Using drug-excipient interactions for siRNA delivery

SiRNA is the trigger of RNA interference, a mechanism discovered in the late 1990s. To release the therapeutic potential of this versatile but large and fragile molecule, excipients are used which either interact by electrostatic interaction, passively encapsulate siRNA or are covalently attached to enable specific and safe delivery of the drug substance. Controlling the delicate balance between protective complexation and release of siRNA at the right point and time is done by understanding excipients-siRNA interactions. These can be lipids, polymers such as PEI, PLGA, Chitosans, Cyclodextrins, as well as aptamers and peptides. This review describes the mechanisms of interaction of the most commonly used siRNA delivery vehicles, and looks at the results of their clinical and preclinical studies.     

Tacrine-silibinin codrug shows neuro- and hepatoprotective effects in vitro and pro-cognitive and hepatoprotective effects in vivo

A codrug of the anti-Alzheimer drug tacrine and the natural product silibinin was synthesized. The codrug's biological and pharmacological properties were compared to an equimolar mixture of the components. The compound showed potent acetyl- and butyrylcholinesterase inhibition. In a cellular hepatotoxicity model, analyzing the influence on viability and mitochondria of hepatic stellate cells (HSC), the toxicity of the codrug was markedly reduced in comparison to that of tacrine. Using a neuronal cell line (HT-22), a neuroprotective effect against glutamate-induced toxicity could be observed that was absent for the 1:1 mixture of components. In subsequent in vivo experiments in rats, in contrast to the effects seen after tacrine treatment, after administration of the codrug no hepatotoxicity and no induction of the cytochrome P450 system were noticed. In a scopolamine-induced cognitive impairment model using Wistar rats, the codrug was as potent as tacrine in reversing memory dysfunction. The tacrine-silibinin codrug shows high AChE and BChE inhibition, neuroprotective effects, lacks tacrine's hepatotoxicity in vitro and in vivo, and shows the same pro-cognitive effects in vivo as tacrine, being superior to the physical mixture of tacrine and silibinin in all these regards.     

Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies

The presence of oxidized methionine residues in therapeutic monoclonal antibodies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently routinely performed by peptide mapping/liquid chromatography–mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatography as a method of choice for fast and quantitative screening of total Fc methionine oxidation during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G–Fc domains containing oxidized methionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatography, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development.     

Modification of caffeine effects on the affect-modulated startle by neuropeptide S receptor gene variation

Rationale/objectives

Both the neuropeptide S (NPS) system and antagonism at the adenosine A2A receptor (e.g., by caffeine) were found to play a crucial role in the mediation of arousal and anxiety/panic in animal and human studies. Furthermore, a complex interaction of the neuropeptide S and the adenosinergic system has been suggested with administration of the adenosine A2A receptor antagonist caffeine downregulating NPS levels (Lage et al., 2006) and attenuating the stimulatory effects of NPS in rodents (Boeck et al., 2010).

Methods

Thus, in the present study, the impact of the functional neuropeptide S receptor (NPSR) A/T (Asn¹⁰⁷Ile; rs324981) variant on affect-modulated (neutral, unpleasant, and pleasant IAPS pictures) startle response depending on the administration of 300?mg caffeine citrate was investigated in a sample of 124 (m?=?58, f?=?66) healthy probands using a double-blind, placebo-controlled design.

Results

ANOVA revealed a significant interaction between NPSR genotype, challenge condition, and picture valence. Comparing startle magnitudes upon stimulation with neutral or emotional pictures between the placebo and caffeine condition, in AA/AT non-risk genotype carriers no significant difference was discerned, while TT risk genotype carriers showed a significantly increased startle magnitude in response to neutral stimuli (p?=?.02) and a significantly decreased startle magnitude in response to unpleasant stimuli (p?=?.02) in the caffeine condition as compared to the placebo condition.

Conclusions

In summary, the present findings ?? extending previous evidence from rodent studies ?? for the first time provide support for a complex, non-linear interaction of the neuropeptide S and adenosinergic systems affecting the affect-modulated startle response as an intermediate phenotype of anxiety in humans.     

Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte-derived dendritic cells

Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides - which are the most frequent cause of adverse drug reactions - were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIR and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines.     

A Convenient Method to Produce Close‐ and Non‐close‐Packed Monolayers using Direct Assembly at the Air–Water Interface and Subsequent Plasma‐Induced Size Reduction

An extremely facile approach to produce close‐packed colloidal monolayers over large areas using direct assembly at the air–water interface is presented. The influence of small amounts of sodium dodecyl sulfate (SDS) as well as the influence of the pH value of the subphase on the quality of the resulting monolayer is investigated. It is found that small amounts of SDS at the interface influence capillary forces and form a soft barrier that facilitates the crystallization process. Increased electrostatic repulsion arising from a higher pH of the subphase induced a higher order using carboxylic acid functionalized particles. The deposited close‐packed monolayers were subjected to plasma treatment in order to shrink the colloids and produce non‐close packed monolayers with lattice spacing and symmetry reflecting the order of the initial close‐packed monolayer. A detailed examination of etching conditions and their influence on the shrinkage of the particles was performed, including effects of plasma power, composition, flow rates as well as polymeric‐ and substrate material. The monolayers exhibit vivid coloration, which is determined by their size and packing density. UV–Vis–NIR spectroscopy was used to investigate the change of monolayer color during the size reduction of the individual particles. A simple theoretical model was elaborated to explain the optical properties. Finally, the non‐close‐packed monolayers were used as masks to produce gold nanostructures to exemplify the versatility of the monolayer architectures in nanosphere lithography.