Considerations for the Terminal Sterilization of Oligonucleotide Drug Products.

A primary function of the parenteral drug product manufacturing process is to ensure sterility of the final product. The two most common methods for sterilizing parenteral drug products are terminal sterilization (TS), whereby the drug product is sterilized in the final container following filling and finish, and membrane sterilization, whereby the product stream is sterilized by membrane filtration and filled into presterilized containers in an aseptic processing environment. Although TS provides greater sterility assurance than membrane sterilization and aseptic processing, not all drug products are amenable to TS processes, which typically involve heat treatment or exposure to ionizing radiation. Oligonucleotides represent an emerging class of therapeutics with great potential for treating a broad range of indications, including previously undruggable targets. Owing to their size, structural complexity, and relative lack of governing regulations, several challenges in drug development are unique to oligonucleotides. This exceptionality justifies a focused assessment of traditional chemistry, manufacturing, and control strategies before their adoption. In this article, we review the current state of sterile oligonucleotide drug product processing, highlight the key aspects to consider when assessing options for product sterilization, and provide recommendations to aid in the successful evaluation and development of TS processes. We also explore current regulatory expectations and provide our interpretation as it pertains to oligonucleotide drug products.

Sizing up the Next Generation of Nanomedicines.

During the past two decades the nanomedicine field has experienced significant progress. To date, over sixty nanoparticle (NP) formulations have been approved in the US and EU while many others are in clinical or preclinical development, indicating a concerted effort to translate promising bench research to commercially viable pharmaceutical products. The use of NPs as novel drug delivery systems, for example, can improve drug safety and efficacy profiles and enable access to intracellular domains of diseased cells, thus paving the way to previously intractable biological targets. However, the measurement of their physicochemical properties presents substantial challenges relative to conventional injectable formulations. In this perspective, we focus exclusively on particle size, a core property and critical quality attribute of nanomedicines. We present an overview of relevant state-of-the-art technologies for particle sizing, highlighting the main parameters that can influence the selection of techniques suitable for a specific size range or material. We consider the increasing need, and associated challenge, to measure size in physiologically relevant media. We detail the importance of standards, key to validate any measurement, and the need for suitable reference materials for processes used to characterize novel and complex NPs. This perspective highlights issues critical to achieve compliance with regulatory guidelines and to support research and manufacturing quality control.

Influence of molecular organization and interactions on drug release for an injectable polymer-lipid blend.

An injectable blend composed of a water soluble chitosan (WSC) derivative, egg phosphatidylcholine (ePC), and fatty acid chlorides (FACl) was explored for localized delivery of anticancer agents. The composition-property relationships of the injectable WSC-FACl-ePC blend were determined by investigating the physico-chemical and performance properties of the blend as a function of the ratio of the components, as well as the acyl chain length of the FACl (C10-C16) employed. Thermal and rheological measurements revealed that the melting transitions and viscosities of the blends increased as a function of FACl acyl chain length. FTIR analysis demonstrated that the stability of the blends was attributed to the specific interactions among the molecules. In addition, confocal laser scanning microscopy revealed that the incorporation of C10-C16 FACl altered the molecular organization of ePC and WSC within the blends, which resulted in distinct physico-chemical properties. Specifically, the formation of micro-domains within the blends increased the stability, as well as delayed the release of paclitaxel from the formulation under physiologically relevant conditions. Overall, the interactions identified among the components, and the relationships established between the composition and properties of the blend can be used as a tool to develop advanced injectable drug delivery systems for pharmaceutical applications.

Gold-labeled block copolymer micelles reveal gold aggregates at multiple subcellular sites.

There is increasing interest in the usefulness of block copolymer micelles as drug delivery vehicles. However, their subcellular distribution has not been explored extensively, mostly because of the lack of adequately labeled block copolymers. In a previous study, we showed that fluorescently labeled block copolymer micelles entered living cells and co-localized with cytoplasmic organelles selectively labeled with fluorescent dyes. The details of the observed co-localizations were, however, limited by the resolution of the fluorescence approach, which is ca. 500 nm. Using transmission electron microscopy (TEM), we established time- and concentration-dependent subcellular distributions of gold-labeled micelles within human embryonic kidney (HEK 293) cells and human lung carcinoma (A549) cells. Gold particles were incorporated into poly(4-vinylpyridine)-block-poly(ethylene oxide) (P4VP21-b-PEO45) micelles. Data from dynamic light scattering (DLS) and TEM analyses revealed that the sizes of the gold particles ranged from 4 to 8 nm. The cells survived up to 24 h in the presence of low gold-labeled micelle concentrations (0.73 microg/mL), but cell death occurred at higher concentrations (i.e., kidney cells are more susceptible than lung cells). Over 24 h periods of equivalent exposure, lung cells internalized significantly more gold-incorporated micelles than kidney cells. Although micelles were added to the cell culture media as dispersed colloidal particles, the presence of serum in these media caused aggregation. These aggregates occurred mainly close to the cell plasma membrane at early times (5-10 min); however, at later times (24 h) aggregated particles were seen inside endosomes and lysozomes. Thus, gold-incorporated (labeled) micelles can serve as a valuable extension of the fluorescence approach to visualizing the localization of micelles in subcellular compartments, improving the resolution by at least 20-fold.

Impact of intraperitoneal, sustained delivery of paclitaxel on the expression of P-glycoprotein in ovarian tumors.

Recently, we developed a novel implantable drug delivery system which can provide sustained intraperitoneal (i.p.) delivery of paclitaxel (PTX). As the impact of local sustained delivery on the development of multidrug resistance (MDR) is unknown, the objective of this study was to determine the impact of this drug delivery system on the in vivo expression of MDR1/P-glycoprotein (PGP) in a human ovarian xenograft tumor model. As compared to controls, intermittent i.p. dosing with PTX formulated in Cremophor EL (PTX(CrEL)) induced a two-fold increase in mRNA levels of MDR1 after a 14-day dosing period. On the other hand, sustained i.p. delivery of PTX with the implant system (PTX(film)) did not significantly affect MDR1 expression. Immunodetection of PGP in isolated xenografts supported the mRNA data. Histological analysis by H&E staining demonstrated a dose-dependent increase in tumor necrosis in the PTX(film) treated animals. Further, in vitro studies in human ovarian carcinoma cells also demonstrated a significant induction in the efflux activity of PGP with intermittent dosing schedules to PTX(CrEL) whereas this was not seen in cells dosed with PTX(film). Our findings suggest that sustained i.p. administration with PTX(film) attenuates development of MDR, suggesting that sustained, localized delivery of chemotherapeutic agents may improve current treatment strategies for ovarian cancer.

Active loading and tunable release of doxorubicin from block copolymer vesicles.

Vesicles are spherical bilayers that offer a hydrophilic reservoir, suitable for the incorporation of water-soluble molecules, as well as a hydrophobic wall that protects the loaded molecules from the external solution. The permeability of a vesicle wall made from polystyrene can be enhanced by adding a plasticizer such as dioxane. Tuning the wall permeability allows loading and release of molecules from vesicles to be controlled. In this study, vesicles are prepared from polystyrene(310)-b-poly(acrylic acid)(36) and used as model carriers for doxorubicin (DXR), a weak amine and a widely used anticancer drug. To increase the wall permeability, different amounts of dioxane are added to the vesicle solution. A pH gradient is created across the vesicle wall (inside acidic) and used as an active loading method to concentrate the drug inside the vesicles. The results show that a pH gradient of ca. 3.8 units can enhance the loading level up to 10-fold relative to loading in the absence of the gradient. After loading, the release of DXR from vesicles is followed as a function of the wall permeability. The diffusion coefficient of doxorubicin through polystyrene (D) is evaluated from the initial slope of the release curves; the value of D ranges from 8 x 10(-17) to 6 x 10(-16) cm(2)/s, depending on the degree of plasticization of the vesicle wall.

Influence of metalation on the morphologies of poly(ethylene oxide)-block-poly(4-vinylpyridine) block copolymer micelles.

Micellization of a poly(ethylene oxide)-block-poly(4-vinylpyridine) (PEO45-b-P4VP28) copolymer in water during metalation (incorporation of gold compounds and gold nanoparticle formation) with three types of gold compounds, NaAuCl4, HAuCl4, and AuCl3, was studied using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The transformations of the PEO45-b-P4VP28 block copolymer micelles in water were found to depend on a number of parameters including the thermal history of the as-prepared block copolymer, the type of the metal compound, and the metal loading. For the HAuCl4-filled PE045-b-P4VP28 micelles, the subsequent reduction with hydrazine hydrate results in a significant fraction of rodlike micelles, suggesting that slow nucleation (confirmed by the formation of the large gold nanoparticles) and facilitated migration of gold ions yields the ideal conditions for sphere-to-rod micellar transition.