical translation.     

Inhibition of repair of bleomycin-induced DNA strand breaks by 2'-deoxycoformycin and its effect on antitumor activity in L5178Y lymphoblasts.

We have observed previously that treatment of plateau-phase L5178Y murine lymphoblasts in vitro with 2'-deoxycoformycin plus deoxyadenosine (dCF/dAdo) can inhibit the repair of X-irradiation-induced DNA single-strand breaks (SSB) in these cells and that this effect is associated with synergistic cell kill. In this study we examined the effect of a combination treatment of plateau-phase L5178Y cells with bleomycin (BLM) plus dCF/dAdo. Incubation of BLM-treated cells with dCF/dAdo resulted in significant inhibition of the repair of BLM-induced DNA SSB. However, an additive, but not a synergistic, increase in cell kill was observed when cells were treated with a combination of BLM plus dCF/dAdo.     

胡椒碱与棉酚联用对前列腺癌DU145细胞生长抑制的协同作用

目的研究胡椒碱与棉酚联合用药对激素非依赖型前列腺癌DU145细胞生长的抑制作用及其机制。方法 MTS比色法检测细胞活性;流式细胞术分析细胞周期;免疫印迹法检测细胞周期相关蛋白以及CYP3A4的表达。结果胡椒碱本身的细胞毒性较小,但与棉酚联用时,表现出协同作用。棉酚对细胞的半数抑制浓度IC50由单用时的21.00μmol·L-1下降到联合用药时的8.07μmol·L-1;两药相互作用指数CDI<1。胡椒碱与棉酚联合用药,可引起细胞G0/G1期阻滞和亚二倍体峰(凋亡峰)增加;同时上调细胞周期抑制蛋白p21Cip1的表达,并下调Cyclin D1、Cyclin A、CyclinB1以及药物代谢酶CYP3A4的表达。结论胡椒碱与棉酚联用,可能是通过诱导细胞周期阻滞,增强凋亡以及抑制CYP3A4药物代谢酶活性,发挥其协同抗前列腺癌作用。     

Synergistic and antagonistic interactions of methotrexate and 1-β-D-arabinofuranosylcytosine in hepatoma cells: The modulating effect of purines

A 4-hr pretreatment with methotrexate antagonized the cytotoxic effect of subsequent arabinosylcytosine treatment in rat hepatoma cells of lines N₁S₁ and 3924A, but in the hepatoma line 8999R and the fibroblast line BF5, MTX pretreatment was synergistic with the arabinosylcytosine treatment. Measurement of cellular deoxyribonucleoside triphosphate concentrations showed that in those lines in which antagonism was found the dCTP increased, whereas in the lines where the drugs were synergistic the dCTP pool was decreased. Conversely, dATP levels were high when the drugs were synergistic and low when antagonism was obtained. Although methotrexate pretreatment antagonized arabinosylcytosine in N₁S₁ and 3924A cells, pretreatment of these cells with the combination of methotrexate plus a purine (either hypoxanthine or 2′-deoxyadenosine) resulted in synergism with arabinosylcytosine. Deoxynucleotide pool measurements showed that methotrexate in combination with either hypoxanthine or 2′-deoxyadenosine increased dATP and decreased dCTP in the N₁S₁ and 3924A hepatoma cells. In N₁S₁ cells, pretreatment with 2′-deoxyadenosine alone for 4 hr was synergistic with arabinosylcytosine. It was concluded that elevated dATP pools enhanced arabinosylcytosine cytotoxicity by depleting the dCTP pool, through feedback inhibition of ribonucleotide reductase, thus causing greater inhibition of DNA biosynthesis and greater incorporation of AraCTP into nucleic acid. Methotrexate was synergistic in those cell lines where dATP accumulated and dCTP was decreased, but when methotrexate had a potent antipurine effect dCTP pools increased and arabinosylcytosine was antagonized. The synergistic interaction was more marked at cytocidal drug concentrations than it was at growth-inhibitory doses.     

Assessing combinations of cytotoxic agents using leukemia cell lines

The mainstay of clinical anti-neoplastic chemotherapy is multi-agent combinations, most of which were developed empirically. To speed research and decrease costs, there is increasing interest in moving new drugs into clinical trials in potentially active combinations based upon pre-clinical testing data. Because testing drug combinations in animals is expensive and complex, defining drug combinations initially in cell culture assays is essential. For in vitro testing we employ a panel of well-characterized cell lines and DIMSCAN, a semi-automatic fluorescence-based digital image microscopy system that quantifies relative total (using a DNA stain) or viable [using fluorescein diacetate (FDA)] cell numbers in tissue culture multi-well-plates ranging from 6 to 384 wells per plate. DIMSCAN is a rapid and efficient tool for conducting in vitro cytotoxicity assays across a 4 log dynamic range. The specificity of detecting viable cells with FDA is achieved by use of digital image processing and chemical quenching of fluorescence in non-viable cells with eosin Y. Cytotoxicity measured by DIMSCAN was found to be comparable to manual trypan blue dye exclusion counts or colony formation in soft agar, but with a significantly wider dynamic range, that enables drug combination studies used to detect synergistic or antagonistic interactions in cell lines from both solid tumors and leukemias. While different mathematical models have been proposed for evaluating drug interactions, which can be classified as synergistic (combinations demonstrating greater than the additive activity expected from each agent alone), additive, or antagonistic (drugs showing less activity in combination than expected from the sum of each agent alone), we generally find the Combination Index method (as developed by Chou, et al.) to be the most suitable for evaluating of drug interactions in cell culture assays.     

Development of a combination drug-eluting bead: towards enhanced efficacy for locoregional tumour therapies

Drug-eluting beads (DEBs) are becoming a mainstay locoregional therapy for hepatic malignancies but are currently loaded with single drugs alone. Here, we wished to prepare DEB containing different drug combinations, to screen their efficacy using an in-vitro cell culture assay and to include any promising combinations that demonstrate additive efficacy in an in-vivo model of locoregional tumour treatment. A modified in-vitro assay was used based upon the use of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) with either HepG2 liver cancer or PSN1 pancreatic cancer cell lines. The comparative cytotoxicity of DEB combinations prepared containing doxorubicin, irinotecan, topotecan and rapamycin was evaluated. Those combinations that demonstrated an additive cytotoxicity effect were investigated in vivo using a nude mouse xenograft model of pancreatic cancer. Although many of the DEB combinations showed either no effect or a slight antagonistic effect, the combination of doxorubicin and rapamycin DEBs demonstrated synergistic activity. On the basis of these findings, a method was developed to prepare a doxorubicin/rapamycin dual-loaded DEB, which was shown to possess the same drug-loading capacities, drug elution properties and HepG2 cell cytotoxicity synergy as the single drug-loaded DEB combination. Evaluation of this dual-loaded combination DEB versus the respective single drug-loaded DEBs in a mouse xenograft model of pancreatic cancer showed an equivalent tumour volume reduction as the doxorubicin DEB, but with less toxicity than the rapamycin DEB. The doxorubicin/rapamycin combination DEB offers great potential for enhanced efficacy in the locoregional treatment of malignant tumours.     

Synergistic effect of combining paeonol and cisplatin on apoptotic induction of human hepatoma cell lines

Aim： To investigate whether paeonol （Pae） has synergistic effects with cisplatin （CDDP） on the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and SMMC-7721. Methods： The cytotoxic effect of drugs was measured by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-tetrazolium bromide assay. The coefficient of drug interaction was used to analyze the nature of drug interactions. Morphological changes were observed by acridine orange fluorescence staining. Cell cycle and the apoptosis rate were detected by flow cytometry. Bcl-2 and Bax expression were assayed by immunohistochemical staining. Results： Pae or CDDP had antiproliferative effect on the 2 cell lines in a dose-dependent manner, with different sensitivities to drugs. More interestingly, a synergistic inhibitory effect on the viability of the 2 cell lines was observed after treatment with a combination of Pae （15.63, 31.25, and 62.5 mg/L） with various concentrations of CDDP. Further study showed typical morphological changes of apoptosis if the cells were exposed to the two agents for 24 h. The apoptotic rate of the cells with combination treatment was significantly higher than that of cells treated with Pae or CDDP alone. The expression of Bcl-2 decreased and that of Bax increased in the treated groups, especially in the combination group, with the ratio of Bcl-2/Bax decreasing correspondingly. Additionally, a combination of Pae with CDDP resulted in a stronger S phase arrest, compared with Pae or CDDP alone. Conclusion： Pae, in combination with CDDP, had a significantly synergistic growth-inhibitory and apoptosis-inducing effect on the 2 human hepatoma cell lines, which may be useful in hepatoma treatment.     

Melphalan resistance and photoaffinity labelling of P-glycoprotein in multidrug-resistant Chinese hamster ovary cells: reversal of resistance by cyclosporin A and hyperthermia.

The multidrug resistance phenotype is often associated with overexpression of P-glycoprotein, an energy-dependent efflux pump responsible for decreased intracellular accumulation of chemotherapeutic agents. The role of P-glycoprotein in the mechanism of cross-resistance to melphalan in multidrug-resistant Chinese hamster ovary cells (CH(R)C5) was investigated by photoaffinity labelling of P-glycoprotein using [3H]azidopine. We investigated whether the chemosensitiser cyclosporin A and hyperthermia, either used alone or combined, could reverse melphalan resistance and alter transport processes for [14C]melphalan in CH(R)C5 cells. Melphalan inhibited azidopine photolabelling of P-glycoprotein, implicating drug efflux mediated by P-glycoprotein in the mechanism of melphalan resistance in CH(R)C5 cells. Azidopine photolabelling also was inhibited by the chemosensitiser cyclosporin A, which binds to P-glycoprotein. Cyclosporin A alone reversed melphalan resistance in CH(R)C5 cells, but had no effect in drug-sensitive AuxB1 cells. Hyperthermia (40-45 degrees) alone increased melphalan cytotoxicity in both cell lines. When hyperthermia was combined with cyclosporin A, a large increase in melphalan cytotoxicity occurred, but only in CH(R)C5 cells. This effect increased with temperature and exposure time. Sensitisation to melphalan cytotoxicity by heat and cyclosporin A in CH(R)C5 cells appeared to be explained by altered drug transport processes. Lower accumulation of melphalan occurred in CH(R)C5 cells than in drug-sensitive cells. At 37 degrees, cyclosporin A increased drug accumulation in CH(R)C5 cells, but not in AuxB1 cells, by slowing drug efflux from cells. Heat alone increased both melphalan uptake and drug efflux for both cell lines. Our findings suggest that the combination of cyclosporin A and hyperthermia could be very useful in overcoming melphalan resistance by increasing intracellular drug accumulation in multidrug-resistant cells.     

The combination of artesunate and carboplatin exerts a synergistic anti-tumour effect on non-small cell lung cancer

Carboplatin (CBP) is a widely used targeted anticancer therapeutic drug; however, multi-drug resistance induced by the accumulation of CBP eventually causes diseases progression. The anti-malarial drug artesunate (ART) also exerts anticancer effects in various cancers; however, the combined effect of ART and CBP on non-small cell lung cancer (NSCLC) remains unclear. In the present study, the NSCLC cell line A549 was pretreated with various concentrations of CBP, ART and gemcitabine (GEM). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were conducted to detect cell viability. Cell apoptosis was evaluated by both flow cytometry and TUNEL apoptotic assay. The expression profiles of cell cycle-related proteins and apoptotic proteins were determined by western blot. Cell clone numbers were visualized using crystal violet staining. Here, we found that both CBP and ART suppressed cell viability, and promoted cell apoptosis, and the combined application of ART and CBP at a lower concentration exhibited synergistic effects. Specifically, the combination of ART and CBP at a lower concentration suppressed cell clone numbers, promoted cell cycle arrest at the G₂/M phase, and induced the expression of the cell cycle and apoptosis-related proteins BAX, p21, p53, and Caspase-3, while decreasing Bcl-2 and Cyclin B1 expression. Based on these results, we concluded that combined application of ART and CBP exerts synergistic anti-tumour effects on NSCLC by enhancing cell apoptosis in a mitochondria-dependent manner.     

Effect of a combination of S-1 and gemcitabine on cell cycle regulation in pancreatic cancer cell lines

In a previous study, we showed that a combination of an oral fluoropyrimidine anticancer agent (S-1) and gemcitabine (GEM) had synergistic effects on cell growth and cell cycle arrest in the pancreatic cancer cell line MIA PaCa-2. Therefore, we conducted further mechanistic studies using the pancreatic cancer cell lines MIA PaCa-2 and SUIT-2. The combined effect of S-1 and GEM in SUIT-2 cells was evaluated using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the effects of S-1, GEM and S-1 plus GEM on cell cycle regulation were assessed using flow cytometry. We also examined the expression of several cell cycle regulatory proteins in both MIA PaCa-2 and SUIT-2 cells by western blotting. Classical isobolographic analysis of the MTT assay results showed that the combination of S-1 and GEM had a synergistic effect in SUIT-2 cells, and flow cytometric analysis of the cell cycle showed that the combination of S-1 plus GEM induced S-phase arrest to a greater degree than did either S-1 or GEM alone. Also, the combination of S-1 and GEM resulted in the downregulation of cyclin D1 expression and upregulation of cyclin A, p21 and p27 expression levels. Treatment of MIA PaCa-2 and SUIT-2 cells with a combination of both drugs also led to the increased phosphorylation of checkpoint kinase 1. Combined treatment with S-1 and GEM resulted in more prolonged S-phase arrest than with either treatment alone. This difference is shown to be potentially due to the higher levels of phosphorylated checkpoint kinase 1 in pancreatic cancer cell lines treated with the two agents.     

Relationship of deoxynucleotide changes to inhibition of DNA synthesis induced by the antiretroviral agent 3'-azido-3'-deoxythymidine and release of its monophosphate by human lymphoid cells (CCRF-CEM)

The metabolism and cytostatic effects of 3'-azido-3'-deoxythymidine (AZT), one of the most effective agents being used in the treatment of acquired immunodeficiency syndrome, were investigated in the CCRF-CEM line of human T lymphoid cells. The concentration of drug required to inhibit cell growth by 50% (CD50) was significantly lower when the cells were exposed to AZT for 24 hr (CD50 = 50 microM), as compared with 48 or 96 hr (CD50 = 225 and greater than 300 microM, respectively). AZT at 25 microM blocked the progression of cells in S phase for about 12 hr, but this effect was reversed by 24 hr, despite the continued presence of drug in the medium. At this drug concentration, the level of dTTP decreased to about 75% of the control level by 4 hr but rebounded to 30% above normal by 8 hr of drug exposure. dGTP and dATP pool sizes were unchanged, whereas the dCTP pool increased 5-fold. The time course of these biochemical changes indicated that the onset of S phase arrest was not directly related to the decrease in deoxynucleoside triphosphate pools. CCRF-CEM cells incubated with 25 microM AZT accumulated about 0.9 mM 5'-monophosphate (AZTMP) after 4 hr whereas levels of the 5'-di- and 5'-triphosphates (AZTDP and AZTTP) plateaued at about 2 and 5 microM, respectively. After this period, there was a rapid decrease in AZTMP accumulation, to one third its initial level by 24 hr, whereas AZTDP and AZTTP pools decreased to only about 70%. The loss in AZT nucleotide formation with time of drug exposure was associated with a concomitant accumulation of AZTMP in the medium. Cellular excretion of AZTMP was not associated with any detectable cell lysis or leakage of other cellular metabolites. The ability of CCRF-CEM cells to excrete AZTMP may be an important factor limiting the biochemical and biological effects of the drug.     

Isobolographic analysis of the analgesic interactions between ketamine and tramadol

Owing to different mechanisms of analgesia, we hypothesized that the combination of ketamine and tramadol could produce synergistic or additive antinociceptive effects. Swiss albino mice were administered intraperitoneally with ketamine, tramadol, a combination of ketamine and tramadol, or saline, and the resulting antinociceptive effects were tested in the mouse tail-flick and formalin tests. The potencies of the two drugs alone or in combination were obtained by fitting data to the Sigmoid Emax equation. Isobolographic analysis was performed to evaluate the interaction. CNS depression was also monitored. Results showed that tramadol exhibited apparent dose-dependent effects in the tail-flick test, and in phase 1 and phase 2 of the formalin test. Ketamine dose-dependently inhibited the phase 2 responses, but failed to modify the phase 1 and tail-flick responses. Combination of tramadol and ketamine produced significant synergistic interactions only in phase 2 of the formalin test (P < 0.05). The synergistic combinations also displayed less CNS depression than when an equianalgesic dose of ketamine was administered alone. We conclude that in the acute thermal or chemical pain model, ketamine is not effective and the net effect of ketamine and tramadol in combination was simply additive after systemic administration. However, the coadministration produced synergistic antinociception in the chemical-induced persistent pain model.     

Interactions between depressants (alcohol-type) and stimulants (amphetamine-type)

The rotarod disruption in rats by 1.5 g/kg of ethanol was prolonged by combining the depressant with 2 or 8 mg/kg d-amphetamine, but not after combinations with 4 or 6 mg/kg of the stimulant. The combination with 8 mg/kg d-amphetamine also induced a prolonged coma and lethality. Cocaine or methylphenidate in combination form with ethanol also showed prolonged disruption of rotarod performance, but severe depression and lethality were not observed at any dose combination. d-Amphetamine in combination with pentobarbital or diazepam also increased the duration of rotarod impairment. Amphetamine plus methaqualone did not prolong rotarod disruption, but rather showed a trend toward antagonism. These combinations of 8 mg/kg d-amphetamine with depressants other than alcohol did not cause prolonged coma and lethality. Lower doses of ethanol (0.25 and 0.5 g/kg) plus 8 mg/kg d-amphetamine induced a delayed impairment of rotarod performance in rats as well as a comatose state and lethality. Mice showed a similar trend for these interactions between alcohol and d-amphetamine but the influence was much less predictable. Analysis of alcohol levels in rat serum and brain indicated little effect of d-amphetamine on the rate of elimination of ethanol. On the other hand, 1.5 g/kg of ethanol prolonged the d-amphetamine decay from brain and serum. This latter interaction was not observed in rats treated with 8 mg/kg d-amphetamine plus 0.5 g/kg ethanol. Mice treated with 8 mg/kg d-amphetamine plus 2.25 g/kg alcohol showed little trend for changes in rate of elimination of either drug. The behavioral effects of the combination of d-amphetamine and ethanol cannot be explained adequately on the basis of altered pharmacokinetics of either drug.     

In vitro activity of the novel cytotoxic agent CHS 828 in childhood acute leukemia

CHS 828, a pyridyl cyanoguanidine, is a new drug candidate now in phase I/II trials, that has shown promising anticancer activity in experimental tumor models and primary cultures of cancer cells from patients. In this study the fluorometric microculture cytotoxicity assay was used for evaluation of CHS 828 in primary cell cultures from children with acute leukemia. The activity of and interaction with the standard drugs, doxorubicin, melphalan, etoposide and cytosine arabinoside (Ara-C), were also assessed. Samples from 65 patients, 42 with acute lymphocytic leukemia (ALL) and 23 with acute myelocytic leukemia (AML) were tested with 72-h continuous drug exposure. There was 50% cell kill at very low CHS 828 concentrations; median IC50 was 0.01 microM in ALL and 0.03 in AML samples (NS) with large interindividual variability in both groups. ALL samples were significantly more sensitive than AML samples to melphalan, doxorubicin and etoposide, but not to Ara-C. In AML samples, combinations between CHS 828 and each of the four standard drugs resulted in significantly lower cell survival than either drug alone. This was also observed in ALL samples, except for Ara-C. Using the additive interaction model, CHS 828 showed a synergistic effect with melphalan in 67%, doxorubicin in 47%, etoposide in 38% and Ara-C in 14% of AML samples. In most ALL samples subadditive effects were found. Further exploration of CHS 828 in childhood leukemia is warranted, especially in AML.