Adsorption of Small Cationic Nanoparticles onto Large Anionic Particles from Aqueous Solution: A Model System for Understanding Pigment Dispersion and the Problem of Effective Particle Density.

The present study focuses on the use of copolymer nanoparticles as a dispersant for a model pigment (silica). Reversible addition-fragmentation chain transfer (RAFT) alcoholic dispersion polymerization was used to synthesize sterically stabilized diblock copolymer nanoparticles. The steric stabilizer block was poly(2-(dimethylamino)ethyl methacrylate) (PDMA) and the core-forming block was poly(benzyl methacrylate) (PBzMA). The mean degrees of polymerization for the PDMA and PBzMA blocks were 71 and 100, respectively. Transmission electron microscopy (TEM) studies confirmed a near-monodisperse spherical morphology, while dynamic light scattering (DLS) studies indicated an intensity-average diameter of 30 nm. Small-angle X-ray scattering (SAXS) reported a volume-average diameter of 29 ± 0.5 nm and a mean aggregation number of 154. Aqueous electrophoresis measurements confirmed that these PDMA71-PBzMA100 nanoparticles acquired cationic character when transferred from ethanol to water as a result of protonation of the weakly basic PDMA chains. Electrostatic adsorption of these nanoparticles from aqueous solution onto 470 nm silica particles led to either flocculation at submonolayer coverage or steric stabilization at or above monolayer coverage, as judged by DLS. This technique indicated that saturation coverage was achieved on addition of approximately 465 copolymer nanoparticles per silica particle, which corresponds to a fractional surface coverage of around 0.42. These adsorption data were corroborated using thermogravimetry, UV spectroscopy and X-ray photoelectron spectroscopy. TEM studies indicated that the cationic nanoparticles remained intact on the silica surface after electrostatic adsorption, while aqueous electrophoresis confirmed that surface charge reversal occurred below pH 7. The relatively thick layer of adsorbed nanoparticles led to a significant reduction in the effective particle density of the silica particles from 1.99 g cm-3 to approximately 1.74 g cm-3, as judged by disk centrifuge photosedimentometry (DCP). Combining the DCP and SAXS data suggests that essentially no deformation of the PBzMA cores occurs during nanoparticle adsorption onto the silica particles.

Combination therapy for treating breast cancer using antiestrogen, ERA-923, and the mammalian target of rapamycin inhibitor, temsirolimus.

The effect of combinations of a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, and an estrogen receptor-alpha (ERalpha) antagonist, ERA-923, on breast carcinoma in culture and in a xenograft model has been studied. Phase III trials are underway using temsirolimus for several cancers. ERA-923 was studied in a phase I trial for tamoxifen refractory metastatic breast cancer and was shown to have good safety profiles. Combination of noninhibitory doses of temsirolimus with suboptimal doses of ERA-923 synergistically inhibited the growth of MCF-7 cells. Synergy was found across a wide range of doses and could also be achieved by combining temsirolimus with other antiestrogens such as raloxifene and 4-hydroxytamoxifen. In vivo combination of temsirolimus and ERA-923 at certain doses and schedules completely inhibited tumor growth, while individual agents were only partially effective. Although the mechanism underlying the synergism remains to be understood, the results were associated with the ability of temsirolimus to block the transcriptional activity mediated by ERalpha as well as an increase in G1 arrest when it was combined with ERA-923. Results demonstrated for the first time that the combination of temsirolimus and a pure antiestrogen has excellent anticancer activity in preclinical models and, therefore, may have clinical use in treating hormone-dependent tumors.

Luminescence techniques and characterization of the morphology of polymer latices 2. Fluorescence lifetime, phosphorescence and fluorescence anisotropy studies.

Five poly(n-butyl methacrylate), PBMA, latex dispersions have been prepared, each incorporating a different fluorescent label, via a two-stage seeded emulsion polymerization. The resultant latices contain ca. 35% by weight total solids and are of 80 (+/-10) nm diameter as determined by photon correlation spectrometry. Luminescence spectroscopic techniques, namely fluorescence (and phosphorescence) excited state lifetime measurements in addition to time-resolved anisotropy experiments have provided useful information regarding the morphology, microviscosity and water permeability of the resultant particles. A picture of the PBMA colloid emerges of an interior which is highly viscous and water impermeable in nature. Indeed, the environment is protective enough to sustain room temperature stabilized phosphorescence from both an acenaphthylene and 9-phenanthrylmethyl methacrylate labeled dispersion through simple nitrogen purging of the solutions. However, the current spectroscopic measurements should be viewed with the knowledge that each luminescent label may fashion its own distinctive microenvironment within the latex during polymerization.

Luminescence techniques and characterization of the morphology of polymer latices. 3. An investigation of the microenvironments within stabilized aqueous latex dispersions of poly(n-butyl methacrylate) and polyurethane.

Fluorescence techniques (including time-resolved anisotropy measurements, TRAMS) have been used to probe differences in morphology between two stabilized aqueous latex dispersions (poly(n-butyl methacrylate), PBMA, and polyurethane, PU). Use of the emission characteristics of probes such as pyrene and phenanthrene dispersed within particles reveals that the PU latices are more heterogeneous in nature: evidence exists, particularly from quenching measurements and TRAMS, that voids and channels of water permeate the PU structure, resulting in a relatively soft, open particle, swollen by ingress of the bulk aqueous phase. Fluorescence measurements indicate that PBMA colloids, however, are composed of relatively hard, hydrophobic particles. In addition, TRAMS are considered to be a valuable tool both for probing the morphological characteristics of such dispersions and in estimating the average particle size.

A new antiestrogen, 2-(4-hydroxy-phenyl)-3-methyl-1-[4-(2-piperidin-1-yl-ethoxy)-benzyl]-1H-indol-5-ol hydrochloride (ERA-923), inhibits the growth of tamoxifen-sensitive and -resistant tumors and is devoid of uterotropic effects in mice and rats.

PURPOSE: Tamoxifen is an antiestrogen used in women who have estrogen receptor (ER)-alpha-positive breast cancer. Unfortunately, resistance to tamoxifen is common in women with metastatic disease and side effects, including increased risk of endometrial cancer, exist. Here we describe the activity of a new selective ER modulator, ERA-923, in preclinical models focused on these limitations. EXPERIMENTAL DESIGN: The ability of ERA-923, 4-OH tamoxifen, or raloxifene to inhibit estrogen-stimulated growth was evaluated in cell-based and xenograft assays with tumor cells that are sensitive or resistant to tamoxifen. Uterine effects of selective ER modulators were compared in rodents. RESULTS: ERA-923 potently inhibits estrogen binding to ER-alpha (IC(50), 14 nM). In ER-alpha-positive human MCF-7 breast carcinoma cells, ERA-923 inhibits estrogen-stimulated growth (IC(50), 0.2 nM) associated with cytostasis. In vitro, a MCF-7 variant with inherent resistance to tamoxifen (10-fold) or 4-OH tamoxifen (>1000-fold) retains complete sensitivity to ERA-923. Partial sensitivity to ERA-923 exists in MCF-7 variants that have acquired profound tamoxifen resistance. In tumor-bearing animals, ERA-923 (10 mg/kg/day given p.o.) inhibits 17beta-estradiol-stimulated growth in human tumors derived from MCF-7, EnCa-101 endometrial, or BG-1 ovarian carcinoma cells, including a MCF-7-variant that is inherently resistant to tamoxifen. Raloxifene is inactive in the MCF-7 xenograft model. Unlike tamoxifen, droloxifene, or raloxifene, ERA-923 is not uterotropic in immature rats or ovariectomized mice. Consistent with this, tamoxifen, but not ERA-923, stimulates the growth of EnCa-101 tumors. CONCLUSIONS: In preclinical models, ERA-923 has an improved efficacy and safety compared with tamoxifen. Clinical trials with ERA-923 are in progress.

Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C.

We selected a human colon carcinoma cell line in increasing concentrations of mitoxantrone to obtain a resistant subline, S1-M1-3.2, with the following characteristics: profound resistance to mitoxantrone; significant cross-resistance to doxorubicin, bisantrene, and topotecan; and very low levels of resistance to Taxol, vinblastine, colchicine, and camptothecin. This multidrug resistance (MDR) phenotype, which was not reversed by verapamil or another potent P-glycoprotein (Pgp) inhibitor, CL 329,753, was dependent, in part, upon an energy-dependent drug efflux mechanism. Pgp and the multidrug resistance protein (MRP) were not elevated in the resistant cells relative to the drug-sensitive parent, suggesting that resistance was mediated by a novel pathway of drug transport. A cell-based screen with S1-M1-3.2 cells was used to identify agents capable of circumventing this non-Pgp, non-MRP MDR. One of the active agents identified was a mycotoxin, fumitremorgin C. This molecule was extremely effective in reversing resistance to mitoxantrone, doxorubicin, and topotecan in multidrug-selected cell lines showing this novel phenotype. Reversal of resistance was associated with an increase in drug accumulation. The compound did not reverse drug resistance in cells with elevated expression of Pgp or MRP. We suggest that fumitremorgin C is a highly selective chemosensitizing agent for the resistance pathway we have identified and can be used as a specific pharmacological probe to distinguish between the diverse resistance mechanisms that occur in the MDR cell.

alpha-(3,4-dimethyoxyphenyl)-3,4-dihydro-6,7-dimethoxy-alpha- [(4-methylphenyl)thio]-2(1H)-isoquinolineheptanenitrile (CL 329,753): a novel chemosensitizing agent for P-glycoprotein-mediated resistance with improved biological properties compared with verapamil and cyclosporine A.

Agents that inhibit P-glycoprotein may restore sensitivity to some antitumor drugs in cancer patients. Optimization of the specificity and potency of one class of chemosensitizing agents related to verapamil has led to the identification of alpha-(3,4-dimethyoxyphenyl)-3,4-dihydro-6, 7-dimethoxy-alpha-[(4-methylphenyl) thio]-2(1H)-isoquinolineheptanenitrile, designated CL 329,753. In vitro, 0.1 to 2.0 microM CL 329,753 restored sensitivity to drugs in the multidrug resistance (MDR) phenotype in cell lines that overexpress P-glycoprotein. CL 329,753 was greater than 10-fold more potent and efficacious than cyclosporine A or verapamil in vitro, particularly in cells that express high levels of P-glycoprotein. The enhanced activity of CL 329,753 may be related to its inability to be transported by P-glycoprotein, since low drug accumulation of cyclosporine or verapamil but not CL 329,753 was found in P-glycoprotein-containing cells, yet all three agents inhibited vinblastine binding to membranes containing P-glycoprotein and inhibited photoaffinity labeling of P-glycoprotein. In vivo, CL 329,753 resensitized drug-resistant tumors to vinblastine or doxorubicin in an ascitic or solid tumor model, respectively. No alteration in the plasma pharmacokinetic profile of doxorubicin by CL 329,753 has been found. Furthermore, the compound had 70-fold less calcium channel antagonistic activity compared with verapamil.

P-glycoprotein mediates profound resistance to bisantrene.

Bisantrene, mitoxantrone, and anthracyclines are anthracene derivatives that interact with DNA and are used for the treatment of cancers. The mechanisms of resistance to bisantrene are unknown. Here we show that cells that overexpress low levels of P-glycoprotein or are transfected with human MDR1 have approximately 10-fold greater resistance to bisantrene compared to vinblastine, doxorubicin, or colchicine. Furthermore, bisantrene can be used to select for high-level P-glycoprotein-mediated multiple drug resistance in a human colon carcinoma cell line, LS 174T, and the drug blocks photoaffinity labeling of P-glycoprotein. The data suggest that bisantrene is an excellent substrate for P-glycoprotein. These findings could influence subsequent clinical evaluation of bisantrene for the treatment of cancer.