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.

Patient Centricity Driving Formulation Innovation: Improvements in Patient Care Facilitated by Novel Therapeutics and Drug Delivery Technologies.

Innovative formulation technologies can play a crucial role in transforming a novel molecule to a medicine that significantly enhances patients' lives. Improved mechanistic understanding of diseases has inspired researchers to expand the druggable space using new therapeutic modalities such as interfering RNA, protein degraders, and novel formats of monoclonal antibodies. Sophisticated formulation strategies are needed to deliver the drugs to their sites of action and to achieve patient centricity, exemplified by messenger RNA vaccines and oral peptides. Moreover, access to medical information via digital platforms has resulted in better-informed patient groups that are requesting consideration of their needs during drug development. This request is consistent with health authority efforts to upgrade their regulations to advance age-appropriate product development for patients. This review describes formulation innovations contributingto improvements in patient care: convenience of administration, preferred route of administration, reducing dosing burden, and achieving targeted delivery of new modalities.

Technical Considerations for Use of Oligonucleotide Solution API.

The most common approach for the manufacture of oligonucleotides includes isolation of the active pharmaceutical ingredient (API) via lyophilization to provide a solid product, which is then dissolved to provide an aqueous formulation. It is well known from the development and manufacture of large molecules ("biologics") that API production does not always require isolation of solid API before drug product formulation, and this article provides technical considerations for the analogous use of oligonucleotide API in solution. The primary factor considered is solution stability, and additional factors such as viscosity, concentration, end-to-end manufacturing, microbiological control, packaging, and storage are also discussed. The technical considerations discussed in this article will aid the careful evaluation of the relative advantages and disadvantages of solution versus powder API for a given oligonucleotide drug substance.

Stability and compatibility of Basmisanil granules co-administered with soft food.

Co-administration of solid oral dosage forms with soft food or beverages is commonly used to facilitate administration and to improve compliance in the paediatric and geriatric population and in patient groups with swallowing difficulties. The present case study was conducted to investigate the compatibility, stability and dissolution of Basmisanil administered as granules mixed with different soft food matrices. The data were generated to justify dosing instructions, according which Basmisanil should be sprinkled on or mixed with one tablespoon of soft food to aid swallowing. Different soft food types were selected to cover a broad range of various food components (e.g. fat, protein, carbohydrates, fiber and water) and pH. Active content and degradation products of the active substance were determined after mixing the granules with the semisolid food matrix and after two hours of storage under ambient conditions, respectively. In-vitro dissolution tests of granule/food mixtures were also conducted. Furthermore, the stability of the API polymorph was evaluated. Basmisanil shows good chemical stability when the granules are mixed with soft food and consumed within two hours. No polymorphic conversion (anhydrate to monohydrate) could be detected in the granule/food mixtures after preparation and after storage up to 24 h. The in-vitro dissolution of the API from the granules was not adversely affected by the presence of the food matrix. All results were comparable regardless of the tested food matrix. The results do not prohibit the administration of the granules with soft food to the patient.

Development of stable liquid formulations for oligonucleotides.

Oligonucleotide-based therapeutics have been implemented as a new therapeutic modality in biotech industry, which offers the opportunity to develop formulation platforms for robust parenteral formulations. The aim of this study was to gain a better understanding of stabilizing/de-stabilizing effects of different formulation parameters on unconjugated and N-acetylgalactosamine (GalNAc) conjugated single stranded oligonucleotides with locked nucleic acid modifications (LNA SSO), as model oligonucleotides. Various buffer systems, pH levels and different excipients were evaluated to optimize conditions for LNA SSO in liquid formulations. LNA SSO were exposed to different temperature conditions, mechanical stress as well as oxidative conditions, and the maximum feasible LNA SSO concentrations regarding handling and processing were determined. Finally, options for terminal sterilization of LNA SSO were evaluated. Results show that the tested LNA SSO were most stable under slightly alkaline conditions. A decrease in viscosity was best accomplished in the presence of spermine and lysine. Heat treatment and gamma irradiation caused high levels of degradation of the LNA SSO. Crucial formulation parameters, as identified in this study, should contribute to a significant increase in future productivity in drug product development for single-stranded oligonucleotides.

Metabolically stable dibenzo[b,e]oxepin-11(6H)-ones as highly selective p38 MAP kinase inhibitors: optimizing anti-cytokine activity in human whole blood.

Five series of metabolically stable disubstituted dibenzo[b,e]oxepin-11(6H)-ones were synthesized and tested in a p38α enzyme assay for their inhibition of tumor necrosis factor-α (TNF-α) release in human whole blood. Compared to the monosubstituted dibenzo[b,e]oxepin-11(6H)-one derivatives, it has been shown that the additional introduction of hydrophilic residues at position 9 leads to a substantial improvement of the inhibitory potency and metabolic stability. Using protein X-ray crystallography, the binding mode of the disubstituted dibenzoxepinones and the induction of a glyince flip in the hinge region were confirmed. The most potent compound of this series, 32e, shows an outstanding biological activity on isolated p38α, with an IC50 value of 1.6 nM, extraordinary selectivity (by a factor >1000, Kinase WholePanelProfiler), and low ATP competitiveness. The ability to inhibit the release of TNF-α from human whole blood was optimized down to an IC50 value of 125 nM. With the promising dibenzoxepinone inhibitor 3i, a pharmacokinetic study in mice was conducted.

Dibenzosuberones as p38 mitogen-activated protein kinase inhibitors with low ATP competitiveness and outstanding whole blood activity.

p38α mitogen-activated protein (MAP) kinase is a main target in drug research concerning inflammatory diseases. Nevertheless, no inhibitor of p38α MAP kinase has been introduced to the market. This might be attributed to the fact that there is no inhibitor which combines outstanding activity in biological systems and selectivity. Herein an approach to the development of such inhibitors on the basis of the highly selective molecular probe Skepinone-L is described. Introduction of a "deep pocket" moiety addressing the DFG motif led to an increased activity of the compounds. Hydrophilic moieties, addressing the solvent-exposed area adjacent to hydrophilic region II, conserved a high activity of the compounds in a whole blood assay. Combined with their outstanding selectivity and low ATP competitiveness, these inhibitors are very interesting candidates for use in biological systems and in therapy.

Tissue stretch induces nuclear remodeling in connective tissue fibroblasts.

Studies in cultured cells have shown that nuclear shape is an important factor influencing nuclear function, and that mechanical forces applied to the cell can directly affect nuclear shape. In a previous study, we demonstrated that stretching of whole mouse subcutaneous tissue causes dynamic cytoskeletal remodeling with perinuclear redistribution of alpha-actin in fibroblasts within the tissue. We have further shown that the nuclei of these fibroblasts have deep invaginations containing alpha-actin. In the current study, we hypothesized that tissue stretch would cause nuclear remodeling with a reduced amount of nuclear invagination, measurable as a change in nuclear concavity. Subcutaneous areolar connective tissue samples were excised from 28 mice and randomized to either tissue stretch or no stretch for 30 min, then examined with histochemistry and confocal microscopy. In stretched tissue (vs. non-stretched), fibroblast nuclei had a larger cross-sectional area (P < 0.001), smaller thickness (P < 0.03) in the plane of the tissue, and smaller relative concavity (P < 0.005) indicating an increase in nuclear convexity. The stretch-induced loss of invaginations may have important influences on gene expression, RNA trafficking and/or cell differentiation.

Alpha smooth muscle actin distribution in cytoplasm and nuclear invaginations of connective tissue fibroblasts.

Alpha smooth muscle actin (alpha-SMA) was recently shown to be present in mouse subcutaneous tissue fibroblasts in the absence of tissue injury. In this study, we used a combination of immunohistochemistry and correlative confocal scanning laser and electron microscopy to investigate the structural organization of alpha-SMA in relation to the nucleus. Furthermore, we explored colocalization analysis as a method for quantifying the amount of alpha-SMA in close approximation to the nucleic acid marker, 4',6-diamidino-2-phenyl-indole, dihydrochloride. Our findings indicate the presence of alpha-SMA within nuclear invaginations in close proximity to the nuclear membrane, but not in the nucleoplasm. Although the function of these alpha-SMA-rich nuclear invaginations is at present unknown, the morphology of these structures suggests their possible involvement in cellular and nuclear mechanotransduction as well as nuclear transport.

Fibroblast spreading induced by connective tissue stretch involves intracellular redistribution of alpha- and beta-actin.

Mechanical stretching of connective tissue occurs with normal movement and postural changes, as well as treatments including physical therapy, massage and acupuncture. Connective tissue fibroblasts were recently shown to respond actively to short-term mechanical stretch (minutes to hours) with reversible cytoskeletal remodeling, characterized by extensive cell spreading and lamellipodia formation. In this study, we have examined the effect of tissue stretch on the distribution of alpha- and beta-actin in subcutaneous tissue fibroblasts ex vivo. Normal fibroblasts uniformly exhibited alpha-smooth muscle actin (alpha-SMA) immunoreactivity. Unlike cultured fibroblasts and smooth muscle cells, alpha-SMA in these fibroblasts was not in F-actin form (indicated by lack of phalloidin co-localization) nor was it organized into distinct stress fibers. The lack of stress fibers and fibronexus was confirmed by electron microscopy, indicating that these cells were not myofibroblasts. In unstretched tissue, the pattern of alpha-actin was diffuse and granular. With tissue stretch (30 min), alpha-actin formed a star-shaped pattern centered on the nucleus, while beta-actin extended throughout the cytoplasm including lamellipodia and cell cortex. This dual response pattern of alpha- and beta-actin may be an important component of cellular mechanotransduction mechanisms relevant to physiologic and therapeutic mechanical forces applied to connective tissue.