Search

Trimidox (3,4,5-trihydroxybenzamidoxime), a newly synthesized analog of didox (N,3,4-trihydroxybenzamide) reduced the activity of ribonucleotide reductase (EC 1.17.4.1) in extracts of L1210 cells by 50% (50% growth-inhibitory concentration, IC50) at 5 microM, whereas hydroxyurea, the only ribonucleotide reductase inhibitor in clinical use, exhibited an IC50 of 500 microM. Ribonucleotide reductase activity was also measured in situ by incubating L1210 cells for 24 h with trimidox at 7.5 microM, a concentration that inhibits cell proliferation by 50% (IC50) or at 100 microM for 2 h; these concentrations resulted in a decrease in enzyme activity to 22% and 50% of the control value, respectively. Trimidox and hydroxyurea were cytotoxic to L1210 cells with IC50 values of 7.5 and 50 microM, respectively. Versus ribonucleotide reductase, trimidox and hydroxyurea yielded IC50 values of 12 and 87 microM, respectively. A dose-dependent increase in life span was observed in mice bearing intraperitoneally transplanted L1210 tumors. Trimidox treatment (200 mg/kg; q1dx9) significantly increased the life span of mice bearing L1210 leukemia (by 82% in male mice and 112% in female mice). The anti-tumor activity appeared more pronounced in female mice than in male mice. Viewed in concert, these findings suggest that trimidox is a new and potent inhibitor of ribonucleotide reductase and that it is a promising candidate for the chemotherapy of cancer in humans. [ABSTRACT FROM AUTHOR]

Background: Amidox (AX) is a novel anticancer drug currently undergoing pre-clinical studies. Though AX shows activity against various tumor cells, its biotransformation is still unknown., Material and Methods: Livers of male Wistar and mutant TR- rats were perfused with AX in a single pass system and bile and effluent perfusate analyzed by HPLC for AX and its metabolites., Results: In bile, seven biotransformation products but not AX could be quantified though their total excretion into bile was low. However, the cumulative efflux of AX metabolites into the effluent perfusate was high with about 55% of AX applied to the liver during perfusion. Biliary excretion of AX metabolites in TR- rats was substantially reduced compared to Wistar rats, indicating that metabolites are substrates of the canalicular multispecific organic anion transporter cmoat., Conclusion: Metabolism of AX in rat liver is high and has to be considered during cancer therapy of patients.

BACKGROUND-Prostate-specific antigen (PSA), acid phosphatase (AP), and prostatic acid phosphatase (PAP) are serum markers for adenocarcinoma of the prostate gland. Previous studies indicated that prostatic ischemia may also produce elevations of PSA. Cardiopulmonary resuscitation (CPR) is frequently associated with profound tissue hypoperfusion. The present study investigated whether PSA, AP, and PAP are influenced by prolonged CPR. METHODS AND RESULTS-PSA, AP, and PAP were assessed immediately, 12 hours, 24 hours, 2 days, 3 days, 5 days, and 7 days after prolonged CPR (>5 minutes) in 14 male and 5 female patients. No changes were noted in women. In men, serum levels increased significantly after CPR and gradually decreased to near baseline values after 7 days. PSA, AP, and PAP values above the normal range were observed in 63%, 71%, and 64% of all patients, respectively. Compared with survivors, nonsurvivors exhibited higher peak serum levels of PSA (98.6+/-14.3 versus 1.1+/-2.2 mcg/L; P<0.03), AP (57.0+/-71 versus 8.6+/-8.8 U/L; P<0.05), and PAP (47.0+/-62 versus 5.7+/-8.0 U/L; P=NS). Patients with poor neurological outcome exhibited higher peak serum levels of PSA (86.4+/-135.5 versus 12.0+/-23.8 mcg/L; P<0.05), AP (50.9+/-68.1 versus 8.7+/-9.6 U/L; P=NS), and PAP (41.6+/-59.5 versus 5.8+/-8.8 U/L; P=NS) than patients with good neurological outcome. CONCLUSIONS-Prolonged CPR is frequently associated with increases of PSA, AP, and PAP serum levels. Therefore, PSA cannot be used for diagnosis of adenocarcinoma of the prostate during the first weeks after CPR. Further evaluation of these parameters as additional prognostic markers after CPR is warranted.

Ribonucleotide reductase is the rate limiting enzyme of deoxynucleoside triphosphate synthesis and is considered to be an excellent target of cancer chemotherapy. Trimidox, a newly synthesized compound, inhibits this enzyme and has in vitro and in vivo antitumour activity. As trimidox was able to upregulate the expression of the transferrin receptor in HL-60 human promyelocytic leukaemia cells, we have now investigated the capability of trimidox to interfere with iron metabolism. We show by photometric and polarographic methods that trimidox is able for form an iron complex. However, its cytotoxic action cannot be circumvented by addition of iron-saturated transferrin or iron-ammonium citrate, indicating that the iron complexing capacity is not responsible for the mechanism of action of this compound. When HL-60, K562 or L1210 leukaemia cells were incubated with the trimidox-iron complex itself, we could observe increases of the 50% growth inhibitory capacity of the complex in comparison with trimidox alone. We conclude that trimidox is able to form an iron complex, but in contrast to other agents, the anticancer activity cannot be contributed to this effect alone. Further studies will have to elucidate the molecular mechanism of action of this new and promising anticancer agent.

The success of chemotherapy of colon tumours is currently limited. We have therefore used the human colon tumour cell line HT-29 to evaluate the cytotoxic effects of various drug combinations. Trimidox (3,4,5-trihydroxybenzamidoxime), a recently patented inhibitor of ribonucleotide reductase was combined with cytosinearabinoside (Ara-C) or 2',2'-difluorodeoxycytidine (DFDC) in order to inhibit both pyrimidine de novo and salvage pathways. Synergistic cytotoxic effects were observed. When HT-29 cells were sequentially treated with trimidox (20 microM for 24 h) and Ara-C (2 microM for 2 h), colony numbers decreased to 71% of the value calculated for additive cytotoxicity. When cells were simultaneously treated with trimidox (10 microM and 15 microM) and DFDC (0.2 nM), synergistic inhibition of colony formation was likewise noted (colony numbers decreased to values as low as 73% or 71% of the values calculated for additive cytotoxicity). On the other hand, we combined tiazofurin, an inhibitor of the guanylate de novo pathway, with allopurinol, which inhibits the guanylate salvage pathway by increasing intracellular hypoxanthine concentrations, leading to inhibition of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Synergistic cytotoxic effects were observed under these conditions too. When cells were treated with 10 microM tiazofurin and 400 microM or 800 microM allopurinol the number of colonies decreased to 69% and 27%, respectively, of the values calculated for additive effects. Our data suggest these drug combinations to be promising options in the treatment of human colon cancer.

Cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3) recruit quiescent cells into the cell cycle and sensitize these cells towards cell cycle specific chemotherapeutic agents. We examined the in vitro effects of GM-CSF on HL-60 cells and tested its modulatory influence on biochemical and cytotoxic effects seen with tiazofurin, a potent and specific inhibitor of IMP dehydrogenase. Incubation of HL-60 cells with 500 U/ml GM-CSF for 4 d enhanced cell proliferation, which was accompanied by a significant increase in IMP dehydrogenase activity (from 2.22 in control cells to 3.70 nmol/mg/h in cells pretreated with GM-CSF). When HL-60 cells were incubated with 100 microM tiazofurin for 2 h, intracellular GTP decreased to 46% of untreated control cells. In HL-60 cells pretreated with GM-CSF, GTP pools decreased to 38% of control after incubation with tiazofurin which is 69% of the predicted value for additive effect. The MTT chemosensitivity assay yielded significantly decreased IC50 values for tiazofurin in HL-60 cells, preincubated with GM-CSF (IC50 decreased from 13 microM to 10 microM). Therefore our results suggest that combination therapy with GM-CSF and tiazofurin may be beneficial for the treatment of refractory leukaemia patients.

The influence of conjugation of doxorubicin to holotransferrin on the receptor-mediated endocytosis of and on the iron uptake from transferrin was studied using K562 cells. 125I-labelled transferrin and doxorubicin-transferrin conjugates were used in the binding, dissociation, and ligand-exchange experiments at 0 degree C, and 59Fe,125I-labelled (double-labelled) ligands were used in the endocytosis, iron uptake, and recycling experiments at 37 degrees C. The binding affinity of conjugates was about half of that of transferrin. Binding of 125I-labelled ligands was blocked by both unlabelled ligands to the same degree, however, it was not blocked at all by an 8000-fold excess of doxorubicin. After saturation bindings, slightly more 125I-labelled conjugates dissociated from the surface of cells than transferrin. Exchange of 125I-labelled ligands for unlabelled ligands resulted in different EC50 values (defined as the concentration of unlabelled ligand at which half as much radioligand is exchanged for unlabelled ligand as would be exchanged at infinitely high concentration of unlabelled ligand under similar assay conditions). While transferrin exchanged transferrin with an EC50 value close to the binding affinity, conjugates exchanged conjugates with much lower efficiency. The heterolog exchange experiments yielded EC50 values inbetween the two extrema. For studying iron uptake, K562 cells were loaded with the double-labelled ligands either at 37 degrees C (endosome-loading only) or at 0 degree C (surface-loading only). Results obtained for the endocytosis of, the iron uptake from, and the recycling of double-labelled ligands indicate that (a) the rate of iron uptake is smaller from conjugates than from transferrin, (b) there are at least two parallel recycling processes for both ligand.receptor complexes, and (c) each time constant characterizing the different steps of iron uptake via receptor-mediated endocytosis is smaller for conjugates than for transferrin (or, the half times characterizing the different steps are higher for conjugates than for transferrin). Endocytosis and iron uptake were unaffected by free doxorubicin (12.5 microM) or colchicine (1 mM). Benzyl alcohol (30 mM) slowed down the rate of both endocytosis and iron uptake, while dithiothreitol (5 mM) decreased the rate of iron uptake and increased the rate of endocytosis. N-Ethylmaleimide (1 mM) completely stopped both endocytosis and iron uptake. The results suggest that the binding of conjugates to the surface of cells is governed by the binding of the transferrin part of conjugates to the transferrin receptor. However, conjugation of doxorubicin to transferrin seems to influence all properties of transferrin.(ABSTRACT TRUNCATED AT 400 WORDS)

The concentration of atrial natriuretic peptide was measured in order to evaluate its importance in patients suffering from a variety of cardiac diseases. There was a correlation between plasma concentrations of atrial natriuretic peptide and its "second messenger" cyclic guanosine monophosphate (cGMP) in all of the cases examined. We investigated the relationship between atrial natriuretic peptide and cGMP plasma concentrations during rest and exercise in comparison with the scintigraphically assessed left- and right-ventricular ejection fraction in patients with chronic heart disease (n = 20), and after orthotopic heart transplantation (n = 16); plasma concentrations were also measured in healthy controls (n = 14). Atrial natriuretic peptide and cGMP concentrations showed a similar correlation during rest and exercise with r = 0.74 and r = 0.81, respectively. With the exception of patients after heart transplantation, a significant negative correlation was seen between the left ventricular ejection fraction and atrial natriuretic peptide or cGMP plasma concentrations during rest conditions (r = 0.76 or 0.58, respectively). No correlation was apparent between plasma concentrations of atrial natriuretic peptide or cGMP and the left- or right ventricular ejection fraction during exercise. The concentrations of atrial natriuretic peptide and cGMP in plasma differed significantly between healthy controls and patients during rest and exercise. It is noteworthy that atrial natriuretic peptide and cGMP concentrations were markedly higher in patients after heart transplantation than in patients suffering from chronic heart disease. Our results indicate that plasma atrial natriuretic peptide and cGMP concentrations are sensitive markers of cardiac impairment.

Conjugates of adriamycin coupled to transferrin by glutaraldehyde are cytotoxic to human promyelocytic (HL-60) and erythroleukemic (K562) cells. Growth inhibition of adriamycin-sensitive cells, as evaluated by thymidine incorporation and the MTT-assay, was higher for conjugates than for free adriamycin. The cytotoxicity toward adriamycin-resistant K562 and HL-60 cells was 3-fold and more than 10-fold higher, respectively, for the transferrin-adriamycin conjugate than for the free drug. The effect of the conjugate was dependent on its adriamycin content, i.e., on its conjugation number.

Reperfusion of the ischemic myocardium is associated with the extension of already existing injury. Investigation of the reperfused myocardium under different conditions revealed ultrastructural changes as well as functional disturbances which were identified as free radical-mediated damage. Free radical scavengers and antioxidant agents have been shown to reduce the size of the infarct, in which a temporary regional ischemia was followed by reperfusion. Elaborately designed in vitro and animal models strongly suggest that reperfusion injury might be of clinical significance in operations involving extracorporeal circulation, as in coronary by-pass surgery. However, there is limited information on the relationship between reperfusion injury and subsequent clinical conditions. In our study, we determined lipid peroxide concentrations in timed serum samples of patients who had undergone coronary by-pass surgery. Preoperative lipid peroxide concentrations were 2.50 +/- 0.50 mumol/l. This value increased to 3.30 +/- 1.00 mumol/l after six hours and showed a second increase to 4.20 +/- 0.80 mumol/l 48 hours after the onset of reperfusion. These results suggest that postoperative increases in lipid peroxide concentrations can be of multifactorial origin and may be an indication of reperfusion injury to the myocardium. Further studies are under way to elucidate the relevance of this increase in different groups and clinical conditions.

The activity of IMP dehydrogenase (IMP DH), the rate-limiting enzyme of de novo GTP biosynthesis, was shown to be increased in cancer cells. Tiazofurin, an inhibitor of IMP dehydrogenase, proved to be an effective agent in the treatment of refractory granulocytic leukemia. To examine the cell cycle dependent alterations of GTP synthesis and sensitivities to tiazofurin, we measured IMP DH activities and GTP pools, as well as the effects of tiazofurin on cell cycle phase enriched HL-60 cells. We now show that IMP DH activities and GTP concentrations are increased in S-phase enriched fractions of HL-60 cells. Moreover, the depletion of GTP concentrations by tiazofurin is most effective in S-phase enriched HL-60 cells. These results may be utilized in cancer chemotherapy to combine tiazofurin with biologic response modifiers which recruit quiescent leukemic cells into the cell cycle.