Potential Cytostatic Drug Research Articles

Cytotoxic effect of efavirenz is selective against cancer cells and associated with the cannabinoid system

Recently, a regression of precancerous lesions in HIV-1-infected patients after initiation of HAART was reported. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) as efavirenz (EFV) might be mediators of this effect, as they are known to have a cytotoxic effect on tumour cells. A potential mechanism involved in this effect may be the activation of the cannabinoid receptor to mediate tumour toxicity. Several tumour-derived and fibroblast cell lines were studied. Cytotoxicity of EFV was evaluated by Annexin-Pi staining. The expression of the cannabinoid receptors CB1, CB2 and GPR55 was analysed by western blot, quantitative reverse transcriptase (qRT-PCR) and fluorescence activated cell sorting. The influence of the cannabinoid agonists and antagonists on the effects of EFV was investigated. Furthermore, the effect of EFV on the phosphorylation state of the growth factors Erk, Akt and the tumour suppressor protein p53 was tested. EFV revealed a selective cytotoxic effect on several tumour cell lines, whereas primary fibroblasts were not affected. The cytotoxic effect was associated with the expression of CB1. The combination of EFV with cannabinoid agonists showed an increase in toxicity. The phosphorylation state of Erk and Akt was not affected by EFV, whereas p53 showed an increased phosphorylation. EFV has a selective cytotoxic effect on several tumour cells. Furthermore, EFV led to an activating phosphorylation of the tumour suppressor protein p53 going in line with earlier reports that EFV may be antitumourigenic and a potential cytostatic drug. The observed synergistic effect with cannabinoid agonists implicates an involvement of the cannabinoid system.

Reduction of the potential anticancer drug oracin in the rat liver in-vitro.

Studies on the metabolism of the potential cytostatic drug oracin have shown that a principal metabolite of oracin is 11-dihydrooracin (DHO). We conducted in-vitro experiments to investigate the extent of oracin carbonyl reduction in microsomal or cytosolic fractions and to find out the enzymes involved under these conditions. Among several inducers of rat cytochrome P450 only 3-methylcholanthrene caused a significant (P < 0.01) stimulation (1.9 times) of DHO production in microsomal fraction and the specific P4501A inhibitor alpha-naphthoflavone significantly (P < 0.01) decreased (twice) the induced reduction activity. Cytochrome P4501A participates in oracin reduction in microsomes. 18beta-Glycyrrhetinic acid, a specific inhibitor of hydroxysteroid dehydrogenase, significantly (P < 0.01) inhibited the production of DHO in the microsomal fraction (>95% inhibition) in comparison with the non-inhibited reaction. Statistically significant (P < 0.01) inhibition (95%) of DHO formation was caused by metyrapone, which is also the substrate of 11-hydroxysteroid dehydrogenase. The main microsomal enzyme which catalyses the carbonyl reduction of oracin is probably 11beta-hydroxysteroid dehydrogenase. Important oracin reduction to DHO in the cytosolic fraction was found. According to its specific sensitivity towards quercitrin (inhibition by 99%, P < 0.01), the enzyme responsible for DHO formation in the rat liver cytosol is postulated to be carbonyl reductase.

2-Methoxyestradiol as a Potential Cytostatic Drug in Gliomas?

Gliomas of astrocytic origin show only a limited chemotherapy response. Chemoresistance is most pronounced in glioblastoma multiforme, the most common and most malignant glioma, with median survival times not much longer than one year. Failure of chemotherapy partly relies on protective mechanisms against the commonly used DNA alkylating agents, but also on the constitutive activation of the pro-survival PI3K-Akt pathway in glioma cells, which inhibits apoptosis. Therefore, new drugs with an alternative mechanism, independent of DNA alkylation, are required. The microtubule targeting drug 2-methoxyestradiol (2-ME) efficiently induces mitotic arrest, apoptosis, but also autophagic cell death in glioma cells in vitro. Moreover, it may be able to inhibit vascularization of the highly vascular gliobastomas, because the drug influences blood vessel sprouting via a HIF-1-dependent mechanism. Although high doses of i.p. injected 2-ME were recently shown to be effective in an orthothopic rat glioma model, clinical phase I/II trials revealed low oral bioavailability. One of the most exciting future perspectives will be the currently ongoing development of improved 2-ME analogs. Compounds, sulphamoylated at positions 3 and 17, combine sufficient toxicity against tumor cells with resistance against metabolic degradation and sufficient plasma levels in experimental animals. They were found to be superior in some animal models of tumor growth and vascularization, following oral application.

Inhibiting wild-type and C299S mutant AKR1B10; a homologue of aldose reductase upregulated in cancers

AKR1B10 is an aldose reductase (AR) homologue overexpressed in liver cancer and various forms of that enzyme in carcinomas catalyze the reduction of anticancer drugs, potential cytostatic drug, and dl-glyceraldehyde but do not catalyze the reduction of glucose. Kinetic parameters for wild-type and C299S mutant AKR1B10 indicate that substitution of serine for cysteine at position 299 reduces the affinity of this protein for dl-glyceraldehyde and enhances its catalytic activity. Fibrates suppress peroxisome proliferation and the development of liver cancer in human. Here we report the potency of fibrate-mediated inhibition of the carbonyl reduction catalyzed by wild-type and C299S mutant AKR1B10 and compare it with known AR inhibitors. Wild-type AKR1B10-catalyzed carbonyl reduction follows pure non-competitive inhibition kinetics using zopolrestat, EBPC or sorbinil, whereas fenofibrate, Wy 14,643, ciprofibrate and fenofibric acid follow mixed non-competitive inhibition kinetics. In contrast, catalysis of reaction by the C299S AKR1B10 mutant is not inhibited by sorbinil and EBPC. Despite these differences, the C299S AKR1B10 mutant still manifests kinetics similar to the wild-type protein with other fibrates including zopolrestat, fenofibrate, Wy 14,346, gemfibrozil and ciprofibrate that show mixed non-competitive inhibition kinetics. The reaction of the mutant AKR1B10 is inhibited by fenofibric acid, but manifests pure non-competitive inhibition kinetics that are different from those demonstrated for the wild-type enzyme.

Phase-II Trial Using Gemcitabine as Monochemotherapy in Patients with Metastasized Pancreatic Carcinoma

BACKGROUND: The aim of this phase-II study was to evaluate the efficacy of gemcitabine monochemotherapy in patients with metastasized pancreatic carcinoma known to have a poor overall tumor response rate to chemotherapy in order to achieve an improvement in the quality of life. METHODS: In 28 patients with metastasized pancreatic carcinoma (mean age, 63.7 years; range, 37 to 77 years; sex ratio, 13 males, 15 females), systemic chemotherapy with gemcitabine (dose, 1000 mg/m2) was administered on day 1, 8 and 15. After one further week (day 29), the cycle was repeated. After each 2nd cycle, extension of tumor growth (restaging) including radiological imaging (ultrasound, computed tomography, plain film of the thorax) and laboratory analysis (tumor marker) was performed. Frequency, severity and spectrum of side effects were assessed according to WHO grading prior to each treatment. Quality of life was evaluated using standardized questionnaires. RESULTS: All in all, 106 chemotherapeutic cycles were administered in 28 patients (range, 1–18 cycles; mean, 3.78). While in no patient complete remission was observed, 2 out of 28 patients showed partial remission (7.2 %). In 11 out of 28 patients, stable tumor disease was detected (39.2 %). Fifteen out of 28 patients (53.6 %) showed progressive tumor growth. Four out of 28 patients lived longer than 1 year (1-year survival rate, 14.3 %). Side effects of the chemotherapy were only moderate. Only in 26 of 106 cycles (26.5 %), side effects were documented. Significant improvement in the quality of life was reported in 25 % of the treated patients. CONCLUSIONS: Chemotherapy using gemcitabine is a well tolerable treatment option with a minimal rate of side effects in the case of metastasized pancreatic carcinoma. However, overall response rate is low. Even considering the acceptable median survival time of 9.1 months most likely caused by second-line chemotherapy, optimization of gemcitabine monotherapy appears to be required using a combination with a further potential cytostatic drug.

Stereochemical aspects of carbonyl reduction of the original anticancer drug oracin by mouse liver microsomes and purified 11β-hydroxysteroid dehydrogenase type 1

Oracin, 6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11 H-indeno[1,2- c] isoquinoline, is a potential cytostatic drug for oral use and presently in phase II of clinical trials. Major advantages of this novel chemotherapeutic are the possibility of oral administration, its negative results in the Ames test on mutagenicity, and the lack of cardiotoxicity. Metabolic studies on oracin have revealed that the principal metabolite in all laboratory animals is 11-dihydrooracin (DHO), which is produced by carbonyl reduction of the parent compound. Since the carbonyl moiety of oracin is a pro-chiral centre, reduction may lead to the two stereoisomer forms (+)-DHO and (−)-DHO. The aim of the present study was to infer if 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD 1) is responsible for carbonyl reduction of oracin in mouse liver and if this enzyme exhibits stereospecificity in DHO formation. 11β-HSD 1 was purified from mouse liver microsomes, and the kinetics and stereospecificity regarding DHO formation were determined and compared to values obtained from the whole microsomal fraction. We could show that purified mouse liver 11β-HSD 1 catalyzes the stereospecific carbonyl reduction of oracin, thereby following a sigmoidal dose–response kinetics. Due to a different ratio of (+)-DHO and (−)-DHO (93:7) formed by purified 11β-HSD 1 compared to that produced in whole microsomes (70:30), the existence of at least one other oracin carbonyl reducing enzyme can be expected in mouse liver microsomes. This suggestion is further supported by the fact that the Hill coefficient of 2 for purified 11β-HSD 1 (which is supporting earlier data on the cooperativity of this dimeric enzyme) changes to a Hill coefficient of 3 in whole microsomes (which is indicative for another enzyme participating in oracin carbonyl reduction).

Central composite design as a powerful optimisation technique for enantioresolution of the rac-11-dihydrooracin—the principal metabolite of the potential cytostatic drug oracin

Three types of chiral stationary phase were used to achieve chromatographic resolution of enantiomers of rac-11-dihydrooracin (DHO), the principal metabolite of a potential cytostatic drug oracin. Chiralcel OD-R as a chiral stationary phase with mobile phase comprising acetonitrile (modifier) and sodium perchlorate (buffering component) proved to be the most suitable system. Chemometric optimisation based on the Box–Wilson central composite design was employed to find the optimum resolution. The optimum factor space was defined by three parameters: temperature, modifier concentration and buffer concentration. Newly designed chromatographic response functions based on a combination of resolution R S and retention time of the last component eluted t RL were employed to evaluate the resolution with regard to quality and quantity. Optimum values predicted from those models of response surfaces were in excellent agreement with the experimental results. The chromatographic resolution of DHO enantiomers is suitable for xenobiochemical studies on stereoselectivity and stereospecificity of biotransformation enzymes.

Biphasic cytotoxic mechanism of extracellular ATP on U-937 human histiocytic leukemia cells: involvement of adenosine generation

Since extracellular ATP can exhibit cytotoxic activity in vivo and in vitro, its application has been proposed as an alternative anticancer therapy. In this study we investigated the mechanisms of ATP-induced cytotoxicity in a human leukemic cell line (U-937). ATP added as a single dose exceeding 50 μM was cytostatic or even cytotoxic for U-937 cells. Interestingly, growth inhibition by ATP (50–3500 μM) showed a biphasic dose response. Up to 800 μM, ATP was cytotoxic in a dose-dependent manner (EC 50 90 μM). In a range between 800 and 2500 μM, cell count was markedly higher despite the higher ATP concentrations. The cytotoxic effect of ATP could be antagonized by addition of uridine as a pyrimidine source and, alternatively, by addition of the nucleoside transmembrane inhibitor dipyridamole. The apoptosis-inducing adenosine A 3 receptor was not involved in measurable quantities, since (1) adenosine did not lead to an elevation of intracellular calcium levels, and (2) an unselective A 1–3 antagonist (ULS-II-80) could not abrogate the cytotoxic effect. Experiments monitoring extracellular nucleotide metabolism confirmed the assumption that the long-term production and continuous uptake of adenosine, which is extracellularly generated by degradation of ATP, led to an intracellular nucleotide imbalance with pyrimidine starvation. The biphasic dose response to higher ATP concentrations could be explained by the rapid degradation of lower ATP concentrations (300 μM) to adenosine by serum-derived enzymes, whereas higher concentrations (900 μM) only produced small amounts of adenosine due to forward inhibition of AMP hydrolysis by prolonged high ADP levels. FACS analysis revealed that at lower adenosine concentrations (300 μM) a reversible G 1 phase arrest of the cell cycle was induced, whereas higher concentrations (1000 μM) triggered apoptosis. Considering ATP as a potential cytostatic drug, our data have important implications concerning metabolic interactions of administered nucleotides.

High-performance liquid chromatography study of stereospecific microsomal enzymes catalysing the reduction of a potential cytostatic drug, oracin: Interspecies comparison

One of the main metabolites of oracin (I) {6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2-c]isoquinoline}, a potential cytostatic drug, is 11-dihydrooracin (II) {(+),(−)-6-[2-(2-hydroxyethyl)aminoethyl]-5-oxo-11-hydroxy-5,6-dihydro-11H-indeno[1,2-c]isoquinoline}, a metabolite formed by the reduction of oracin's pro-chiral centre on C 11. This metabolite has been found in all laboratory species in vitro and in vivo and it constitutes the main metabolite in man. The stereospecificity of reducing enzymes participating in the oracin biotransformation pathway was investigated using microsomal preparations from standard laboratory animals. Enzyme stereospecificity has been defined as preferential formation by the enzyme of the (+) or (−) stereoisomer of II. Significant interspecies differences were observed in the stereospecificity of the respective biotransformation enzymes. HPLC quantitative determinations of both enantiomers were performed using a Chiralcel OD-R column as chiral stationary phase with excellent resolution and stability.

High-performance liquid chromatographic assay for the separation and characterization of metabolites of the potential cytostatic drug oracine

Oracine ( I), a potential cytostatic drug, is enzymically converted to a number of metabolites whose formation has been studied in vitro and in vivo. The metabolites were separated by reversed-phase HPLC and characterized by UV spectra. Preparative TLC served for the isolation of the individual metabolites to allow their identification. Two metabolites were identified by Fourier transform NMR as 11-dihydrooracine ( II) and a phenolic product ( III). Two further metabolites ( IV,V) were characterized. Some minor, presumably 11-dihydro metabolites and an 11-oxo metabolite produced in vitro and in vivo were revealed.