Antineoplastic drug NSC631570 modulates functions of hypoxic macrophages.

Hypoxia is an important factor in the macrophages microenvironment. Many physiological and pathological processes including solid tumor development are characterized by both low oxygen content and presence of macrophages. Tumor-associated hypoxia causes alternative polarization of macrophages in tumor tissue and transformation of these cells into the allies of a malignant neoplasm. The aim of the work was to investigate the effect of NSC631570, a cancer-selective drug that is known to selectively accumulate in the tumor tissue, on hypoxic macrophage function. Murine peritoneal macrophages (PMs) were subjected to hypoxia (3% O2). Nitrite level was assayed by the Griess reaction. Arginase activity was measured by colorimetric method. ROS generation and phagocytosis was estimated by flow cytometry. O2(-) generation was assayed by the NBT reduction method. HMGB1 expression was determined by ELISA. 42 h hypoxia caused alternative polarization of murine PMs with significant arginase prevalence. NSC631570 repolarized arginine metabolism of hypoxic macrophages to NOS dominant and activated their pro-inflammatory functions: recovered ROS production and increased alarmin release NSC631570 can restore pro-inflammatory functions of macrophages, alternatively polarized by hypoxia.

Modulation of immune effector cell cytolytic activity and tumour growth inhibition in vivo by Ukrain (NSC 631570).

Ukrain is a semisynthetic compound consisting of alkaloids from Chelidonium majus L. conjugated to thiophosphoric acid, with immunomodulatory and therapeutic properties in cancer patients. The present in vitro studies demonstrate that Ukrain is an effective biological response modifier augmenting, by up to 48-fold, the lytic activity of splenic lymphocytes obtained from alloimmunized mice. The lytic activities of interleukin-2 (IL-2) treated spleen cells and peritoneal exudate lymphocytes were also significantly increased by the addition of Ukrain to the cell mediated lysis (CML) assay medium. The highest Ukrain-induced enhancement of splenic lymphocytolytic activity in vitro was found to occur at day 18 after alloimmunization was dose-dependent and specific for the immunizing P815 tumour cells. Since Ukrain was present only during the CML assays, its mode of action is thought to be via direct activation of the effector cells' lytic mechanism(s). The effect of Ukrain on the growth of Balb/c syngenic mammary adenocarcinoma was also evaluated. Intravenous, but not subcutaneous or intraperitoneal, administration of this drug was found to be effective in delaying tumour growth in an actual therapeutic protocol initiated five days after tumour implantation. No deleterious side-effects were observed using these in vivo treatment modalities. The role of macrophages in the observed retardation of tumour development was investigated, using peritoneal exudate macrophages (PEM) in cytotoxicity assays. Previous studies showed that PEM of mammary tumour-bearing mice lose their capacity to kill a variety of tumour target cells including the in vitro cultured homologous tumour cells (DA-3). Pretreatment of PEM from normal mice with 2.5 microM Ukrain for 24 h, followed by stimulation with either IFN-gamma or with lipopolysaccharide (LPS) plus IFN-gamma enhanced their cytotoxic activity. Treatment of PEM from tumour-bearing mice with 2.5 microM Ukrain and LPS results in a reversal of their defective cytotoxic response against DA-3 target cells. Furthermore, Ukrain alone, in the absence of a secondary signal, induced the activation of tumouricidal function of PEM from tumour-bearing, but not from normal, mice. These data indicate that Ukrain's in vivo effects against the development of mammary tumours may be due, at least in part, to its ability to restore macrophage cytolytic function.

In vitro effects of Chelidonium majus L. alkaloid thiophosphoric acid conjugates (Ukrain) on the phenotype of normal human lymphocytes.

Several plant-derived drugs are used in medical oncology (1-3). The thiophosphoric acid-alkaloid derivative from the plant Chelidonium majus L. (Ukrain) has been produced since 1978 by Nowicky. (Austrian Patent No. 354644, Vienna, 25 January 1980). It has been reported that this drug may exert therapeutic effects in cancer patients by modulating their immune systems (4, 5-8). The aim of this study was to evaluate in vitro the possibility that Ukrain may influence the phenotype of lymphocyte subpopulations isolated from healthy donors.

Mitogenic properties of Ukrain (NSC-6315170) on human peripheral blood monocytes: clinical implications.

Using a cell-proliferation assay, the authors examined and compared the mitogenic effects of Ukrain and phytohaemagglutinin (PHA) on human peripheral blood mononuclear cells (PBMCs), as well as their synergic effects. It was found that even a short period of pretreatment of the cell with Ukrain had a potent synergic effect on PHA mitogenesis resulting in significantly higher cell stimulation indices than those of PHA alone. Moreover, it was found that a short period of PHA treatment of the cells is almost imperative for Ukrain to exert its mitogenic effects. The mitogenic effect of Ukrain on human PBMCs is consistent with a previous clinical report which found that circulating lymphocytes were significantly increased in cancer patients treated with Ukrain. Thus the in vitro assay used in these studies may serve as a prognostic assay for potential patient responsiveness to Ukrain treatment, as well as a clinical parameter during Ukrain therapy.

Influence of Ukrain on immunological blood parameters in vitro and in vivo.

Immunological changes are often seen in subjects suffering from oncological disease. Decreased NK activity, alterations of the T4/T8 quotient, decreased phagocytic activity, low reactivity and decreased recognition of the "foreign" are common features seen in these patients. Cytostatic therapy (chemotherapy and radiation therapy) very often enhance these negative properties, thus limiting therapeutic possibilities by highly toxic sequels. Ukrain, being cytostatic and immune-stimulating and modulating, has no adverse effects on the organism.

Inhibitory effect of thiophosphoric acid alkaloid derivatives from Chelidonium majus L. (Ukrain) on ovalbumin antigenicity and antiovalbumin IgE antibody response in mice.

The ability of the Chelidonium majus L. alkaloid derivative Ukrain (UK) to inhibit ovalbumin-induced sensitization was tested in BALB/c and F1(BALB/c x C57BL/6J) mice. UK introduced into the mice in the mixture with antigen (ovalbumin) and adjuvant (alum) inhibited the sensitization of mice, reflected in lower anti-OA IgE antibody response and decreased antigen-induced histamine release from mast cells isolated from peritoneal cavities of sensitized mice. The effect of UK on the antigenicity of ovalbumin (OA) in anaphylaxis was tested in heterologous passive cutaneous anaphylactic (PCA) reaction on rats. The results show that the OA prepared in the mixture with UK had a decreased ability to react with anti-OA IgE antibodies raised against native OA in mice and fixed on the surface of rat mast cells in heterologous PCA reactions. The results suggest that UK pretreatment of OA may affect its antigenic property and the ability to react with anti-OA IgE antibodies raised against the native IgE molecules.

Biophysiological effects of Ukrain therapy in a patient with breast cancer (case report).

The effects on different biophysiological parameters and subjective impressions were studied in a patient with breast cancer who was not previously given any therapy before receiving Ukrain. Daily measurements of pulse, blood pressure, temperature and various laboratory examinations were carried out. Development and course of subjective and objective phenomena seem to be typical for patients in whom Ukrain could induce long-term complete remission. The patient described here has had to data 12 years without any oncopathological symptoms.

Influence of Ukrain on the nuclear thyroid hormone receptors after short-term gamma-irradiation.

The ability of Ukrain, a semi-synthetic alkaloid thiophosphoric acid derivative (NSC-631579), to influence thyroid hormone levels in plasma and nuclear thyroid hormone receptors in female rat liver, as well as to modify the effects of short-term whole body gamma-irradiation on nuclear thyroid-hormone receptors in the liver was evaluated after intraperitoneal administration of the drug at 0.4 mg/kg body weight. Ukrain had no effect on the concentration of thyroid hormones in rat blood and increased the concentration of thyroid hormone receptors in the liver of intact rats during the first 2 months after administration. Ukrain normalized the level of nuclear thyroid hormone receptors influenced by short-term whole body gamma-irradiation of rats with 1 Gy, beginning from the first day after administration of the drug. Thus, Ukrain can minimize the consequences of irradiation in the endocrine system of experimental animals.

Radiomodification effects of Ukrain, a cytostatic and immunomodulating drug, on intracellular glucocorticoid reception during short-term gamma-irradiation.

The ability of Ukrain, a cytostatic and immunomodulating semisynthetic compound of thiophosphate-modified alkaloids of Chelidonium majus L., to modify the effects of irradiation on intracellular glucocorticoid reception in female rat liver was evaluated after intraperitoneal administration of the drug at 0.4 mg/kg of body weight. Ukrain caused the normalization of the intracellular glucocorticoid-receptor system, influenced by short-term whole-body gamma-irradiation of rats with 1 Gy, beginning on the 10th day after administration of the drug. It was found that Ukrain minimized the consequences of irradiation in the endocrine system of the experimental animals.

Selective inhibition of in vitro cell growth by the anti-tumour drug Ukrain.

The inhibitory effect of Ukrain on malignant cells and on normal cells, in vitro, has been compared. To obtain a 50% inhibition of cell growth, a tenfold concentration had to be used with normal endothelial cells compared to a human osteosarcoma cell line. Hybrids of the two cell types showed nearly the same sensitivity as normal cells. A laser scanning microscope showed a high uptake of Ukrain in malignant cells, while the content in normal cells under the same experimental conditions was substantially lower.

Induction of bimodal programmed cell death in malignant cells by the derivative Ukrain (NSC-631570).

Selective induction of malignant cell death is one of the major goals of effective and safe chemotherapy. Recent developments in the understanding of programmed cell death (PCD) or apoptosis are expected to provide new leads for a safer chemotherapy. The authors investigated whether the semisynthetic alkaloid thiophosphoric acid derivative Ukrain (NSC-631570) could induce PCD or apoptosis in human K562 leukaemia cells. Results showed that Ukrain induced two distinct modalities of cell death programmes. One modality corresponded morphologically to classical apoptosis or PCD characterized by blebbing and shedding of membrane vesicles with concomitant 51Cr release; however, the Ukrain-induced apoptosis was not associated with the characteristic nuclear DNA fragmentation. Higher concentrations of Ukrain induced a second cell death programme characterized by cell surface blister formation, high specific 51Cr release and extensive DNA polyploidy. These two cell death programmes are distinct from each other in that they are interphased by a silent period characterized by normal cell morphology and reduced specific 51Cr release.

Influence of Ukrain on DNA, RNA and protein synthesis in malignant cells.

3H labelled thymidine, uridine and leucine were used to evaluate the synthesis of DNA, RNA and proteins in malignant cells and normal cells incubated with Ukrain in different concentrations. Compared with the controls, the inhibiting effects of Ukrain are demonstrated in guinea pig hepatocytes, CIL hepatocytes, human tonsil cells, two murine lymphomas, murine myeloma, Yoshida cells, two HeLa strains, EsB- and EB- (murine) lymphomas. YAC-1, P815 and human WiDr cells. Ukrain inhibits the DNA, RNA and protein synthesis in malignant cell lines at relatively high concentrations and to a small extent in normal cells.

Influence of Ukrain on human xenografts in vitro.

Tumour cells were taken from human tumour xenografts (HTX) and serially transplanted into nude mice. These cells were used in a colony-forming assay in vitro. Tumour cells were incubated continuously for at least one week with several concentrations of the drug Ukrain. This was done with six different types and the colony formation was scored for each tumour. The drug effects were reported as percent T/C (Test/Control) for the numbers of colonies formed. Activity is considered to be present at T/C < 30%. A clear dose-response effect was found for Ukrain.

Effect of six-month treatment with Ukrain on early osteoporosis induced by ovariectomy in rats. Part III. Preliminary studies of some hormone levels.

The effect of six-month treatment with Ukrain on the level of some hormones was investigated in ovariectomized rats. The serum levels of prolactin, adrenocorticotropic hormone (ACTH), corticosterone, aldosterone and progesterone were measured by radioimmunoassay (RIA). Six months after ovariectomy, a decrease in prolactin and progesterone levels was observed. No changes were noted in the levels of ACTH, corticosterone and aldosterone in the serum of the ovariectomized animals. On the other hand, six-month treatment with Ukrain increased the levels of prolactin and progesterone and decreased the levels of corticosterone and aldosterone in the ovariectomized rats. The progesterone level in the treated group was four times higher than in the ovariectomized group. The six-month treatment with Ukrain had no effect on the level of ACTH of the serum of the ovariectomized rats. The present results show that prolonged treatment with Ukrain significantly affects hormone secretion after ovariectomy.

Synergic influence of Ukrain and protoporphyrin amino acid conjugates on human malignant cell lines.

Simultaneous cytotoxicity of Ukrain and protoporphyrin amino-acid derivatives was tested on four malignant cell lines and the cytotoxic effect after laser irradiation was compared with cytotoxicity of Ukrain and protoporphyrin amino-acid derivatives applied on similar cell lines separately. It was found that Ukrain and protoporphyrin amino-acid derivatives act synergistically as cytotoxic substances on the malignant cell lines.

Does the Ukrain preparation protect mice against lethal doses of bacteria?

Ukrain, a semi-synthetic preparation obtained from Chelidonium majus L, is used in the treatment of cancer diseases. It has been observed to exert a protective influence in mice infected by influenza viruses. Recently, the influence of the preparation on the survival of mice infected by lethal doses of E. coli and S. aureus has been estimated. This preparation was administered to Balb/c mice subcutaneously in doses of 0.04, 0.4 and 4.0 mg/kg of body weight. Ukrain was given every second day during 20 days, or a short-term before-and-after method at 48, 24 and 2 h before the infection and or 2, 24 and 48 h after the infection of mice. The mice were infected intraperitoneally with E. coli or S. aureus in doses equivalent to 2LD50. Increased survival of mice, depending on the dose of the preparation and the kind of infecting bacterium was observed. The highest survival (50%) occurred in mice infected with E. coli and receiving the amount of the preparation corresponding to 0.4 mg/kg. The lowest survival was observed in mice infected by S. aureus and receiving the preparation in the amount of 4.0 mg/kg. Higher protective effectiveness of the Ukrain preparation was observed in mice when the preparation had been administered during 20 days as compared to the short-term before-and-after regime.

Estimation of direct influence of Ukrain preparation on influenza viruses and the bacteria E. coli and S. aureus.

Ukrain is a semisynthetic drug with immunomodulatory properties derived from Chelidonium majus L. alkaloids and thiophosphoric acid. It acts selectively in a lytic way on cancer cells. Its protective properties have been shown in mice infected by influenza viruses. In this paper, the studies made on the estimation of the direct activity of Ukrain preparation on viruses and bacteria E. coli and S. aureus are described. Viruses of different haemagglutination titres were incubated with different concentrations of the preparation during period of 1, 2 and 24 h. Afterwards the samples were collected and used for the infection of the allantoic cavity obtained from 10-day-old hen embryos. A second method was based on the introduction of the Ukrain preparation into the allantoic cavity of embryos before infection with influenza viruses and after the infection of embryos. In both the described methods, the embryos were incubated within 48 hours. Then the presence of influenza viruses in allantoic fluid was estimated using a haemagglutination reaction with 30% hen blood cells. The influence of the preparation on hen embryo was also studied. In order to estimate the antibacterial activity the following procedure was used. To the preparation diluted with the growth medium from 500 micrograms/ml to 1 microgram/ml a definite amount of the bacteria S. aureus or E. coli was added, and after 24 and 48 h of incubation at 37 degrees C the results were read off. In the second method, the bacteria were added to 1, 10, 100 and 500 micrograms of the preparation in 1.0 ml of 0.85% NaCl, and after 1, 2 and 24 of incubation at room temperature the samples were collected and inoculated on solid Mueller-Hinton medium. The presence of bacterial growth or medium turbidity after 24 and 48 h of incubation was taken as a positive result. Our studies have revealed that the above mentioned preparation does not exert any negative influence on hen embryos that could make it difficult to estimate replication of influenza viruses. This preparation did not show any direct influence on the inactivation of influenza viruses and the bacteria E. coli and S. aureus.

Potential therapeutic efficacy of Ukrain (NSC 631570) in AIDS patients with Kaposi's sarcoma.

In this study two case reports are presented of therapy with Ukrain, a semi-synthetic thiophosphoric acid compound of alkaloids isolated from Chelidonium majus L, for the treatment of AIDS patients with Kaposi's sarcoma. Ukrain was injected i.v. in the dose of 5 mg every other day for a total of 10 injections. During treatment the Kaposi's sarcoma lesions diminished in size, showed decolouration and no lesion appeared in the 30-day interval after the beginning of treatment. Both patients tolerated Ukrain well and showed an improved immunohaematological status: an increase in total leukocytes, T-lymphocytes and T-suppressor numbers. In one case T-helper lymphocytes were also increased. Future investigations are necessary for the evaluation of Ukrain efficacy in the treatment of this type of patients.

Clinical aspects of cancer treatment and new biochemical mechanisms of the drug Ukrain.

A random group of 50 patients in tumor stages T1-3N0-2M0 was selected from breast cancer patients and given Ukrain therapy by intravenous injection. Twenty-five patients received a total dose of 50 mg Ukrain (5 mg every second day, 10 injections altogether). Twenty-five patients received a total dose of 100 mg Ukrain (10 mg every second day, 10 injections altogether).

Modification of antinociceptive action of morphine by Ukrain in rodents.

Morphine-induced (0.1 mg/kg s.c.)antinociceptive action was determined in rodents by using the writhing syndrome, hot plate and tail-flick tests. Ukrain given i.p. in doses equivalent to 0.05 and 0.1 LD50 did not affect the reactivity of the mice in the writhing syndrome test. Only in a dose of 0.1 LD50 did Ukrain produce an antinociceptive effect in the hot-plate and tail-flick tests. Ukrain significantly enhanced the antinociceptive effect of morphine in the hot-plate and tail-flick tests. The action of morphine was significantly suppressed by Ukrain in the writhing syndrome test in mice. These results indicated that Ukrain modified the antinociceptive action of morphine.