A Nitrite Excipient Database: A Useful Tool to Support N-Nitrosamine Risk Assessments for Drug Products.

N-Nitrosamine risk assessment and control have become an integral part of pharmaceutical drug product development and quality evaluation. Initial reports of nitrosamine contamination were linked with the drug substance and its manufacturing process. Subsequently, the drug product and aspects of the formulation process have shown to be relevant. Regarding specific formulation contributions to nitrosamine content in a product, one risk lies in possible interactions between nitrosating agents, derived from nitrite in excipients, and vulnerable amines, either present as moieties of the active molecule or as impurities / degradants. However, the limited validated information on nitrite levels in excipients available until now, has been an obstacle for scientists to assess the risk of nitrosamine formation in pharmaceutical products. This has driven the creation of a database to store and share such validated information. The database, maintained by Lhasa Limited, constitutes a central platform to hold the data donated by the pharmaceutical company members on the nitrite concentrations in common excipients measured with validated analytical procedures. The goal of this data sharing initiative is to provide a common framework to contextualize and estimate the risk posed by presence of nitrites to contribute to the formation of nitrosamines in drug products. The major findings from the database analyses are: (1) average nitrite content and batch to batch variance differ among excipients, (2) for solid dosage forms, the nitrite contribution is dominated by the highest formula % excipients, e.g., the fillers (diluents), which are typically used in larger proportion, and are characterized by low nitrite levels and low variability, leading to an average value of 1 µg/g nitrite in a typical formulation, (3) substantial differences in average nitrite content in batches from different excipient vendors potentially reflecting differences in source materials or processing methods for excipient manufacturing. That final point suggests that future selection of raw materials or processing by excipient manufacturers may help reduce nitrite levels in finished drug product formulations, and thus the overall risk of nitrosamine formation in cases where the product contains vulnerable amines.

Development of a Binational Framework for Active and Healthy Ageing (AHA) Bridging Austria and Slovenia in a Thermal Spa Region.

In view of ongoing demographic developments resulting in a longer life expectancy of the European population, the creation of "age-friendly" environments represents an initiative picked up by the European Union and its Member States to enable active and healthy ageing. The present study aims at the co-creation of a cross-border framework model to deploy a healthy ageing region linking Austria and Slovenia, building on previous work dealing with the development of an integrated regional ecosystem for active and healthy ageing. A qualitative, community-based action research method based on focus group discussions allowed the development of an exemplary framework model for active and healthy ageing building on cross-border collaboration in the region of Promura. Within the project group, twelve cross-border regional key assets were identified. In the course of further open discussions, an exemplary model for the deployment of a cross-border healthy ageing region was developed, comprising underlying fundamental environmental aspects, regional structures in the field of health and care as well as crosscutting features spreading across all levels. This article presents a promising, strategic co-creation approach on how to span a model on active and healthy ageing across two cross-border regions with similar characteristics and assets.

Determining the deformation and resulting coupling efficiency degradation of ultrastable fiber-coupled optical benches under load.

Fiber-coupled optical benches are an integral part of many laser systems. The base of such an optical bench is usually a slab of solid material, onto which optical components are fixed. In many environments, the ability to retain high fiber coupling efficiency under mechanical loads is essential. In this article, we study the fiber-to-fiber coupling efficiency under the application of static mechanical loads experimentally and theoretically: We constructed a simple three-point bending setup to interferometrically measure the deformation of an optical bench under load. Using the same setup, we further recorded the resulting coupling efficiency variations. The examined optical benches are based on Zerodur optical benches used in sounding rockets and International Space Station missions. We also developed an analytical model that incorporates an Euler-Bernoulli beam deformation model and a simple model for calculating the coupling efficiency, to which the experimentally obtained data are compared. Furthermore, we use a finite element method simulation to compare to the recorded deformation data. Recorded data, the analytical model, and simulations show good agreement. We also show how the presented analytical model can easily be expanded to contain more complex beam paths and, thus, be used to estimate coupling losses for experimentally relevant optical benches under load.

Compound selection for development - is salt formation the ultimate answer? Experiences with an extended concept of the "100mg approach".

In order to select the best candidates for development, physicochemical criteria such as solubility, chemical and physical stability, hygroscopicity, and thermal characteristics need to be evaluated as early as possible and balanced against other important criteria such as pharmacology or pharmacokinetics. It could be shown, that our miniaturized pharmaceutical profiling concept ("100mg approach"), is capable to reliably identify potential development issues of drug candidates, which, therefore, can be approached early on. Salt formation is a well established strategy to improve unfavorable properties, in particular poor solubility. This article describes our stepwise approach on salt screening, including selection criteria, and summarizes the observations we had during compound investigation. Considering a data base of 337 compounds (salts and uncharged substances), experiences with various counterions evaluated over the last 10years are discussed. We realized that salt formation usually improves poor solubility of a given candidate, but this is often at the cost of other attributes being relevant for pharmaceutical development. Surprisingly, in more than 50% of all cases the "free form" was finally selected after carefully weighing all compound characteristics. Therefore, we conclude that an early salt selection strategy is of utmost importance to predict potential development issues and to enable the provision of alternative physical forms. However, salt formation itself is not necessarily the best solution to meet all development requirements. The selection of a free form (acid or base) in combination with advanced formulation strategies should always be considered, sometimes as best compromise.

Role of anisotropy for protein-protein encounter.

Protein-protein interactions comprise both transport and reaction steps. During the transport step, anisotropy of proteins and their complexes is important both for hydrodynamic diffusion and accessibility of the binding site. Using a Brownian dynamics approach and extensive computer simulations, we quantify the effect of anisotropy on the encounter rate of ellipsoidal particles covered with spherical encounter patches. We show that the encounter rate k depends on the aspect ratios xi mainly through steric effects, while anisotropic diffusion has only a little effect. Calculating analytically the crossover times from anisotropic to isotropic diffusion in three dimensions, we find that they are much smaller than typical protein encounter times, in agreement with our numerical results.

MYC is a metastasis gene for non-small-cell lung cancer.

BACKGROUND: Metastasis is a process by which cancer cells learn to form satellite tumors in distant organs and represents the principle cause of death of patients with solid tumors. NSCLC is the most lethal human cancer due to its high rate of metastasis. METHODOLOGY/PRINCIPAL FINDINGS: Lack of a suitable animal model has so far hampered analysis of metastatic progression. We have examined c-MYC for its ability to induce metastasis in a C-RAF-driven mouse model for non-small-cell lung cancer. c-MYC alone induced frank tumor growth only after long latency at which time secondary mutations in K-Ras or LKB1 were detected reminiscent of human NSCLC. Combination with C-RAF led to immediate acceleration of tumor growth, conversion to papillary epithelial cells and angiogenic switch induction. Moreover, addition of c-MYC was sufficient to induce macrometastasis in liver and lymph nodes with short latency associated with lineage switch events. Thus we have generated the first conditional model for metastasis of NSCLC and identified a gene, c-MYC that is able to orchestrate all steps of this process. CONCLUSIONS/SIGNIFICANCE: Potential markers for detection of metastasis were identified and validated for diagnosis of human biopsies. These markers may represent targets for future therapeutic intervention as they include genes such as Gata4 that are exclusively expressed during lung development.

Polycomb group protein Bmi1 is required for growth of RAF driven non-small-cell lung cancer.

BACKGROUND: We have previously described a RAF oncogene driven transgenic mouse model for non small cell lung cancer (NSCLC). Here we examine whether tumor initiation and growth requires the stem cell self-renewal factor Bmi1. PRINCIPAL FINDINGS: In order to evaluate Bmi1 function in NSCLC two founder lines that differ in incidence and latency of tumor formation were compared. Ablation of Bmi1 expression in both lines had a dramatically decreased tumor growth. As the line with shorter latency matched the life span of Bmi1 knock out mice, these mice were chosen for further study. The absence of Bmi1 did not decrease the number of tumor initiation in these mice as only the size and not the number of tumors decreased. Reduction in tumor growth resulted from an increase in cell death and decrease in cell cycle progression that corresponded with up-regulation of the p16(INK4a) and p19(ARF). SIGNIFICANCE: The data identifies Bmi1 as an important factor for expansion but not initiation of RAF driven NSCLC.

Stochastic simulations of cargo transport by processive molecular motors.

We use stochastic computer simulations to study the transport of a spherical cargo particle along a microtubule-like track on a planar substrate by several kinesin-like processive motors. Our newly developed adhesive motor dynamics algorithm combines the numerical integration of a Langevin equation for the motion of a sphere with kinetic rules for the molecular motors. The Langevin part includes diffusive motion, the action of the pulling motors, and hydrodynamic interactions between sphere and wall. The kinetic rules for the motors include binding to and unbinding from the filament as well as active motor steps. We find that the simulated mean transport length increases exponentially with the number of bound motors, in good agreement with earlier results. The number of motors in binding range to the motor track fluctuates in time with a Poissonian distribution, both for springs and cables being used as models for the linker mechanics. Cooperativity in the sense of equal load sharing only occurs for high values for viscosity and attachment time.

Structural basis for stable DNA complex formation by the caspase-activated DNase.

We describe a structural model for DNA binding by the caspase-activated DNase (CAD). Results of a mutational analysis and computational modeling suggest that DNA is bound via a positively charged surface with two functionally distinct regions, one being the active site facing the DNA minor groove and the other comprising distal residues close to or directly from helix alpha4, which binds DNA in the major groove. This bipartite protein-DNA interaction is present once in the CAD/inhibitor of CAD heterodimer and repeated twice in the active CAD dimer.

Interaction of DNA fragmentation factor (DFF) with DNA reveals an unprecedented mechanism for nuclease inhibition and suggests that DFF can be activated in a DNA-bound state.

DNA fragmentation factor (DFF) is a complex of the DNase DFF40 (CAD) and its chaperone/inhibitor DFF45 (ICAD-L) that can be activated during apoptosis to induce DNA fragmentation. Here, we demonstrate that DFF directly binds to DNA in vitro without promoting DNA cleavage. DNA binding by DFF is mediated by the nuclease subunit, which can also form stable DNA complexes after release from DFF. Recombinant and reconstituted DFF is catalytically inactive yet proficient in DNA binding, demonstrating that the nuclease subunit in DFF is inhibited in DNA cleavage but not in DNA binding, revealing an unprecedented mode of nuclease inhibition. Activation of DFF in the presence of naked DNA or isolated nuclei stimulates DNA degradation by released DFF40 (CAD). In transfected HeLa cells transiently expressed DFF associates with chromatin, suggesting that DFF could be activated during apoptosis in a DNA-bound state.

Pharmaceutical evaluation of early development candidates "the 100 mg-approach".

Early development candidates are often selected for pre-clinical and clinical development based primarily on pharmacological and toxicological data. In order to choose the best compounds from a biopharmaceutical point of view, physicochemical parameters such as solubility, dissolution rate, hygroscopicity, lipophilicity, pKa, stability, polymorphism and particle characteristics need to be evaluated as early as possible and above all with the highest accuracy. However, the low amounts of drug substance available in early development often compromise data quality, and therefore, hamper an early pharmaceutical assessment. This article summarises the Aventis approach on early pharmaceutical compound profiling with the aim of providing a high quality assessment requiring not more than 100 mg of drug substance. In particular, the evaluation criteria, process and miniaturised analytical technology that can be applied for this purpose are discussed.

Experimental evidence for a beta beta alpha-Me-finger nuclease motif to represent the active site of the caspase-activated DNase.

The caspase-activated DNase (CAD) is an important nuclease involved in apoptotic DNA degradation. Results of a sequence comparison of CAD proteins with beta beta alpha-Me-finger nucleases in conjunction with a mutational and chemical modification analysis suggest that CAD proteins constitute a new family of beta beta alpha-Me-finger nucleases. Nucleases of this family have widely different functions but are characterized by a common active-site fold and similar catalytic mechanisms. According to our results and comparisons with related nucleases, the active site of CAD displays features that partly resemble those of the colicin E9 and partly those of the T4 endonuclease VII active sites. We suggest that the catalytic mechanism of CAD involves a conserved histidine residue, acting as a general base, and another histidine as well as an aspartic acid residue required for cofactor binding. Our findings provide a first insight into the likely active-site structure and catalytic mechanism of a nuclease involved in the degradation of chromosomal DNA during programmed cell death.

The effect of ICAD-S on the formation and intracellular distribution of a nucleolytically active caspase-activated DNase.

We show here that co-expression of murine CAD with either ICAD-L or ICAD-S in Escherichia coli as well as mammalian cells leads to a functional DFF complex, which after caspase-3 activation releases a nucleolytically active DNase. The chaperone activity of ICAD-S is between one and two orders of magnitude less effective than that of ICAD-L, as deduced from cleavage experiments with different activated recombinant DFF complexes produced in E.coli. With nucleolytically active EGFP fusion proteins of CAD it is demonstrated that co-expression of ICAD-S, which lacks the C-terminal domain of ICAD-L, including the NLS, leads to a homogeneous intracellular distribution of the DNase in transfected cells, whereas co-expression of human or murine ICAD-L variants lacking the NLS leads to exclusion of EGFP-CAD from the nuclei in approximately 50% of cells. These results attribute a particular importance of the NLS in the long isoform of the inhibitor of CAD for nuclear accumulation of the DFF complex in living cells. It is concluded that ICAD-L and ICAD-S in vivo might function as tissue-specific modulators in the regulation of apoptotic DNA degradation by controlling not only the enzymatic activity but also the amount of CAD available in the nuclei of mammalian cells.

Involvement of conserved histidine, lysine and tyrosine residues in the mechanism of DNA cleavage by the caspase-3 activated DNase CAD.

The caspase-activated DNase (CAD) is involved in DNA degradation during apoptosis. Chemical modification of murine CAD with the lysine-specific reagent 2,4,6-trinitrobenzenesulphonic acid and the tyrosine-specific reagent N-acetylimidazole leads to inactivation of the nuclease, indicating that lysine and tyrosine residues are important for DNA cleavage by this enzyme. The presence of DNA or the inhibitor ICAD-L protects the enzyme from modification. Amino acid substitution in murine CAD of lysines and tyrosines conserved in CADs from five different species leads to variants with little if any catalytic activity, but unaltered DNA binding (K155Q, K301Q, K310Q, Y247F), with the exception of Y170F, which retains wild-type activity. Similarly, as observed for the previously characterised H242N, H263N, H308N and H313N variants, the newly introduced His-->Asp/Glu or Arg exchanges lead to variants with <1% of wild-type activity, with two exceptions: H313R shows wild-type activity, and H308D at pH 5.0 exhibits approximately 5% of wild-type activity at this pH. Y170F and H313R produce a specific pattern of fragments, different from wild-type CAD, which degrades DNA non-specifically. The recombinant nuclease variants produced in Escherichia coli were tested for their ability to form nucleolytically active oligomers. They did not show any significant deviation from the wild-type enzyme. Based on these and published data possible roles of the amino acid residues under investigation are discussed.