Pharmacological characterization of chemically synthesized monomeric phi29 pRNA nanoparticles for systemic delivery.

Previous studies have shown that the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor folds into a compact structure, constituting a RNA nanoparticle that can be modularized with functional groups as a nanodelivery system. pRNA nanoparticles can also be self-assembled by the bipartite approach without altering folding property. The present study demonstrated that 2'-F-modified pRNA nanoparticles were readily manufactured through this scalable bipartite strategy, featuring total chemical synthesis and permitting diverse functional modularizations. The RNA nanoparticles were chemically and metabolically stable and demonstrated a favorable pharmacokinetic (PK) profile in mice (half-life (T(1/2)): 5-10 hours, clearance (Cl): <0.13 l/kg/hour, volume of distribution (V(d)): 1.2 l/kg). It did not induce an interferon (IFN) response nor did it induce cytokine production in mice. Repeat intravenous administrations in mice up to 30 mg/kg did not result in any toxicity. Fluorescent folate-pRNA nanoparticles efficiently and specifically bound and internalized to folate receptor (FR)-bearing cancer cells in vitro. It also specifically and dose-dependently targeted to FR(+) xenograft tumor in mice with minimal accumulation in normal tissues. This first comprehensive pharmacological study suggests that the pRNA nanoparticle had all the preferred pharmacological features to serve as an efficient nanodelivery platform for broad medical applications.

Synthesis and characterization of bioactive tamoxifen-conjugated polymers.

Macromolecular conjugates of tamoxifen could perhaps be used to circumvent some of the limitations of the extensively used breast cancer drug. To test the feasibility of these conjugates, a 4-hydroxytamoxifen analogue was conjugated to a diaminoalkyl linker and then conjugated to activated esters of a poly(methacrylic acid) polymer synthesized by atom transfer radical polymerization. A polymer conjugated to the 4-hydroxytamoxifen analogue with a six-carbon linker showed high affinity for both estrogen receptor alpha and estrogen receptor beta and potent antagonism of the estrogen receptor in cell-based transcriptional reporter assays. These results suggest that the conjugation of 4-hydroxytamoxifen to a polymer results in a macromolecular conjugate that can display ligand in a manner that can be recognized by estrogen receptor and still act as a potent antiestrogen in cells.

Tamoxifen stimulates calcium entry into human platelets.

The anti-estrogenic drug tamoxifen, which is used therapeutically for treatment and prevention of breast cancer, can lead to the development of thrombosis. We found that tamoxifen rapidly increased intracellular free calcium [Ca2+]i in human platelets from both male and female donors. Thus 10 microM tamoxifen increased [Ca2+]i above the resting level by 197 +/- 19%. Tamoxifen acted synergistically with thrombin, ADP, and vasopressin to increase [Ca2+]i. The anti-estrogen ICI 182780 did not attenuate the effects of tamoxifen to increase [Ca2+]i; however, phospholipase C inhibitor U-73122 blocked this effect. 4-hydroxytamoxifen, a major metabolite of tamoxifen, also increased [Ca2+]i, but other tamoxifen metabolites and synthetic derivatives did not. Three hydroxylated derivatives of triphenylethylene (corresponding to the hydrophobic core of tamoxifen) which are transitional structures between tamoxifen (Ca agonist) and diethylstilbestrol (Ca antagonist) increased [Ca2+]i slightly (6% to 24%) and partially inhibited thrombin-induced [Ca2+]i elevation (68% to 79%). Therefore the dimethylaminoethyl moiety is responsible for tamoxifen being a Ca agonist rather than antagonist. 4-Hydroxytamoxifen and polymer-conjugated derivatives of 4-hydroxytamoxifen increased [Ca2+]i, with similar efficacy. The ability of tamoxifen to increase [Ca2+]i in platelets, leading to platelet activation, and its ability to act synergistically with other platelet agonists may contribute to development of tamoxifen-induced thrombosis.

Pharmacological characterization of 4-hydroxy-N-desmethyl tamoxifen, a novel active metabolite of tamoxifen.

The antiestrogen tamoxifen is extensively metabolized in patients to form a series of compounds with altered affinity for estrogen receptors (ERs), the primary target of this drug. Furthermore, these metabolites exhibit a range of partial agonist and antagonist activities for ER mediated effects that do not depend directly on their absolute affinity for ERs. Thus, clinical response to tamoxifen therapy is likely to depend on the aggregate effect of these different metabolites resulting from their abundance in the patient, their affinity for the receptors, and their agonist/antagonist profile. A recent study has shown that plasma concentrations of the tamoxifen metabolite 4-hydroxy- N -desmethyl tamoxifen (endoxifen), in patents undergoing tamoxifen therapy, are dependent on the cytochrome p450 (CYP) 206 ge notype of the patient and that medications commonly prescribed to patients on tamoxifen therapy can also inhibit endoxifen production. In this study we characterized the properties of this metabolite with respect to binding to ERs, ability to inhibit estrogen stimulated breast cancer cell proliferation and the regulation of estrogen responsive genes. We demonstrate that endoxifen has essentially equivalent activity to the potent metabolite 4-hydroxy tamoxifen (4-OH-tam) often described as the active metabolite of this drug. Since plasma levels of endoxifen in patients with functional CYP2D6 frequently exceed the levels of 4-OH-tam, it seems likely that endoxifen is at least as important as 4-OH-tam to the overall activity of this drug and suggests that CYP2D6 status and concomitant administration of drugs that inhibit CYP2D6 activity have the potential to affect response to tamoxifen therapy.