Container Closure Integrity of a Glass Prefillable Syringe in Deep Frozen Storage Conditions.

Development of novel pharmaceutical drug modalities has created a need for frozen storage and transportation. Accurate and easy assessment of container closure integrity (CCI) in frozen conditions remains a challenge. Thus, container closure systems (CCS) suitable for low temperatures have been primarily restricted to vials despite the growing popularity of prefillable syringes (PFS) for parenteral administration. A new dye ingress test method, suitable for testing at low temperatures, was developed and applied to PFS across a range of deep-frozen temperatures. The method is versatile and can easily be extended to other common CCS formats over a wide range of temperatures including storage on dry ice (-80 °C). This new method was paired with an orthogonal technique, laser-based CO2 headspace gas analysis, to evaluate the CCI of a glass PFS at temperatures from -50 °C to -80 °C. Both test methods showed comparable results and consistent CCI failure below a temperature of -70 °C. The primary mode of failure was the plunger-to-barrel interface, likely attributable to dimensional changes and loss of elasticity. This study demonstrates the temperature dependent CCI behavior of glass PFS and underscores the importance of thorough characterization of package integrity for deep frozen drug products.

Method Comparison Study-One Step Forward toward Robust Silicone Layer Thickness Measurements.

In the past decades, the silicone layer thickness and its distribution on the inner glass barrels of prefilled syringes have been characterized in several studies. However, the limited number of adequate methods to characterize thin baked-on silicone layers and the destructive nature of some analytical techniques suggest challenges to the inter-lab reproducibility of some methods. In this study, the measured silicone layer thickness of baked-on siliconized syringes was compared between two laboratories, both equipped with white light reflectometry coupled to laser interferometry instrumentation (Bouncer, LE UT 1.0, LE UT 2.0). The quantity of silicone oil of a subset of those syringes was measured by Fourier transform infrared spectroscopy. Glide force tests were realized as complementary measurements on both syringes analyzed by white light reflectometry coupled to laser interferometry instrumentation and on non-analyzed identical syringes from the same lot. Silicone profiles of all prefilled syringes including the limit of detection results replaced with 20 nm were comparable, but values were slightly lower when measured with the Bouncer instrument. An increase of the layer thickness from the finger flange to the needle side was found for all syringes with all instruments (20 nm to 130-140 nm). Glide force results were similar except for a difference in peak width in the break loose region between the laboratories. The mean quantities of silicone oil found by both laboratories were similar (64 µg/syringe and 69 µg/syringe). Overall, comparable results between laboratories suggest a good reproducibility of the thickness measurement method as a result of thorough method understanding and defining key method parameters. Hence this study presents a robust inter-lab comparison between silicone layer thickness measurements that has been a lack in the literature up to now.

Modulation of relaxation activity of human topoisomerases by Pt(II)-based complexes.

The clinical efficiency of Pt(II)-based drugs is founded on articulate mechanisms of action. Indeed it depends on a balanced combination of metal ion reactivity towards proteins and nucleic acids. Here we analysed the effect of two trans-platinum planar amines in comparison to cisplatin and transplatin on the DNA processivity by human topoisomerases I and IIα. Each tested metal complex produces DNA adducts with unique geometrical features and, consistently, they exert different effects on the activity of tested enzymes. Moreover, our results highlighted more subtle consequences on the enzymatic activity by the tested metal complexes which derive from a combination of preferential DNA or protein platination. Moreover, we observed that it is not possible to predict the overall output based only on the cis- vs trans- geometry of the tested metal complexes. This variable behaviour reflects the chemical reactivity profile of each single metal complex and can be usefully addressed to describe their different properties in the complex physiological environment.

Investigating the Effects of the Chemical Composition on Glass Corrosion: A Case Study for Type I Vials.

Glass is the favorite material for parenteral packaging because of its physico-chemical properties. Type I borosilicate glass is worldwide use at this scope, but it may have some issues related to breakage, corrosion and delamination that might compromise the drug quality, safety and efficacy. These issues can be mitigated and avoided starting from the appropriate selection of the most suitable raw material at the early stage of the glass container design. In this study, Type I borosilicate glass vials manufactured using two glass tubes having different chemical compositions, were studied and compared in terms of their resistance to corrosion. Testing design was applied with the aim to select the best practice approach comparing different storage simulation conditions: ageing treatment through autoclaving and stability testing (real-time and accelerated). Clear differences were found between the different glass types in terms of hydrolytic and corrosion resistance that highlighted the relation between chemical composition and glass chemical durability. Non-negligible differences were also observed using different storage conditions.

Quinolino[3,4-b]quinoxalines and pyridazino[4,3-c]quinoline derivatives: Synthesis, inhibition of topoisomerase IIα, G-quadruplex binding and cytotoxic properties.

The quinoline motif fused with other heterocyclic systems plays an important role in the field of anticancer drug development. An extensive series of tetracyclic quinolino[3,4-b]quinoxalines N-5 or C-6 substituted with basic side chain and a limited number of tricyclic pyridazino[4,3-c]quinolines N-6 substituted were designed, synthesized and evaluated for topoisomerase IIα (Topo IIα) inhibitory activity, ability to bind and stabilize G-quadruplex structures and cytotoxic properties against two human cancer cell lines (HeLa and MCF-7). Almost all of the tested agents showed a high activity as Topo IIα inhibitors and G-quadruplex stabilizers. Among all the derivatives studied, the quinolino[3,4-b]quinoxalines 11 and 23, N-5 and C-6 substituted respectively, stand out as the most promising compounds. Derivative 11 resulted a selective binder to selected G-quadruplex sequences, while derivative 23 displayed the most interesting Topo IIα inhibitory activity (IC50 = 5.14 µM); both showed high cytotoxic activity (IC50 HeLa = 2.04 µM and 2.32 µM, respectively).

Hit Identification of a Novel Dual Binder for h-telo/c-myc G-Quadruplex by a Combination of Pharmacophore Structure-Based Virtual Screening and Docking Refinement.

It is well known that G-quadruplexes are targets of great interest for their roles in crucial biological processes, such as aging and cancer. Hence, a promising strategy for anticancer drug therapy is the stabilization of these structures by small molecules. We report a high-throughput in silico screening of commercial libraries from several different vendors by means of a combined structure-based pharmacophore model approach followed by docking simulations. The compounds selected by the virtual screening procedure were then tested for their ability to interact with human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and fluorescence intercalator displacement. Our approach resulted in the identification of a 13-[(dimethylamino)methyl]-12-hydroxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one derivative as a novel promising stabilizer of G-quadruplex structures within the human telomeric and the c-myc promoter sequences.

Low Abundant N-linked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites.

Although wild-type hen egg white lysozyme (HEL) is lacking the consensus sequence motif NX(S/T), in 1995 Trudel et al. (Biochem. Cell Biol. 1995, 73, 307-309) proposed the existence of a low abundant N-glycosylated form of HEL; however, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel's initial work, we report here a comprehensive characterization by means of mass spectrometry of N-glycosylation in wild-type HEL. Our analytical approach comprised ZIC-HILIC enrichment of N-glycopeptides from HEL trypsin digest, deglycosylation by (18)O/PNGase F as well as by various endoglycosidases, and LC-MS/MS analysis of both intact and deglycosylated N-glycopeptides engaging multiple techniques of ionization and fragmentation. A novel data interpretation workflow based on MS/MS spectra classification and glycan database searching enabled the straightforward identification of the asparagine-rich N-glycopeptide [34-45] FESNFNTQATNR and allowed for compositional profiling of its modifying N-glycans. The overall heterogeneity profile of N-glycans in HEL comprised at least 26 different compositions. Results obtained from deglycosylation experiments provided clear evidence of asparagine residues N44 and N39 representing active glycosylation sites in HEL. Both of these sites do not fall into any known N-glycosylation-specific sequence motif but are localized in rarely observed nonconsensus sequons (NXN, NXQ).

Water-soluble isoindolo[2,1-a]quinoxalin-6-imines: in vitro antiproliferative activity and molecular mechanism(s) of action.

Water-soluble isoindoloquinoxalin (IIQ) imines and the corresponding acetates were conveniently prepared from the key intermediates 2-(2'-aminophenyl)-2H-isoindole-1-carbonitriles obtained by a Strecker reaction between substituted 1,2-dicarbaldehydes and 1,2-phenylenediamines. Both series were screened by the National Cancer Institute (Bethesda, MD) and showed potent antiproliferative activity against a panel of 60 human tumor cell lines. Several of the novel compounds showed GI50 values at a nanomolar level on the majority of the tested cell lines. Among IIQ derivatives, methoxy substituents at positions 3 and 8 or/and 9 were especially effective in impairing cell cycle progression and inducing apoptosis in cancer cells. These effects were associated to IIQ-mediated impairment of tubulin polymerization at pharmacologically significant concentrations of tested compounds. In addition, impaired DNA topoisomerase I functions and perturbation in telomere architecture were observed in cells exposed to micromolar concentrations of IIQ derivatives. The above results suggest that IIQ derivatives exhibit multi-target cytotoxic activities.

Exploring the chemical space of G-quadruplex binders: discovery of a novel chemotype targeting the human telomeric sequence.

Recent findings have unambiguously demonstrated that DNA G-rich sequences can adopt a G-quadruplex folding in living cells, thus further validating them as crucial targets for anticancer therapy. Herein, to identify new potent G4 binders as antitumor drug candidates, we have targeted a 24-nt G4-forming telomeric sequence employing a receptor-based virtual screening approach. Among the best candidates, in vitro binding experiments allowed identification of three novel G4 ligands. Among them, the best compound features an unprecedented binding selectivity for the human telomeric DNA G-quadruplex with no detectable binding for other G4-forming sequences present at different genomic sites. This behavior correlates with the detected ability to generate DNA damage response in tumor cells at the telomeric level and efficient antiproliferative effect on different tumor cell lines at low micromolar concentrations.

Platinum-based drugs and proteins: reactivity and relevance to DNA adduct formation.

The mechanism of action of clinically used Pt-based drugs is through the formation of stable DNA adducts occurring at the nitrogen in position 7 of guanine (N7) and involving one or two spatially closed residues. Nevertheless, proteins can represent alternative targets since in particular sulfur groups, present in cysteine or methionine residues, can efficiently coordinate platinum. Here we have characterized the reactivity profile of cisplatin, transplatin and of two trans-platinum amine derivatives (TPAs) towards three different proteins, bovine α-lactalbumin (α-LA), hen egg lysozyme (LYS) and human serum albumin (HSA). Our results demonstrate that generally the tested metal complexes react with the selected target causing protein oligomerization, likely through a cross-linking reaction. Interestingly, the extent of such a process is largely modulated by the target protein and by the chemical features of the metal complex, TPAs being the most efficient platinating agents. From a structural point of view the resulting reaction products turned out to be depending on the nature of the metal complexes. However, in all instances, a transfer reaction of the metal complex to DNA can also occur, maintaining the relevance of nucleic acids as a biological target. These results can be used to better rationalize the different pharmacological profiles reported for cisplatin and TPAs and can help in designing more predictive SARs within the series.

α-Lactalbumin forms with oleic acid a high molecular weight complex displaying cytotoxic activity.

α-Lactalbumin (LA) forms with oleic acid (OA) a complex which has been reported to induce the selective death of tumor cells. However, the mechanism by which this complex kills a wide range of tumor cell lines is as yet largely unknown. The difficulty in rationalizing the cytotoxic effects of the LA/OA complex can be due to the fact that the molecular aspects of the interaction between the protein and the fatty acid are still poorly understood, in particular regarding the oligomeric state of the protein and the actual molar ratio of OA over protein in the complex. Here, the effect of LA addition to an OA aqueous solution has been examined by dynamic light scattering measurements and transmission electron microscopy. Upon protein addition, the aggregation state of the rather insoluble OA is dramatically changed, and more water-soluble and smaller aggregates of the fatty acid are formed. A mixture of LA and an excess of OA forms a high molecular weight complex that can be isolated by size-exclusion chromatography and that displays cellular toxicity toward Jurkat cells. On the basis of gel filtration data, cross-linking experiments with glutaraldehyde, and OA titration, we evaluated that the isolated LA/OA complex is given by 4-5 protein molecules that bind 68-85 OA molecules. The protein in the complex adopts a molten globule-like conformation, and it interacts with the fatty acid mostly through its α-helical domain, as indicated by circular dichroism measurements and limited proteolysis experiments. Overall, we interpret our and previous data as indicating that the cellular toxicity of a LA/OA complex is due to the effect of a protein moiety in significantly enhancing the water solubility of the cytotoxic OA and, therefore, that the protein/OA complex can serve mainly as a carrier of the toxic fatty acid in a physiological milieu.

Characterization of linear forms of the circular enterocin AS-48 obtained by limited proteolysis.

AS-48 is a 70-residue circular peptide from Enterococcus faecalis with a broad antibacterial activity. Here, we produced by limited proteolysis a protein species carrying a single nicking and fragments of 55 and 38 residues. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that circularization is not required for the bactericidal activity, but it is important to stabilize the native structure. Moreover, it is possible to reduce the sequence to a minimal AS-48 domain without causing inactivation of this bacteriocin.