Synthesis, characterization, antitumor activity and safety testing of novel polyphosphoesters bearing anthracene-derived aminophosphonate units.

Novel polyphosphoesters containing anthracene-derived aminophosphonate units, poly(oxyethylene aminophosphonate)s (4 and 5) and poly[oxyethylene(aminophosphonate-co-H-phosphonate)]s (6 and 7), were synthesized via an addition of poly(oxyethylene H-phosphonate)s to 9-anthrylidene-p-toluidine. The IR, NMR ((1)H, (13)C and (31)P) and fluorescence emission spectral data of the polymers are presented. The copolymers 6 and 7 were tested for in vitro antitumor activity on a panel of seven human epithelial cancer cell lines. Safety testing was performed both in vitro (3T3 NRU test) and in vivo on ICR mice for genotoxicity and antiproliferative activity. The copolymer 7 showed excellent antiproliferative activity to HBL-100, MDA-MB-231, MCF-7 and HepG2 cell lines. However, the in vitro safety testing revealed significant toxicity to Balb/c 3T3 mouse embryo cells. In contrast, the copolymer 6 showed complete absence of cytotoxicity to Balb/c 3T3 cells, but inhibited the growth of breast cancer cells, cervical carcinoma cells (HeLa) and hepatocellular carcinoma cell cultures after prolonged (72h) exposure. The polymers (4-6) exhibited low (4 and 6) to moderate (5) clastogenicity in vivo and slightly inhibited bone marrow cell division, compared to Mitomycin C. The subcellular distribution of the copolymers 6 and 7 were studied in model cell culture systems. The tested polyphosphoesters are expected to act in vivo as prodrugs of aminophosphonates and could be valuable as a new class of biodegradable polymer drug carriers.

Synthesis, antiproliferative activity and genotoxicity of novel anthracene-containing aminophosphonates and a new anthracene-derived Schiff base.

A new Schiff base, 9-anthrylidene-furfurylamine and three novel anthracene-containing α-aminophosphonates, [N-methyl(dimethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine, [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine and [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]furfurylamine were synthesized. The compounds have been characterized by elemental analysis, TLC, IR, NMR and fluorescent spectra. The aminophosphonates and their synthetic precursors were tested for in vitro antitumor activity on a panel of seven human epithelial cancer cell lines. Safety testing was performed both in vitro (3T3 NRU test) and in vivo on ICR mice for genotoxicity and antiproliferative activity. 9-Anthrylidene-furfurylamine and [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]furfurylamine were most potent cytotoxic agents towards colon carcinoma cell line HT-29. The latter compound exhibited also antiproliferative activity to HBL-100, MDA-MB-231 and 647-V cells. The aminophosphonate [N-methyl(dimethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine and its synthetic precursor 9-anthrylidene-p-toluidine were found to be cytotoxic to HBL-100 and HT-29 tumor cell lines, respectively. Moderate genotoxic and antiproliferative activity in vivo and low toxicity to Balb/c 3T3 (clone 31) mouse embryo cells were observed for all tested compounds. The subcellular distribution of two tested compounds in a tumor cell culture system was also studied.

Isopathic versus enantiomeric inhibition of U-50488 HCl toxicity--experimental studies.

BACKGROUND: Previous studies have investigated toxicity inhibition of optically active compounds by potentized preparations of their enantiomers. It was hypothesised that inhibition of toxicity may be stereospecific. This paper presents 2 studies investigating stereoisomer potencies in terms of their ability to counteract toxicity of the (-) stereoisomer. The stereoisomers used were (-)-trans-(1S,2S)-U-50488 HCl and (+)-trans-(1R,2R)-U-50488 HCl. MATERIALS & METHODS: Designs were prospective, blind, randomised, intention-to-treat and compared the efficacy of 2 indistinguishable treatments. The outcome was the difference in survival. Potency 'chords' consisting of 4th, 12th and 30th approximately centesimal dilutions were prepared, representing concentrations of 1.08 x 10(-10) M. One study compared inhibition of (-)-U-50488 toxicity injected ip at the estimated LD50 into male ICR mice, treated with a potency chord of the same stereoisomer, with control ('isopathic' study). The other study compared inhibition of toxicity by potency chords made from the stereoisomers (+)-U-50488 and (-)-U-50488 ('enantiomer' study), Treatments were administered orally on 11 occasions: twice before and nine times after ip injections. RESULTS: The isopathic study did not yield a significant result. In the enantiomer study, comparison of isopathy with enantiomer potency treatment showed a highly significant difference odds ratio 1.97 (95% CI: 1.23-3.14). CONCLUSION: We conclude that enantiomeric potencies are superior to identically produced isopathic potencies, in inhibiting toxicity of (-)-U-50488 HCl. Homeopathic inhibition of toxicity may be stereospecific.

Synthesis, cytotoxicity and clastogenicity of novel alpha-aminophosphonic acids.

Alpha-ethyl-N-(phosphonomethyl) glycine is synthesized and characterized by NMR and FAB spectroscopy. The cytotoxicity, clastogenic and antiproliferative effect of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, sodium salt of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, alpha-ethyl-alpha-N-(hydroxyethylamino) methylphosphonic acid, alpha-ethyl-N-(phosphonomethyl) glycine, alpha-ethyl-N-(phosphonomethyl) glycine isopropylammonium salt, glyphosate isopropylammonium salt are tested.

Inhibition of (-)-trans-(1S,2S)-U50488 hydrochloride by its enantiomer in white mice -- a placebo-controlled, randomized study.

BACKGROUND: Previous studies have been performed to see if toxicity of optically active compounds may be inhibited by potentized preparations of their enantiomers. The present study is based on the hypothesis that the toxic effects of an optical isomer may be counteracted or reversed by the administration of a potentized preparation of one of its stereoisomers and in particular the enantiomer (patent applied for). METHODS: The design was prospective, blind, randomized, and placebo-controlled. 210 ICR conventional mice were used. 105 mice were administered a mixture of (+)-U50488 hydrochloride homeopathic potencies prior to and during the experiment, and the other 105 were administered indistinguishable placebo. The first 52 mice were used to establish an LD(50) of intraperitoneally administered (-)-U50488 hydrochloride under the conditions of this experiment. The estimated LD(50) was 25 mg/kg. The remaining 158 mice were then administered this LD(50) of (-)-U50488 HCl intraperitoneally. One mouse from the placebo group was excluded from the analysis because it died immediately after the possibly intravenous injection of (-)-U50488 HCl. RESULTS: 67% of homeopathy mice survived compared with 47% of placebo mice. The end point for statistical analysis was the difference in survival between the placebo and homeopathy mice. The analysis was adjusted for mouse weight using a logistic regression (LR) model. The LR treatment odds ratio for survival of treatment mice relative to placebo mice was 2.301 and the LR treatment chi-square was 6.2030 (1 degree of freedom), which has a p-value of 0.0128. Consequently, we reject the null hypothesis of no treatment effect on survival. CONCLUSION: We conclude that toxicity of intraperitoneal injection of (-)-U50488 hydrochloride may be inhibited by administration of a mixture of potencies of its enantiomer.

Inhibition of (-)-propranolol hydrochloride by its enantiomer in white mice--a placebo-controlled randomized study.

BACKGROUND: A previous pilot study was performed to see if toxicity of (S)-(-)-propranolol hydrochloride may be inhibited by a potentized preparation of its enantiomer. The present study is based on the hypothesis that the toxic effects of an optical isomer, may be counteracted or reversed by the administration of a potentized preparation of one of its stereoisomers, and in particular the enantiomer. METHODS: 508 ICR conventional mice were used. 254 mice were administered (R)-(+)-propranolol HCl homeopathic potency prior to and during the experiment, and the other 254 were administered indistinguishable placebo. On the day of the experiment mice were anesthetized with intraperitoneal Rometar. Once sedated the mice were administered the LD50 dose of (-)-propranolol HCl intraperitoneally. RESULTS: The end point for statistical analysis was the difference in survival between the placebo and treatment mice. The odds ratio for survival of treatment mice relative to placebo mice was 1.52. The hypothesis of equal survival proportions gave a chi-square of 5.0429 (1 degree of freedom), which has a p-value of 0.0247. The analysis was then adjusted for mouse weight and intraperitoneal (-)-propranolol dosage using a logistic regression (LR) model. The LR treatment odds ratio was 1.51 and the LR treatment chi-square was 4.8112 (1 degree of freedom), which has a p-value of 0.0283. Consequently, we reject the null hypothesis of no treatment effect on survival. Eleven percent more treatment mice survived than placebo mice. CONCLUSION: We conclude that the toxicity of intraperitoneal (-)-propranolol HCl, may be counteracted by administration of a potency of its enantiomer, in ICR conventional mice which have survived preceding intraperitoneal Rometar injection, and pre-dosing with (+)-propranolol HCl homeopathic potency.

Bioaccumulation and damaging action of polymetal industrial dust on laboratory mice Mus musculus alba .II. Genetic, cell, and metabolic disturbances.

An ecologo-toxicological experiment was carried out with laboratory mice Mus musculus alba of the inbred line BALB/c. The experimental animals (male and female) were exposed for 120 days to polymetal industrial dust containing zinc, copper, lead, and cadmium, which was mixed with conventional animal food. Chromosome aberration frequency and pathological changes in hematological indices, oxygen consumption, body temperature, and body weight were studied in the context of heavy metal bioaccumulation and interactions. Samples for analyses were taken on days 15, 40, 60, and 90. An increased frequency of chromosome aberrations (up to 22%), lead-induced anemia, and significant decreases in body temperature were observed. A strong correlation between hemoglobin content and oxygen consumption (O2/g h) was established. An increase in hematocrit, accompanied by a loss of body weight after day 60, suggests dehydration resulting from lead and cadmium poisoning.

Bioaccumulation and damaging action of polymetal industrial dust on laboratory mice Mus musculus alba. I. Analysis of Zn, Cu, Pb, and Cd disposition and mathematical model for Zn and Cd bioaccumulations.

The concentrations of Zn, Cu, Pb, and Cd in the liver, kidneys, spleen, bones, and carcass of laboratory mice BALB/cy were observed in toxicological experiments. Polymetal industrial dust containing these metals was given to experimental animals at 1% concentration mixed with conventional animal food. Samples for analyses were taken on Days 15, 40, 60, 90, and 120 posttreatment. The experimental data clearly support the established antagonistic interactions among cadmium, zinc, copper, and lead. A mathematical model was proposed to study the main tendencies of heavy metal bioaccumulation under conditions of metal interaction and excessive exposure. The experimental results were assessed on the basis of the model. A rate constant of renal excretion greater than that of hepatic excretion was obtained, which agrees with the observed inversion of cadmium kidney/liver ratio in the conditions of very high exposure.

A comparative analysis of the heavy metal loading of small mammals in different regions of Bulgaria I: monitoring points and bioaccumulation features.

Data on liver and body copper, zinc, lead, and cadmium content of small mammals (rodents and insectivorous) were collected and analyzed. Data comparisons were performed in two aspects: (1) points and years of monitoring; (2) monitor species bioaccumulations. Specific bioaccumulation features were observed in some of the monitor species. A method for comparative evaluation of heavy metal loads in the different species is proposed using data for liver and body contamination. The loads of Clethrionomys glareolus and Apodemus flavicollis were compared, and the data are in agreement with data from other authors in Central Europe. A correlation between heavy metal content in the food and liver of snow vole was established. The data demonstrate that two of the regions investigated in Rila Mountain National Park could be assumed to be background locations. Some possible reasons for the heavy metal contamination of the low-altitude region in Rila are discussed. Not very significant pollution was observed around industrial facilities. Correlations between heavy metal levels in zoomonitors and meteorological factors were established.

A comparative analysis of the heavy metal loading of small mammals in different regions of Bulgaria II: chromosomal aberrations and blood pathology.

Heavy metal content was monitored in small mammals inhabiting mountain ecosystems and two industrial polluted regions in Bulgaria. Rodents (Microtus arvalis, M. rossiaemeridionalis, Clethrionomys glareolus, Pitymys subterraneus, Chionomys nivalis, Apodemus flavicollis, A. sylvaticus, and Mus macedonicus) were used as zoomonitors. Pathological changes in chromosome status, hematological indices, and blood cell morphology were analyzed in the context of heavy metal bioaccumulation. Significant correlations were obtained between the heavy metal load of zoomonitors and the frequency of chromosomal aberrations and pathological changes in erythrocytes (mainly micronuclei and basophilic granulations). It is suggested that mercury is a strong damaging factor for chromosomes and red blood cell apparatus.

Inhibition of (-)-propranolol hydrochloride by its enantiomer in white mice.

BACKGROUND: This study is based on the hypothesis, that the toxic or physiological effects of an optical isomer may be counteracted or reversed by the administration of a potentized preparation of one of its stereoisomers. In the present study the enantiomer was used. METHODS: 154 ICR conventional mice were used. 77 mice were administered (R)-(+)-propranolol HCl homeopathic potency prior to and during the experiment, and the other 77 were administered indistinguishable placebo. On the day of the experiment the mice were sedated with intraperitoneal Rometar. Once sedated they were injected intraperitoneally with the LD50 dose of (S)-(-)-propranolol HCl. RESULTS: The end point for statistical analysis was the difference in survival between the placebo and treatment mice. The odds ratio for survival of treatment mice relative to placebo mice was 1.64. The hypothesis of equal survival proportions gave a chi-square of 2.0916 (1 degree of freedom), which has a p-value of 0.1481. The analysis was then adjusted for mouse weight and intraperitoneal (-)-propranolol dosage using a logistic regression (LR) model. The LR treatment odds ratio was 2.017 and the LR treatment chi-square was 2.8864 (1 degree of freedom), which has a p-value of 0.0893. Consequently we accept the null hypothesis of no treatment effect on survival. The odds ratio estimates show that the treatment mice are 2.02 times more likely to survive than placebo mice, but this was not statistically significant with p = 0.089. Nine percent more treatment mice survived than placebo mice. The investigators accustomed to handling rodents noted that mouse recovery seemed substantially faster in the treatment mice than in the placebo mice.

Cytotoxic efficacy of bendamustine in human leukemia and breast cancer cell lines.

PURPOSE: The purpose of this study was to characterise bendamustine's cytotoxic and apoptotic activity in a panel of leukemia and breast cancer cell lines in comparison to its clastogenicity in murine bone marrow. METHODS: The cytotoxic effect of bendamustine was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-dye reduction assay. Induction of apoptosis was evidenced by DNA gel electrophoresis, nuclear staining, Western blot poly-(adenosine diphosphate-ribose) polymerase (PARP) cleavage, and flow cytometry. As a measure of hematological toxicity, the formation of chromosomal aberrations was investigated in bone marrow cells isolated from mice treated with low non-toxic doses of bendamustine and lomustine. RESULTS: Bendamustine was preferably active against leukemic cells of lymphoid origin and was found to induce apoptosis in SKW-3 and BV-173 cells as shown by oligonucleosomal DNA and nuclear fragmentation, PARP cleavage, and formation of a sub-G1 fraction. Myeloid and breast carcinoma cell lines were resistant towards bendamustine with the exception of HL-60 cells which exhibit an intermediate sensitivity. Bendamustine was found to have a very low clastogenic effect as compared with equimolar doses of lomustine. CONCLUSION: Taken together, the mode of action of bendamustine includes induction of apoptosis. The specific spectrum of activity and the unexpectedly low clastogenicity support the hypothesis that bendamustine in not a typical alkylating agent but exerts an additional mode of action, possibly as a purine antimetabolite.

Genotoxic effect of substituted phenoxyacetic acids.

The potential toxic and mutagenic action of 2,4-dichlorophenoxyacetic acid has been studied in different test systems, and the obtained results range from increased chromosomal damage to no effect at all. We reexamined the effect of this herbicide by simultaneous using three tests based on yeast, transformed hematopoietic, and mouse bone marrow cells. The results obtained demonstrated that 2,4-dichlorophenoxyacetic acid has cytotoxic and mutagenic effects. The positive response of yeast and transformed hematopoietic cells was verified in kinetics and dose-response experiments. The analysis of metaphase chromosomes indicated a statistically proved induction of breaks, deletions, and exchanges after the intraperitoneal administration of 2,4-dichlorophenoxyacetic acid in mice. The study of phenoxyacetic acid and its differently chlorinated derivatives showed that cytotoxicity and mutagenicity are induced by chlorine atoms at position 2 and/or 4 in the benzene ring. The mutagenic effect was abolished by introduction of a third chlorine atom at position 5. Thus 2,4,5-trichlorophenoxyacetic acid was found to have very weak, if any mutagenic effect; however, the herbicide preserved its toxic effect.

Alkylphosphocholines: Effects on human leukemic cell lines and normal bone marrow cells.

The anti-leukemic activity of a series of alkylphosphocholines (APCs) was studied against a panel of human leukemic cell lines (HL-60, K-562, Reh, MOLT-4, Jurkat, Ramos and Raji). Cytotoxic efficacy was measured by the MTT cell survival assay. All cell lines were found to be sensitive, except the multipotential CML-derived K-562 cell line. Flow cytometry of HL-60 cells showed a significant decrease of cells in S phase and the formation of a sub-G fraction. DNA fragmentation typical for programmed cell death was detected by DNA gel electrophoresis in these cells but not in any of the other leukemic lines. At concentrations below the cytotoxic range, mitogenic effects were seen in HL-60 cells after 14-hr exposure. Colony formation by K-562 cells revealed an augmented clonogenicity after exposure to APC with a short alkyl chain. In contrast, cells of lymphoid origin did not undergo DNA fragmentation or show mitogenic stimulation after exposure to APC. Normal bone marrow cells were also investigated for mitogenic and genotoxic effects. No decrease was found in the number of hematopoietic progenitors in long-term bone marrow cell cultures after exposure to APC. On the contrary, a significant increase was found after short exposure. Dodecylphosphocholine, hexadecylphosphocholine (HPC) and (octadecyl-[2-(N-methylpiperidino)-ethyl]phosphate exhibited a mild clastogenicity at equimolar high doses on murine bone marrow cells in vivo, which is unusual for the majority of classical DNA-interacting anti-cancer drugs. In conclusion, APCs are agents with a broad spectrum of in vitro anti-leukemic effects, which lack hematological toxicity.

Antitumor, nephrotoxic and clastogenic effect of cis-DDP with DDTC or NAC.

Diethyldithiocarbamate (DDTC) and N-acetylcysteine (NAC) are nucleophile sulfur-containing compounds which can protect the platinum-induced nephrotoxicity. Combinations of cis-diamminedichloroplatinum(II) (cis-DDP) and DDTC or NAC were tested on the leukemia L1210 and melanoma B 16 tumor models. Nephrotoxicity of cis-DDP alone and in combination with DDTC or NAC was evaluated. On both of the investigated tumor models clastogenic effects in bone marrow cells were detected. DNA synthetic and mitotic activity of L1210 cells in vivo were evaluated by 3H-thymidine incorporation and cytogenetic analysis. Amelioration of the platinum induced nephrotoxicity and preservation of the antitumor activity of cis-DDP through combined application with DDTC or NAC were obtained at the L1210 model. Maximal inhibition of the DNA synthesis in L1210 cells was detected with the cis-DDP treatment. The sulfurcontaining nucleophiles DDTC or NAC could modulate the inhibitory effect of cis-DDP on the incorporation of 3H-thymidine into the nuclei of L1210 cells. Enhanced mitotic activity was detected during cytotoxic therapy with cis-DDP. Cis-DDP alone and in combination with DDTC or NAC caused a significant growth inhibition on the s.c. tumor of the melanoma B16 bearing mice. Two times better therapeutic results at this model were obtained with cis-DDP alone (T/C = 234.09%, T/C = 136.36% for cis-DDP+DDTC and T/C = 151.14% for cis-DDP+NAC). The usefulness of DDTC or NAC as adjuvants in the platinum based chemotherapy of human cancers have been discussed. Clastogenic effect and antitumor activity are probably connected and it is supposed that the reduction of the genotoxicity could lead to a decreased antitumor activity of the platinum complex.

[The synthesis, toxicological and comparative cytogenetic characteristics of the anticoagulant warfarin]. / Sintez, toksikologichna i sravnitelna tsitogenetichna kharakteristika na antikoagulanta varfarin.

4-hydroxy-3-(3-oxo-1-phenyl butyl)-2H-1-benzopyran-2-one (warfarin) has been synthesised by an original method. The influence of a phase-transfer catalyst of ammonium type with alkyl substituents containing eight carbon atoms upon the reaction of Michael addition has been investigated. It has been found out that when elongating the hydrocarbon chain of the substituents to the nitrogen atom in the quaternary ammonium salt the yield of the product decreased. The acute (LD50) and subchronic (lasting for 30 days) toxicity was determined when taking warfarin orally. The experimental data show that LD50 is 500 mg/kg for mice and 420 mg/kg body mass for rats. The subchronic toxicity at experiments made with rabbits (each day taking orally respectively 25 and 100 mg/kg) does not reveal any humoral and tissue toxic influence of warfarin. The results from the comparative cytogenetic analysis of warfarin and Niffcumar are the basis of the conclusion cytogenetic analysis of warfarin and Niffcumar are the basis of the conclusion that warfarin damages chromosomes of mice's marrow cells (used as a model) less than Niffcumar. Moreover warfarin has a slight influence on these cells in the first place changing the orientation of chromosomes one towards the other and unlike other drugs it does not damage nuclear chromatin strongly.

[Experimental pharmacologic and morphologic study of new platinum complexes]. / Eksperimentalno-farmakologichno i morfologichno izsledvane na novi platinovi kompleksi.

Antineoplastic and toxic properties of two newly synthesized complexes of platinum (II)--with isovaleric acid and with cystine, were studied on mice with ascitic tumour of Ehrlich. The studies were compared with cis-diaminodichloroplatinum (II) (cisplatinum). Data were obtained for high antineoplastic activity and inhibiting influence of the synthesis of DNA of cisplatinum. There was therapeutic effect on the animals with ascitic tumour of Ehrlich after usage of cisplatinum. There was therapeutic effect on the animals with ascitic tumour of Ehrlich after usage of the complex of platinum with isovaleric acid and no effect after application of the complex with cystine. Information was obtained by histological examination for reduction in organic toxicity of the newly synthesized compounds against renal tissue. The necessity from multiple parametric strategy of the experiment, creating a possibility for logic direction of the investigation according to the aim-search for metallic complexes with reduced toxicity, is confirmed by the conducted study.