Pharmacokinetic de-risking tools for selection of monoclonal antibody lead candidates

Pharmacokinetic studies play an important role in all stages of drug discovery and development. Recent advancements in the tools for discovery and optimization of therapeutic proteins have created an abundance of candidates that may fulfill target product profile criteria. Implementing a set of in silico, small scale in vitro and in vivo tools can help to identify a clinical lead molecule with promising properties at the early stages of drug discovery, thus reducing the labor and cost in advancing multiple candidates toward clinical development. In this review, we describe tools that should be considered during drug discovery, and discuss approaches that could be included in the pharmacokinetic screening part of the lead candidate generation process to de-risk unexpected pharmacokinetic behaviors of Fc-based therapeutic proteins, with an emphasis on monoclonal antibodies.     

Plasmonic Properties of Gold Nanostructures on Gold Film

This paper reports on a systematic study of the plasmonic properties of periodic arrays of gold cylindrical nanoparticles in contact with a gold thin film. Depending on the gold film thickness, it observes several plasmon bands. Using a simple analytical model, it is able to assign all these modes and determine that they are due to the coupling of the grating diffraction orders with the propagating surface plasmons travelling along the film. With finite difference time domain (FDTD) simulations, it demonstrates that large field enhancement occurs at the surface of the nanocylinders due to the resonant excitation of these modes. By tilting the sample, it also observes the evolution of the spectral position of these modes and their tuning through nearly the whole visible range is possible. Such plasmonic substrates combining both advantages of the propagative and localised surface plasmons could have large applications in enhanced spectroscopies.

     

Bragg-Scattered Surface Plasmon Microscopy: Theoretical Study

We present a new approach to surface plasmon microscopy with high refractive index sensitivity and spatial resolution that is not limited by the propagation length of surface plasmons. It is based on a nanostructured metallic sensor surface supporting Bragg-scattered surface plasmons. We show that these non-propagating surface plasmon modes are excellently suited for spatially resolved observations of refractive index variations on the sensor surface owing to their highly confined field profile perpendicular to as well as parallel to the metal interface. The presented theoretical study reveals that this approach enables reaching similar refractive index sensitivity as regular surface plasmon resonance (SPR) microscopy and offers the advantage of improved spatial resolution when observing dielectric features with lateral size <10???m for the wavelength around 800?nm and gold as the SPR-active metal. This paper demonstrates the potential of Bragg-scattered surface plasmon microscopy for high-throughput SPR biosensing with high-density microarrays.     

Biosurfactant production by Pseudomonas fluorescens 378: growth and product characteristics

Summary An isolate of Pseudomonas fluorescens, strain 378 was shown to produce a novel surface active compound (code name AP-6). The compound is unique in being a high molecular weight compound but has, in some aspects, properties of a low molecular weight surfactant. The product is extracellular and its formation appeared to be partly growth-associated. Using a semisynthetic medium, fermentor cultivations were performed in the pH range 6.8–8.4. The product yield was optimal at pH 8.0 and gave a final concentration of 210 times critical micelle dilution. At higher pH, specific growth rate, final biomass and product concentration decreased. It consists mainly of carbohydrates and protein, the molecular weight is 1×10⁶ and the isoelectric point is pH 9.1.The surface tension of an aqueous solution reached 27 mN/m which is a very low value even compared to other surfactants of considerably lower size and the critical micelle concentration was less than 10 mg/l in 0.9% (w/v) NaCl. The kinetics of the adsorption process at the air-water interface was studied using the drop volume technique, and the reaction was found to be rapid, considering the size of the molecule. A concentration as low as 0.025 g/l reached a surface tension of 30 mN/m within 70 s.     

Effect of St John's wort (Hypericum perforatum) on cytochrome P-450 activity in perfused rat liver

St. John's wort (Hypericum perforatum) is a popular over-the-counter dietary supplement and a herbal antidepressant that has been implicated in drug interactions with substrates of several cytochrome P-450 (CYP) isozymes. The effects of the St. John's wort extract (100 mg/kg, i.p., once daily for 10 days) on metabolic activity of CYP450 were assessed in the system of isolated perfused rat liver. The substrates used in this study were tolbutamide (CYP2C6), dextromethorphan (CYP2D2) and midazolam (CYP3A2). Validated HPLC method was used to quantify all compounds of interest. St. John's wort administration affected CYP activity, causing a significant decline in AUC of dextromethorphan [F(4,31)=1511, p<0.001; PLSD, p<0.001] and AUC of midazolam [F(3,25)=221, p<0.001; PLSD, p=0.035] and a significant increase in AUC of tolbutamide [F(3,26)=200, p<0.001; PLSD, p<0.001]. St. John's wort administration resulted in a significant induction of CYP2D2 and CYP3A2, and in a significant inhibition of CYP2C6 metabolic activities.     

Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide

Cytochrome P450 (P450) 4X1 is one of the so-called 'orphan' P450s without an assigned biological function. Codon-optimized P450 4X1 and a number of N-terminal modified sequences were expressed in Escherichia coli. Native P450 4X1 showed a characteristic P450 spectrum but low expression in E. coli DH5alpha cells (< 100 nmol P450.L(-1)). The highest level of expression (300-450 nmol P450.L(-1) culture) was achieved with a bicistronic P450 4X1 construct (N-terminal MAKKTSSKGKL, change of E2A, amino acids 3-44 truncated). Anandamide (arachidonoyl ethanolamide) has emerged as an important signaling molecule in the neurovascular cascade. Recombinant P450 4X1 protein, co-expressed with human NADPH-P450 reductase in E. coli, was found to convert the natural endocannabinoid anandamide to a single monooxygenated product, 14,15-epoxyeicosatrienoic (EET) ethanolamide. A stable anandamide analog (CD-25) was also converted to a monooxygenated product. Arachidonic acid was oxidized more slowly to 14,15- and 8,9-EETs but only in the presence of cytochrome b(5). Other fatty acids were investigated as putative substrates but showed only little or minor oxidation. Real-time PCR analysis demonstrated extrahepatic mRNA expression, including several human brain structures (cerebellum, amygdala and basal ganglia), in addition to expression in human heart, liver, prostate and breast. The highest mRNA expression levels were detected in amygdala and skin. The ability of P450 4X1 to generate anandamide derivatives and the mRNA distribution pattern suggest a potential role for P450 4X1 in anandamide signaling in the brain.     

Plasmon-Enhanced Fluorescence Biosensors: a Review

Surfaces of metallic films and metallic nanoparticles can strongly confine electromagnetic field through its coupling to propagating or localized surface plasmons. This interaction is associated with large enhancement of the field intensity and local optical density of states which provides means to increase excitation rate, raise quantum yield, and control far field angular distribution of fluorescence light emitted by organic dyes and quantum dots. Such emitters are commonly used as labels in assays for detection of chemical and biological species. Their interaction with surface plasmons allows amplifying fluorescence signal (brightness) that accompanies molecular binding events by several orders of magnitude. In conjunction with interfacial architectures for the specific capture of target analyte on a metallic surface, plasmon-enhanced fluorescence (PEF) that is also referred to as metal-enhanced fluorescence (MEF) represents an attractive method for shortening detection times and increasing sensitivity of various fluorescence-based analytical technologies. This review provides an introduction to fundamentals of PEF, illustrates current developments in design of metallic nanostructures for efficient fluorescence signal amplification that utilizes propagating and localized surface plasmons, and summarizes current implementations to biosensors for detection of trace amounts of biomarkers, toxins, and pathogens that are relevant to medical diagnostics and food control.