Microtubule disruption induced in vivo by alkylation of beta-tubulin by 1-aryl-3-(2-chloroethyl)ureas, a novel class of soft alkylating agents.

We have previously reported that 4-tert-butyl-[3-(2-chloroethyl)ureido] benzene (4-tBCEU), a potent cytotoxic agent, modulates the synthesis of tubulins, suggesting that its cytotoxicity may be mediated through an antimicrotubule mechanism. Indeed, 4-tBCEU and its 4-iso-propyl (4-isopropyl [3-(2-chloroethyl)ureido] benzene) and 4-sec-butyl (4-sec-butyl [3-(2-chloroethyl)ureido] benzene) homologues induced disruption of the cytoskeleton and arrest of the cell cycle in G2 transition and mitosis. To better understand the mechanisms responsible for microtubule disruption by 1-aryl-3-(2-chloroethyl)ureas (CEU), we first examined their cytotoxicity on Chinese hamster ovary cells resistant to vinblastine and colchicine due to the expression of mutated tubulins (CHO-VV 3-2). These cells showed resistance to CEU, e.g., 4-tBCEU having an IC50 of 21.3+/-1.1 microM as compared with an IC50 of 11.6+/-0.7 microM for wild-type cells, suggesting a direct effect of the drugs on tubulins. Western blot analysis confirmed the disruption of microtubules and evidenced the formation of an additional immunoreactive beta-tubulin with an apparent lower molecular weight on SDS polyacrylamide gel. Incubation of MDA-MB-231 cells with [urea-14C]-4-tBCEU revealed the presence of a radioactive protein that coincided with the additional beta-tubulin band, indicating that CEU could covalently bind to the beta-tubulin. The 4-tBCEU-binding site on beta-tubulin was identified by competition of the CEU with colchicine, vinblastine, and iodoacetamide, a specific alkylating agent of sulfhydryl groups of cysteine residues. Colchicine, but not vinblastine, prevented the formation of the additional beta-tubulin band, suggesting that 4-tBCEU alkylates either Cys239 or Cys354 residues near the colchicine-binding site. To determine the cysteine residue alkylated by 4-tBCEU, we incubated the radiolabeled drug with human neuroblastoma cells (SK-N-SH) that overexpress the betaIII-tubulin, an isoform where Cys239 is replaced by a serine residue. The results clearly showed that betaIII-tubulin is not alkylated by [urea-14C]-4-tBCEU, suggesting that cysteine 239 residue is essential for the reactivity of 4-tBCEU with beta-tubulin. Taken together, these findings indicate that the mechanism of cytotoxicity of CEU involves microtubule depolymerization through alkylation of beta-tubulin.

Improving arterial prosthesis neo-endothelialization: application of a proactive VEGF construct onto PTFE surfaces.

The formation of a confluent endothelium on expanded polytetrafluoroethylene (PTFE) vascular prostheses has never been observed. This lack of endothelialization is known to be one of the main reasons leading to the development of thromboses and/or intimal hyperplasia. In this context, several efforts were put forward to promote endothelial cell coverage on the internal surface of synthetic vascular prostheses. The goal of the present study was to immobilize the vascular endothelial growth factor (VEGF) onto Teflon PTFE surfaces to generate a proactive polymer construct favoring interaction with endothelial cells. An ammonia plasma treatment was first used to graft amino groups on PTFE films. Subsequent reactions were performed to covalently bind human serum albumin (HSA) on the polymer surface and to load this protein with negative charges, which allows adsorbtion of VEGF onto HSA via strong electrostatic interactions. X-ray photoelectron spectroscopy (XPS) experiments along with surface derivatization strategies were performed between each synthesis step to ascertain the occurrence of the various molecules surface immobilization. Finally, the electrostatic binding of VEGF to the negatively charged HSA matrix was performed and validated by ELISA. Endothelial cell adhesion and migration experiments were carried out to validate the potential of this VEGF-containing biological construct to act as a proactive media toward the development of endothelial cells.

Comparison of two classes of non-peptide drugs as antagonists of neutrophil receptors for f-Met-Leu-Phe. Pyrazolons and iodinated radiographic contrast agents.

The radiographic contrast agent sodium diatrizoate (DTR) reportedly inhibits f-Met-Leu-Phe-induced chemotaxis in human neutrophils. DTR is also an ingredient of Ficoll-Paque, a density centrifugation medium widely used to purify human polymorphonuclear leukocytes (PMNs). Exposure of PMNs to DTR during preparation had no detrimental effect on subsequent binding characteristics of tritiated f-Met-Leu-Phe, probably owing to a rapid dissociation of DTR from the PMN receptors. DTR competed directly with f-Met-Leu-Phe for receptor binding, but was 160- and 640-fold less potent than phenylbutazone and 1,2-diphenyl-4-[3-(1-naphthyl)-propyl]-3,5-pyrazolidinedione (DPN; an analog of phenylbutazone), respectively. Iohexol and the methylamide of DTR did not compete with [3H]f-Met-Leu-Phe in receptor binding, supporting the existence of a definite interaction between iodinated aromatic molecules and the f-Met-Leu-Phe receptor. DTR did not inhibit prostaglandin synthesis, as did DPN. Both drugs inhibited chemotactic peptide-induced release of superoxide anion in a concentration-dependent manner, and were relatively selective for f-Met-Leu-Phe, as opposed to C5a. Both drugs at 10 microM interfered non-selectively with chemotactic peptide-induced beta-glucuronidase release from PMNs. Available non-peptide antagonists of f-Met-Leu-Phe exhibited other pharmacodynamic properties that could make them unsuitable for future in vivo studies designed to probe the physiological role of the receptor.

Lack of cross-resistance to a new cytotoxic arylchloroethyl urea in various drug-resistant tumor cells.

1-Aryl 3-(2-chloroethyl) ureas (CEUs), a new class of potent antineoplastic agents, were recently developed in our laboratory. These compounds were designed from the aromatic moiety of chlorambucil and the unnitrosated pharmacophore of carmustine. In the present study we investigated the effect of the potent CEU derivative 4-tert-butyl-[3-(2-chloroethyl)ureido] benzene (tBCEU) on tumor cell lines selected for resistance to a wide range of anticancer drugs. The resistance mechanisms found in these cells included increased expression of P-glycoprotein, increased intracellular concentration of glutathione and/or glutathione-S-transferase activity, alteration of topoisomerase II, and increased DNA repair. Whereas the resistant cell lines were found to be highly resistant to a panel of clinically known anticancer drugs, tBCEU was found to be equally cytotoxic to both resistant and parental cells. The nitrobenzylpyridine assay indicated that tBCEU is a weaker alkylating agent than chlorambucil. This lack of cross-resistance in various resistant tumor cells suggests that tBCEU could be potentially useful in the treatment of cancers resistant to conventional anticancer drugs.

Development of an enzyme-linked immunosorbent assay for 2,3-dinor-6-keto-prostaglandin F1 alpha in urine using a monoclonal antibody.

OBJECTIVES: To develop and validate an enzyme-linked immunosorbent assay (ELISA) for measurement of urinary 2,3-dinor-6-keto-prostaglandin F1 alpha (2,3D6KPGF1 alpha) using a monoclonal antibody and a horseradish peroxidase-linked antigen. DESIGN AND METHODS: Assay validation included optimization of the standard curve, antibody cross-reactivity, accuracy and imprecision studies together with preliminary measurement of clinical samples. RESULTS: Optimal conditions of the standard curve (0.078-10.0 micrograms/L) used 2 mg/L of antibody and 3 micrograms/L of peroxidase conjugate in each well, at pH 7.2. The coefficient of variation of various concentrations of the standard curve averaged 6.8%. Antibody cross-reactivity was < 0.01% for related prostanoids. Recovery of known amounts (0.1-5.0 micrograms/L) of 2,3D6KPGF1 alpha added to an urinary sample was 101.2 +/- 6.3%. Imprecision studies with non-pregnant (0.24 microgram/L) and pregnant (2.5 micrograms/L) samples displayed an intraassay variability of 8.9 and 9.9%, and an interassay variability of 9.6 and 10.0%, respectively. Urinary measurements in the non-pregnant and pregnant states were similar to those previously reported. An apparent decreased concentration was observed early in pregnancy in future preeclampsia. CONCLUSION: With similar precision and validity, our assay method is time- and cost-saving. Preliminary urinary measurements show that this analyte may be of interest as an early marker for preeclampsia.

Down-regulation of interleukin-1beta production and PGE2 accumulation by an indomethacin-phenylalanine derivative in human monocytes.

This study was initiated to investigate the mechanism of action of a new indomethacin derivative, indomethacin-phenylalanine (indo-Phe) in human monocytes. We determined the effect of indo-Phe on the induction by LPS of prostaglandin-E2 (PGE2) and interleukin-1beta (IL-1beta) production in human monocytes. Indomethacin and indo-Phe inhibited the PGE2 synthesis in treated and untreated IL-1beta or LPS-treated monocytes. Furthermore, in IL-1beta and LPS-treated monocytes, prostaglandin G/H synthase-1 (PGHS-1) protein expression was down-regulated with indomethacin or its indo-Phe analog whereas the level of the inducible protein (PGHS-2) was up-regulated. We analyzed the effect of indomethacin and indo-Phe on the expression of IL-1beta protein in LPS-treated monocytes and found that indo-Phe blocked the LPS-induction of IL-1beta synthesis while indomethacin did not. These differential effects of indomethacin and indo-Phe suggest that two independent ways are involved in the stimulation of monocytes by LPS: the PGHS-2 protein induction and the IL-1beta secretion.

Membrane interactions of a new class of anticancer agents derived from arylchloroethylurea: a FTIR spectroscopic study.

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and different lipids such as dimyristoylphosphatidylcholine (DMPC) in the absence and presence of 30 mol% of cholesterol, dimyristoylphosphatidylglycerol (DMPG) and a mixture made of 1-palmitoyl-2-oleylphosphatidylcholine (POPC) and DMPC by Fourier transform infrared (FTIR) spectroscopy. The results indicate that the drugs incorporate in the bilayer and cause a decrease of the phase transition temperature and an increase of the conformational disorder of the lipid acyl chains. These effects are dependent on the nature (degree of branching, length of the alkyl chain and presence of a sulfur atom), as well as on the position of the R substituent and are related to the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky branched substituent and those for which the disordering effect on the lipid bilayer is the greatest. On the other hand, the disordering effect is small for the long chain CEUs, such as 4-n-hexadecyl CEU, which have been shown to have weak cytotoxic activity.

Erythrocyte membrane-bound daunorubicin as a delivery system in anticancer treatment.

Drug encapsulation in erythrocytes has been proposed to extend its biological lifetime. Molecules encapsulated this way are protected against rapid cellular metabolism and body elimination. Unfortunately, drugs such as daunorubicin cannot be efficiently entrapped in erythrocytes since drugs diffuse rapidly from the cells. In order to overcome this problem, we have covalently linked daunorubicin to erythrocyte membranes (ghosts) using two different types of linking arms: glutaraldehyde and cis-aconitic acid. Both ghost-daunorubicin conjugates were tested in vitro on mouse leukemia cells (P388D1) and on human osteosarcoma cells (CRL-1427). Results showed a better cytotoxic activity for ghost-glutaraldehyde-daunorubicin conjugate than for ghost-cis-aconityl-daunorubicin conjugate. Both conjugates were also tested in vivo on CDF1 mice bearing P388D1 cells. T/C% of 161 and 103 respectively were observed with ghost-glutaraldehyde-daunorubicin and ghost-cis-aconityl-daunorubicin conjugate at 6.0 mg/kg. Compared to free drug, the increase in survival time could be explained by a slow release of daunomycin in the circulation from the erythrocyte membranes, leading to a more complete absorption by cancer cells.

Nanoerythrosomes, a new derivative of erythrocyte ghost: III. Is phagocytosis involved in the mechanism of action?

We have previously developed a new drug carrier, named nanoerythrosome which is prepared by extrusion of erythrocyte ghosts to produce small vesicles having an average diameter of 100 nm. Daunorubicin (DNR) conjugated to these nanoerythrosomes has a higher antineoplastic index than the free drug. Moreover, since nanoerythrosomes are particles, phagocytosis may be involved in their mechanism of potentiation. In the present study, we have compared the mechanism of penetration between free DNR and conjugate DNR linked to nanoerythrosomes, on cells presenting high phagocytic activity, macrophages, and cells lacking phagocytic activity, the P388 D1 cell line. Our results demonstrate that: 1) The nanoerythrosome-DNR complex is rapidly adsorbed and phagocytosed by the macrophages, but not by the P388 D1 cell line. 2) On the contrary, DNR enters both phagocytic and non phagocytic cells. Furthermore, the cellular distribution of DNR is the same in both cell lines, the nucleus being the target organelle. We conclude that phagocytosis of the nanoerythrosome-DNR complex is not involved in its mechanism of action.

In vitro and in vivo activity of 1-aryl-3-(2-chloroethyl) urea derivatives as new antineoplastic agents.

A few 1-aryl 3-(2-chloroethyl)ureas (CEU) were synthesized and screened in vitro for their cytotoxicity. Some of these derivatives were assayed for their mutagenicity, their in vivo toxicity and their antineoplastic activity. Methyl 4-(p-(3-(2-chloroethyl) ureido) phenyl) butyrate, 4-methyl and 4-tertbutyl (3-(2-chloroethyl) ureido) phenyl) butyrate, 4-methyl and 4-tert-butyl (3-(2-chloroethyl) ureido) benzene had an ID50 of 28, 20 and 4 microM respectively when tested on LoVo cells, while chlorambucil (CBL) and CCNU had an ID50 of 21 and 45 microM. These 3 chloroethyl urea derivatives were not toxic when injected i.p. at doses up to 220 mg/kg, whereas chlorambucil was already toxic at 18.5 mg/kg. The survival time of BDF1 mice bearing L1210 leukemia tumors was significantly enhanced by intraperitoneal injections of CBL and CEU. The most cytotoxic derivative (tert-butyl derivative) gave the best antineoplastic activity with a median survival time 1.77 times that of the control at 10 mg/kg/day and was not toxic, whereas CBL at this concentration enhanced survival time by a factor of 1.6 and presented important side effects. The 4-tert-butyl (3-(2-chloroethyl) ureido) benzene and the methyl 4-(p-(3-(2-chloroethyl) ureido) phenyl) butyrate showed no mutagenicity when assayed on TA-97, TA-98, TA-100 and TA-102, four strains of S. thyphimurium, while CBL had a weak effect on TA-102 and CCNU was highly mutagenic on TA-100 and TA-102.

Effect of an aryl chloroethyl urea on tubulin and vimentin syntheses in a human breast cancer cell line.

A new class of antineoplastic agents, 1-aryl-3-(2-chloroethyl) ureas (CEUs), was recently developed in our laboratory. To optimize the pharmacological and the biological properties of this new class of compounds and to determine its mechanism of action, at the cellular level, we studied the effect of 4-tert-butyl-[3-(2-chloroethyl) ureido] benzene (tBCEU) on MDA-MB-231, a human breast cancer hormone-independent cell line. The effect of tBCEU on protein synthesis and on the accumulation of specific mRNAs was evaluated. The results indicate that tBCEU increases the synthesis of at least two proteins present in the cytoskeleton: tubulin and vimentin. The effect of tBCEU on their transcripts indicates that tBCEU decreases the accumulation of tubulin and vimentin mRNA. These results suggest that the antineoplastic activity of tBCEU is in part related to an alteration in the synthesis pathway of tubulin and vimentin.

Nanoerythrosomes, a new derivative of erythrocyte ghost II: identification of the mechanism of action.

We have recently developed a new drug which is a carrier from red blood cell membrane. This carrier, named nanoerythrosome (nEryt), is prepared by extrusion of erythrocyte ghosts to produce small vesicles having an average diameter of 100 nm. Daunorubicin (DNR) was covalently conjugated to the nEryt (nEryt-DNR) using glutaraldehyde as homobifunctional linking arm. This led to a complex that is more active than free DNR both in vitro and in vivo. In this study, we identified the mechanisms that make the complex nEryt-DNR more active than free DNR. Using fluorescence microscopy and cellular-uptake, we observed that the nEryt-DNR complex cannot diffuse through the cell membrane and do not enter the cell by endocytosis. Our results suggest that the nEryt-DNR is rapidly absorbed onto the cell membrane. Free DNR is then slowly released by hydrolysis of the glutaraldehyde linking arm, producing a high concentration of free DNR in the cell's vicinity over a long period of time.

1-Aryl-3-(2-chloroethyl) ureas: synthesis and in vitro assay as potential anticancer agents.

1-Aryl-3-(2-chloroethyl) ureas and 1-aryl-3-nitroso-3-(2-chloroethyl) ureas, derived from 4-phenylbutyric acid and alkylanilines, were synthesized and their cytotoxicity was evaluated on human adenocarcinoma cells in vitro. Methyl 4-[p-[3-(2-chloroethyl)ureido]-phenyl]butyrate, 4-methyl [3-(2-chloroethyl)ureido]benzene, and 4-butyl[3-(2-chloroethyl)ureido]benzene were found to be at least as cytotoxic as 4-[p-[bis-(2-chloroethyl)amino]phenyl]butyric acid (chlorambucil), while their N-nitroso derivatives were inactive.

Fluorescence determination of microconcentrations of chlorambucil after photoactivation.

A fluorescence assay is described which measures the alkylating activity of chlorambucil or its isocyanate derivative after photoactivation in the presence of dimethyl sulfoxide. This assay has a lower limit of sensitivity of 100 ng/mL and RSD of less than 10% for chlorambucil. The method requires less than 5 micrograms of alkylating agent and the fluorophore produced is stable for at least 24 h.

The interaction of 3,5-pyrazolidinedione drugs with receptors for f-Met-Leu-Phe on human neutrophil leukocytes: a study of the structure-activity relationship.

The 3,5-pyrazolidinedione (3,5-P) drugs, phenylbutazone and sulfinpyrazone, have been reported to bind to receptors for the chemotactic peptide, f-Met-Leu-Phe, and to behave as functional antagonists of f-Met-Leu-Phe in human and rabbit neutrophils. To explore the structure-activity relationship of this family of drugs for f-Met-Leu-Phe receptor binding, 36 drugs with the 3,5-P structure, a structure related to antipyrine, or an unrelated structure were tested as competitors for the binding of f-Met-Leu-Phe-Lys-fluorescein isothiocyanate on human neutrophils by flow cytometric analysis. Only drugs possessing the 3,5-P ring were significant competitors. The five most potent 3,5-Ps behaved as selective antagonists of f-Met-Leu-Phe-induced superoxide anion release by neutrophils. The potency was not correlated to the pKa or to their capacity to inhibit prostaglandin E2 released from culture fibroblasts but instead appeared to be correlated to their apparent octanol-buffer partition coefficients. The most potent f-Met-Leu-Phe antagonist identified, 1,2-diphenyl-4-(3-(1-naphthyl)-propyl)-3,5-pyrazolidinedione (DPN), may also possess an improved pharmacodynamic specificity compared with phenylbutazone and sulfinpyrazone, as it was less potent than phenylbutazone in the inhibition of prostaglandin synthesis and it was not cytotoxic. DPN may be a prototype for a valuable new class of anti-inflammatory drugs.

Synthesis and preliminary in vitro cytotoxic activity of novel cisplatin and carboplatin analogues.

Six new platinum(II) complexes were synthesized from a common triphenylethylene precursor using various diamines. The cytotoxicity of the compounds, evaluated on human breast cancer cell lines (MCF-7 and MDA-MB-231), was greatly influenced by the nature of the diamine ligand. Two derivatives presented cytotoxic activity greater than tamoxifen and, for the first time, as potent as cisplatin.

Porosity and biological properties of polyethylene glycol-conjugated collagen materials.

Collagen-based materials can be designed for use as scaffolds for connective tissue reconstruction. The goal of the present study was to evaluate the behavior of collagen materials as well as cell and tissue reactions after the conjugation of activated polyethylene glycols (PEGs) with collagen. It is known that proteins conjugated with PEGs exhibit a decrease in their biodegradation rate and their immunogenicity. Different concentrations and molecular weights of activated PEGs (PEG-750 and PEG-5000) were conjugated to collagen materials (films or sponges) which were then investigated by collagenase assay, fibroblast cell culture, and subcutaneous implantation. PEG-conjugated collagen sponge degradation by collagenase was delayed in comparison to untreated sponges. In culture, fibroblasts with a normal morphology reached confluency on PEG-conjugated collagen films. In vivo, the porous structure of non-modified sponges collapsed by day 15 with a few observable fibroblasts between the collagen fibers. In PEG-modified collagen sponges, the porous structure remained stable for 30 days. Cell infiltration was particularly enhanced in PEG-750-conjugated collagen sponges. In conclusion, PEGs conjugated onto collagen sponges stabilize the porous structure without deactivating the biological properties of collagen. These porous composite materials could function as a scaffold to organize tissue ingrowth.

Disposition and metabolism of a novel antineoplastic agent, 4-tert-butyl-[3-(2-chloroethyl)ureido]benzene, in mice.

1-Aryl-3-(2-chloroethyl)ureas are new agents that have shown promising cytotoxic and antineoplastic activities. In this work, we studied the disposition and metabolism of one of these molecules, 4-tert-butyl-[3-(2-chloroethyl)ureido]benzene (tBCEU). tBCEU was labeled with 14C and 13C in the urea function and in the chloroethyl moiety. After ip administration of the molecule labeled in the urea function, radioactivity was widely distributed in the whole organism, including the brain. HPLC analysis of plasma showed that tBCEU was extensively metabolized, with <20% being found in the plasma as unchanged tBCEU 1 hr after administration. One main metabolite was identified by NMR and MS analysis as N-[4-(2-hydroxy-1, 1-dimethylethyl)phenyl]urea, widely conjugated to glucuronic acid. The same metabolite was found in the urine. After administration of tBCEU labeled in the chloroethyl moiety, the same tissue affinities were observed, but the decrease of total radioactivity in blood and tissues was slower than that observed for the molecule labeled in the urea function. HPLC analysis of urine showed the presence of two main metabolites, identified by MS as thiodiacetic acid and its sulfoxide. From these results, we can deduce that the metabolic pathway of tBCEU involves N-dealkylation of the urea portion of the molecule and hydroxylation of the tert-butyl group. The strong cytochrome P450 reactivity of the carbon adjacent to the urea portion of tBCEU is probably related to particular sensitivity to oxidation at this position, based on the chemical structure of tBCEU. These results can explain the fact that the cytotoxic effect of tBCEU is not due to DNA alkylation, in contrast to that of its parent molecule, chloroethylnitrosourea.