Effects of the combined arginase and canavanine treatment on leukemic cells in vitro and in vivo.

It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. Administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. Thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities.

Measuring single-cell density.

We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g mL(-1). We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here, we demonstrate this with four examples: identifying Plasmodium falciparum malaria-infected erythrocytes in a culture, distinguishing transfused blood cells from a patient's own blood, identifying irreversibly sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single-cell density will provide valuable insights into cell state for a wide range of biological processes.

In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry.

Quantitation of circulating tumor cells (CTCs) constitutes an emerging tool for the diagnosis and staging of cancer, assessment of response to therapy, and evaluation of residual disease after surgery. Unfortunately, no existing technology has the sensitivity to measure the low numbers of tumor cells (<1 CTC per ml of whole blood) that characterize minimal levels of disease. We present a method, intravital flow cytometry, that noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves i.v. injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs. Studies in mice with metastatic tumors demonstrate that CTCs can be quantitated weeks before metastatic disease is detected by other means. Analysis of whole blood samples from cancer patients further establishes that human CTCs can be selectively labeled and quantitated when present at approximately 2 CTCs per ml, opening opportunities for earlier assessment of metastatic disease.

Dietary docosahexaenoic acid levels influence the outcome of arabinosylcytosine chemotherapy in L1210 leukemic mice.

The purpose of this study was to investigate whether dietary supplementation with the n-3 fatty acid docosahexaenoic acid (DHA) in combination with arabinosylcytosine (AraC) chemotherapy could prolong the life expectancy of mice bearing L1210 leukemia. The four control diets included rodent chow, a diet containing 5% of a blended oil mimicking the fatty acid composition of rodent chow, and diets containing 5% or 10% fat with safflower oil as the main oil source. The two DHA-supplemented diets provided 1.5% or 3.5% DHA and 5% or 10% total fat, respectively. After tumor cell inoculation, mice were treated with AraC for 10 days. Mice fed the 5% safflower oil diet (30.1 -/+ 4.1 days), but not those fed the 10% safflower oil diet, survived longer than the chow-fed animals (22.1 -/+ 3.1 days, P = 0.05). The 1.5%-/+ DHA diet (average intake 1.8 g DHA/kg/day) was associated with a longer life span (33.3 -/+ 3.4 days, P < 0.01 vs. chow-fed) and no incidence of death due to drug toxicity. Further increasing DHA intake (4.5 g DHA/kg/day) resulted in shortened survival time (26.5 -/+ 2.0 days), increased circulating tumor cell burden, and lowered red blood cell concentrations. These data suggest that a modest level of dietary DHA or linoleic acid supplementation may improve the antineoplastic efficacy of AraC. However, overconsumption of DHA reverses the beneficial effect of DHA intake on drug sensitivity.

Effective chemo-immunotherapy of L1210 leukemia in vivo using interleukin-12 combined with doxorubicin but not with cyclophosphamide, paclitaxel or cisplatin.

It has been well established that chemo-immunotherapy using cytotoxic drugs and appropriate cytokines offers a new approach to increasing the therapeutic index in the treatment of neoplastic diseases. This study investigates the efficacy of combinations of interleukin-12 with cyclophosphamide, paclitaxel, cisplatin or doxorubicin in the murine L1210 leukemia model. Mice inoculated i.p. with 1 x 10(3) or 1 x 10(5) leukemia cells were treated with interleukin-12 and/or chemotherapeutics, and were observed daily for survival. Immunosuppression with X-irradiation or macrophage depletion with injections of silica were used to examine the dependence of the therapeutic effects on the efficiency of the immune system. Treatment with interleukin-12 or one of the studied chemotherapeutics given alone resulted in moderate antileukemic effects. Combination of interleukin-12 with cyclophosphamide or paclitaxel produced no augmentation of anti-leukemic effects in comparison with these agents given alone. Combination of interleukin-12 with cisplatin resulted in prolongation of the survival time; however, in the experiment with mice inoculated with 1 x 10(5) leukemia cells, no long-term survivors (>60 days) were observed; on the contrary, combination of interleukin-12 with doxorubicin resulted in 100% long-term survivors. This effect was completely abrogated either by X-irradiation of mice or by macrophage depletion. We also found that doxorubicin augments IL-12-stimulated production of interferon-gamma in vivo. Our observations demonstrating potentiation of the antileukemic effects of the IL-12 and doxorubicin combination suggest that the combined use of these 2 agents could be beneficial in leukemia therapy.

Pharmacokinetic studies in mice of two new thioxanthenones (183577 and 232759) that showed preferential solid tumor activity.

Two new thioxanthenones, 183577 and 232759, have rekindled interest in the development of representatives from this class of structures as useful anticancer agents. Although the mechanism of action is unknown, both compounds demonstrated a similar spectrum of solid tumor selectivity. 232759 was selected for clinical development because it showed no hepatotoxicity in preliminary studies, whereas 183577 showed hepatotoxicity but only at the maximum tolerated dose (MTD). The limiting toxicity for the clinical candidate was myelosuppression in preliminary studies. Plasma and tissue drug levels, as well as protein binding, were studied in mice using optimal administration times at the MTD for each drug (for 183577, this was a 4-h infusion at 1350 mg/m2 and for 232759, it was a 5-min injection at 240 mg/m2), as well as at one-half the MTD for the clinical candidate. The drugs were 96-100% bound by plasma proteins. The peak drug concentrations, half-life, and area under the concentration-time curve in plasma for 183577 were 3483 ng/ml, 465 min, and 2018 microgram/ml. min, respectively. The peak drug concentration, half-life, and area under the concentration-time curve in plasma for 232759 were 5257 ng/ml, 44 min, and 276 microgram/ml. min, respectively, at the MTD and 2810 ng/ml, 40 min, and 110 microgram/ml. min at one-half the MTD. In all instances of simultaneous measurements, drug concentrations were equal or higher in tissues than they were in plasma. Unlike the plasma and kidney concentrations of 183577, the liver concentrations did not show a declining trend over the 8-h observation period. Declines in plasma, liver, kidney, and tumor levels of 232759 were detected over the 8-h observation period. The sustained high 183577 concentration in liver is believed to be responsible for its prolonged half-life and hepatotoxicity. Evidence for metabolism of the parent drugs was based on the finding of additional peaks on the high-pressure liquid chromatography tracings. Future studies will focus on identification and antitumor studies of these presumed metabolites in hopes of a better understanding of the solid tumor activity profiles and toxic effects of these compounds.

Reversal by cytidine of cyclopentenyl cytosine-induced toxicity in mice without compromise of antitumor activity.

Among nine compounds surveyed, cytidine was found to be the most effective in reversing the antiproliferative effects of cyclopentenyl cytosine (CPEC) on human T-lymphoblasts (MOLT-4) in culture. Cytidine, at concentrations of 1-25 microM, enabled cells to maintain normal logarithmic growth when added up to 12 hr after exposure to a 200 nM concentration of the oncolytic nucleoside, CPEC. The most abundant CPEC metabolite, CPEC-5'-triphosphate, is a potent [K1 approximately 6 microM] inhibitor of CTP synthetase (EC 6.3.4.2). Accumulation of this inhibitor resulted in a depletion of CTP levels to 17% of their original cellular concentration. Exogenous cytidine reversed CPEC-induced cellular cytotoxicity by suppressing the formation of CPEC-5'-triphosphate by 70%, and by partially replenishing intracellular CTP to at least 60-70% of its original concentration. In vivo, cytidine (500 mg/kg) administered intraperitoneally 4 hr after each daily dose of CPEC (LD10-LD100) for 9 days reduced the toxicity and abolished the lethality of CPEC to non-tumored mice. Of greater practical importance is the finding that, under these experimental conditions, cytidine did not curtail the antineoplastic properties of CPEC in L1210 tumor-bearing mice. Moreover, the concentration range over which CPEC exhibited antineoplastic activity was extended with cytidine administration.

Identification of vesicle properties that enhance the antitumour activity of liposomal vincristine against murine L1210 leukemia.

The influence of vesicle lipid composition, size and drug-to-lipid ratio on the antitumour activity of liposomal vincristine was assessed in the murine L1210 ascitic leukemia model. A pH gradient-dependent entrapment procedure was used to encapsulate vincristine and allowed such vesicle properties to be independently varied. Free vincristine delivered i.v. at the maximum tolerated dose (2.0 mg/kg) resulted in a 27.8% increase in the life span (ILS) of mice inoculated i.p. with L1210 cells. Encapsulation of the drug in egg phosphatidylcholine/cholesterol vesicles did not significantly increase the antitumour efficacy of vincristine (ILS, 38.9%). In contrast, administration of vincristine entrapped in vesicles composed of distearoylphosphatidylcholine (DSPC)/cholesterol resulted in ILS values as high as 133%. This enhanced antitumour activity of the DSPC/cholesterol formulations was sensitive to the size of the liposomes; increasing the vesicle size from 100 nm to 1 micron decreased the ILS from 133.3% to 55.6% at a drug dose of 2.0 mg/kg. Decreasing the drug-to-lipid ratio from 0.1:1 to 0.05:1 (w/w) had negligible effects on the activity of liposomal vincristine; however, a further decrease in the drug-to-lipid ratio to 0.01:1 (w/w) decreased the antitumour potency at all drug doses studied. Pharmacology studies indicated that the antitumour activities of free and various liposomal forms of vincristine correlated well with the residence time of the drug in the circulation. These studies indicate that efforts to enhance the therapeutic activity of vincristine through liposome encapsulation must address not only the circulation lifetime of the vesicle systems but also the capacity of the liposomes to retain entrapped drug in vivo.

[Influence of benzyl penicillin on cytotoxicity and level of metabolic activity of cyclophosphamide in mice]. / Wplyw benzylopenicyliny na cytotoksycznosc i poziom aktywnych metabolitów cyklofosfamidu u myszy.

The influence of benzylpenicillin on cytotoxicity of cyclophosphamide (CF) against leukaemia L 1210 was studied. The level of CF metabolites in mouse serum was traced. The benzylpenicillin appeared to enhance cytotoxic activity of CF and leads to increase of active metabolites when CF applied in high doses (300 mg/kg).

Antitumor activity of FTC-092, a masked 5-trifluoromethyl-2'-deoxyuridine derivative.

1-(3-O-Benzyl-2-deoxy-beta-D-ribofuranosyl)-5-trifluoromethyl-2,4(1H,3)- pyrimidinedione (FTC-092), a fluorinated pyrimidine derivative, appeared to be effective against various transplantable tumors in mice following oral administration, and its activity was superior to that of several other antitumor fluorinated pyrimidines. The ED50 value for FTC-092 the dose effective in achieving 50% inhibition of tumor growth against the solid form of sarcoma 180 was 13.3 mg/kg daily, whereas those for 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd), the parent compound of FTC-092, for 1-(2-tetrahydrofuryl)-5-fluorouracil (Tegafur, FT), the prodrug of 5-fluorouracil (FUra), and for FUra were 64.1, 122, and 28 mg/kg daily, respectively. The therapeutic indices (LD10/ED50) of FTC-092, CF3dUrd, FT, and FUra were 4.39, 1.7, 1.35, and 1.65, respectively. FTC-092 itself is not an active agent. After it has been absorbed from the gastrointestinal tract, FTC-092 undergoes a gradual biotransformation, mainly via the action of liver microsomes, releasing CF3dUrd over a long period. The levels of CF3dUrd in the stomach and small intestine of mice after the oral administration of FTC-092 were undetectable, whereas those following the administration of CF3dUrd at the same dose were high for a period of several hours. In contrast, the CF3dUrd level generated in plasma after the administration of FTC-092 remained at a high level for a longer period than did that observed on the administration of CF3dUrd. The low levels of CF3dUrd measured in stomach and small-intestine tissues and the maintenance of CF3dUrd in blood over long periods after the administration of FTC-092 are features that favor the possible clinical application of FTC-092.

The influence of recombinant human tumor necrosis factor-alpha, alone and in combination with cyclophosphamide or methotrexate, on leukemia L1210 and normal hematopoiesis in mice.

We investigated the influence of rh-TNF administered as a single agent or in combination with CY or MTX on the survival time of mice inoculated with lymphoid leukemia L1210 and the effects of similar treatment on normal hematopoiesis in mice. The MST of rh-TNF--treated mice was longer than that of control animals. The longest survivals were observed in mice treated with 250 and 275 micrograms/kg of rh-TNF. Groups of mice receiving a combination of rh-TNF at doses of 225 or 250 micrograms/kg and MTX lived longer than animals treated with these agents separately. We observed the longest survival time of mice treated with combined administration of rh-TNF at a dose of 250 micrograms/kg and CY, but survival time was not significantly prolonged compared with mice receiving only CY. Additional studies were performed to examine the influence of rh-TNF administered as a single agent or in combination with toxic doses of CY or MTX on the number of granulocytes, lymphocytes, erythrocytes with hematocrit values and hemoglobin concentration, and platelets in peripheral blood, and the number of mononuclear cells as well as multipotential stem cells (CFU-GEMM) in bone marrow. Rh-TNF caused dose-dependent suppression of mononuclear cells and multipotential stem cells in bone marrow. The addition of MTX to rh-TNF caused no enhanced suppression of any of the above mentioned hematological parameters. In contrast, the addition of CY to rh-TNF suppressed erythrocytes and hematocrit values, as compared with rh-TNF alone.(ABSTRACT TRUNCATED AT 250 WORDS)

[Study of the DNA structure of peripheral blood leukocytes, proliferating lymphoid and tumor cells according to the rate of alkaline denaturation of DNA lysates]. / Izuchenie struktury DNK leikotsitov perifericheskoi krovi, proliferiruiushchikh limfoidnykh i opukholevykh kletok po skorosti shchelochnoi denaturatsii DNK lizatov.

Direct fluorometry was used to identify quantitative differences in the DNA structure of human peripheral blood leukocytes and proliferating lymphoid cells. The differences should be taken into account in the study of varied damaging actions.

[Quantitative evaluation of the surface membrane receptors of leukemic cells to blood serum transcobalamin-II]. / Kolichestvennaia otsenka retseptorov poverkhnostnoi membrany leikoznykh kletok k transkobalaminu-II syvorotki krovi.

Serum protein transcobalamin II (TC-II) is responsible for transport of cobalamins into mammalian cells. A method of quantitative estimation of plasma membrane receptors of hemopoietic cells to TC-II cobalamin complex is suggested. Analysis of mouse leukemia L1210 cells includes the saturation of radiolabelled ligand-receptor complex with papain. The number of receptors and 57CoCNCbl content in one cell is determined by differentiated radioactivity count of solubilized protein complexes and of cytoplasm.

Increased protective activity against acid precipitation of poly(U) in the serum of tumor-bearing mice.

Transplantation of leukemia L1210 cells into DBA/2 mice and of Ehrlich ascites tumor cells into BALB/C mice resulted in a significant increase of protective activity against acid precipitation of poly (U) in the serum. The increase was observed as early as one day after the tumor transplantation and seems to be connected with cancer growth, since inoculation of L1210 cells into BALB/C mice did not affect the protective activity, evidently as a result of their well established inability to cause cancer in this strain. Furthermore, no increase of activity was observed when bacteria were inoculated into mice, or when the latter were partially hepatectomized. The results suggest that the protective activity against acid precipitation of poly (U) could prove to be a tumor marker for the early detection of cancer growth.

Pharmacology of combination chemotherapy of cytosine arabinoside (ara-C) and uracil arabinoside (ara-U) or tetrahydrouridine (THU) against murine leukemia L1210/0 in tumor-bearing mice.

Deamination of cytosine arabinoside (ara-C) by cytidine deaminase (Cyt DA) is the main mode of the inactivation of this drug in vivo. Tetrahydrouridine (THU) and the deamination product, uracil arabinoside (ara-U) are potent inhibitors of Cyt DA. We investigated whether ara-U or THU pretreatments can protect ara-C from excessive deamination in tumor- (L1210) bearing mice. In order to determine this, plasma concentrations of ara-C, ara-U, and the intracellular levels of ara-CTP, the active anabolite of ara-C, were assayed. The control peak plasma levels of ara-C and ara-U were 3.3 and 0.78 mM and they were eliminated with a half life (t 1/2) of 1.26 and 1.43 hours, respectively. One hour pretreatment with a nontoxic dose of ara-U (single dose of 300 mg/kg intraperitoneally), resulted in increased ara-C levels by 5.9-fold, while ara-U increased 14.3 fold in comparison with controls. A 24-hour (every 8 hours) pretreatment with ara-U increased ara-C plasma levels by 3.0-fold and it was eliminated with a t 1/2 of 1.21 hours. One hour pretreatment with THU (single dose 25 mg/kg intraperitoneally) enhanced ara-C plasma levels by 5.3-fold. In control L1210/0 acid extracts, ara-CTP peaked at 2 hours and reached 2030 +/- 85 microM; ara-CTP was eliminated with a t 1/2 1.47 hours. The ara-CTP cellular concentrations after 1- and 24-hour pretreatments were 1875 +/- 534 and 2624 +/- 429 microM at 4 hours; the t 1/2 were 2.20 and 1.44 hours, respectively. The THU pretreatment resulted in a peak concentration of ara-CTP of 2208 +/- 366 microM at 2 hours and was eliminated with a t 1/2 of 2.54 hours. We concluded that all pretreatments increased both the peak plasma ara-C concentrations and the area under the plasma concentration-time curve (AUC). One hour ara-U pretreatment did not enhance the peak ara-CTP cellular concentration, but did extend the t 1/2. The 24-hour ara-U and the 1-hour THU pretreatments increased, to some extent, the cellular ara-CTP concentrations, but these differences were not statistically significant. THU pretreatment increased the time the peak occurred and the t 1/2 of ara-CTP. The area under the cellular ara-CTP concentration-time curve (AUC) in L1210 cells was either the same or increased by a small amount after the pretreatments.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzyme activity in mice with transplantable leukemia.

Activity of cobalt activated acylase, gamma-glutamyltransferase, leucylaminopeptidase and alanylaminopeptidase in serum and liver of mice with transplantable leukemias (L1210, L1210/ara-C, L1210/CH3-G, AKSL-4, plasmacytoma ADJ-PC-5) were determined. Adenosinotriphosphatase, 5'-nucleotidase and alkaline phosphatase were histochemically localized in lymphatic nodes and spleen. Among the investigated enzymes the rise in serum activity of cobalt activated acylase and gamma-glutamyltransferase was demonstrated. A substantial increase of leucylaminopeptidase and alanylaminopeptidase was shown in the liver. A decrease in the histochemical reactions of all the studied enzymes was observed.

[Non-phosphorylative transglycosylation in the serum and liver of mice with transplantable leukemia P388 and L1210]. / Niefosforylacyjna transglikozylacja w surowicy i w watrobie myszy z przeszczepialna bialaczka limfatyczna P 388 i L 1210.

Nonphosphorylative transglycosylation was determined both against p-nitrophenyl-N-maltoside and maltose with p-nitrophenyl-N-glucoside in serum and liver of mice with transplantable leukemia P 388 and L 1210. Histopathological verification of lymph nodes, spleen and liver of all used mice was carried out. Significant increase of transglycosylating activity in serum of P 388 mice and simultaneously decrease enzymatic activity in liver was observed. Moreover decrease of examined activity in serum and liver of L 1210 mice was demonstrated. The donor properties of some sugar in transglycosylation reaction was investigated.

Tumor-associated changes in plasma samples revealed by two-dimensional macromolecular mapping and selective lectin binding.

Two-dimensional electrophoretic analysis of plasma samples from EL-4 lymphoma-bearing C57 black mice revealed five 75 kd protein species in contrast with the presence of only two comparable components of similar migration in plasma from control animals. In contrast, no comparable alterations were observed in a comparison of plasma samples from L1210 tumor-bearing DBA mice and the corresponding plasma from animals immune-suppressed with antilymphocytic serum or in control plasma from DBA control plasma from DBA control animals. Analysis of selective binding using iodinated lectins revealed significant binding of I125 Lens culinaris in the more cathodic 75 kd component present in the plasma from control C57 black mice and a decreased Lens culinaris binding in the corresponding plasma components from EL-4 tumor-bearing C57 black animals. An identical assay with the same samples using I125 Ricinus communis did not show significant interaction with any 75 kd protein species, revealing instead lectin binding in components with molecular weights of about 70 and 50 kd. Our results suggest the use of combined two-dimensional electrophoretic separation and relative lectin binding in the analysis of tumor-specific and tumor-associated changes in plasma samples from tumor-bearing individuals.