Mechanism-based inhibition of CYP2C8 by gemfibrozil in humans : characterisation of time and dose relationships

Drug-drug interactions may cause serious, even fatal clinical consequences. Therefore, it is important to examine the interaction potential of new chemical entities early in drug development. Mechanism-based inhibition is a pharmacokinetic interaction type, which causes irreversible loss of enzyme activity and can therefore lead to unusually profound and long-lasting consequences. The in vitro in vivo extrapolation (IVIVE) of drug-drug interactions caused by mechanism-based inhibition is challenging. Consequently, many of these interactions have remained unrecognised for many years. The concomitant use of the fibrate-class lipid-lowering agent gemfibrozil increases the concentrations of some drugs and their effects markedly. Even fatal cases of rhabdomyolysis occurred in patients administering gemfibrozil and cerivastatin concomitantly. One of the main mechanisms behind this effect is the mechanism-based inhibition of the cytochrome P450 (CYP) 2C8 enzyme by a glucuronide metabolite of gemfibrozil leading to increased cerivastatin concentrations. Although the clinical use of gemfibrozil has clearly decreased during recent years, gemfibrozil is still needed in some special cases. To enable safe use of gemfibrozil concomitantly with other drugs, information concerning the time and dose relationships of CYP2C8 inhibition by gemfibrozil should be known. This work was carried out as four in vivo clinical drug-drug interaction studies to examine the time and dose relationships of the mechanism-based inhibitory effect of gemfibrozil on CYP2C8. The oral antidiabetic drug repaglinide was used as a probe drug for measuring CYP2C8 activity in healthy volunteers. In this work, mechanism-based inhibition of the CYP2C8 enzyme by gemfibrozil was found to occur rapidly in humans. The inhibitory effect developed to its maximum already when repaglinide was given 1-3 h after gemfibrozil intake. In addition, the inhibition was shown to abate slowly. A full recovery of CYP2C8 activity, as measured by repaglinide metabolism, was achieved 96 h after cessation of gemfibrozil treatment. The dose-dependency of the mechanism-based inhibition of CYP2C8 by gemfibrozil was shown for the first time in this work. CYP2C8 activity was halved by a single 30 mg dose of gemfibrozil or by twice daily administration of less than 30 mg of gemfibrozil. Furthermore, CYP2C8 activity was decreased over 90% by a single dose of 900 mg gemfibrozil or twice daily dosing of approximately 100 mg gemfibrozil. In addition, with the application of physiological models to the data obtained in the dose-dependency studies, the major role of mechanism-based inhibition of CYP2C8 in the interaction between gemfibrozil and repaglinide was confirmed. The results of this work enhance the proper use of gemfibrozil and the safety of patients. The information related to time-dependency of CYP2C8 inhibition by gemfibrozil may also give new insights in order to improve the IVIVE of the drug-drug interactions of new chemical entities. The information obtained by this work may be utilised also in the design of clinical drug-drug interaction studies in the future.

Interaction of Sesbania mosaic virus movement protein with the coat protein - implications for viral spread

Sesbania mosaic virus (SeMV) is a single-stranded positive-sense RNA plant virus belonging to the genus Sobemovirus. The movement protein (MP) encoded by SeMV ORF1 showed no significant sequence similarity with MPs of other genera, but showed 32% identity with the MP of Southern bean mosaic virus within the Sobemovirus genus. With a view to understanding the mechanism of cell-to-cell movement in sobemoviruses, the SeMV MP gene was cloned, over-expressed in Escherichia coli and purified. Interaction of the recombinant MP with the native virus (NV) was investigated by ELISA and pull-down assays. It was observed that SeMV MP interacted with NV in a concentration- and pH-dependent manner. Analysis of N- and C-terminal deletion mutants of the MP showed that SeMV MP interacts with the NV through the N- terminal 49 amino acid segment. Yeast two-hybrid assays confirmed the in vitro observations, and suggested that SeMV might belong to the class of viruses that require MP and NV/coat protein for cell-to-cell movement.

Interaction of Sesbania Mosaic Virus Movement Protein with VPg and P10: Implication to Specificity of Genome Recognition

Sesbania mosaic virus (SeMV) is a single strand positive-sense RNA plant virus that belongs to the genus Sobemovirus. The mechanism of cell-to-cell movement in sobemoviruses has not been well studied. With a view to identify the viral encoded ancillary proteins of SeMV that may assist in cell-to-cell movement of the virus, all the proteins encoded by SeMV genome were cloned into yeast Matchmaker system 3 and interaction studies were performed. Two proteins namely, viral protein genome linked (VPg) and a 10-kDa protein (P10) c v gft encoded by OFR 2a, were identified as possible interacting partners in addition to the viral coat protein (CP). Further characterization of these interactions revealed that the movement protein (MP) recognizes cognate RNA through interaction with VPg, which is covalently linked to the 59 end of the RNA. Analysis of the deletion mutants delineated the domains of MP involved in the interaction with VPg and P10. This study implicates for the first time that VPg might play an important role in specific recognition of viral genome by MP in SeMV and shed light on the possible role of P10 in the viral movement.

Xenon in sodium Y zeolite. 2. Arrhenius relation, mechanism, and barrier height distribution for cage-to-cage diffusion

Various geometrical and energetic distribution functions and other properties connected with the cage-to-cage diffusion of xenon in sodium Y zeolite have been obtained from long molecular dynamics calculations. Analysis of diffusion pathways reveals two interesting mechanisms-surface-mediated and centralized modes for cage-to-cage diffusion. The surface-mediated mode of diffusion exhibits a small positive barrier, while the centralized diffusion exhibits a negative barrier for the sorbate to diffuse across the 12-ring window. In both modes, however, the sorbate has to be activated from the adsorption site to enable it to gain mobility. The centralized diffusion additionally requires the sorbate to be free of the influence of the surface of the cage as well. The overall rate for cage-to-cage diffusion shows an Arrhenius temperature dependence with E(a) = 3 kJ/mol. It is found that the decay in the dynamical correction factor occurs on a time scale comparable to the cage residence time. The distributions of barrier heights have been calculated. Functions reflecting the distribution of the sorbate-zeolite interaction at the window and the variations of the distance between the sorbate and the centers of the parent and daughter cages are presented.

Mechanism of ?-cleavage in the excited states of keto sulfides

The beta-cleavage process in photoexcited ketones of structure RCOCH2X (X = CH2CH3, OCH3, SCH3; R = CH3, Ph) has been studied using the configuration interaction procedure within the framework of MINDO/3. The results explain qualitatively why the beta-cleavage process is faster than the alpha-cleavage process in keto sulfides while in keto ethers the reverse is true.

Realtime kinetic analysis of antigen-antibody interaction using solid phase binding: Transformation of hCG-monoclonal antibody complex

Kinetic constants of MAb-hCG interactions have been determined using solid phase binding of I-125[hCG] to immobilized MAb. While association has been shown to follow the expected pattern, dissociation consists of at least two reversible steps, one with a rate constant of 0.0025 min(-1), and a second with a rate constant of 0.00023 min(-1). Validity of affinity constant measurements in the light of the complex reaction kinetics is discussed, A comparison between the method of surface plasmon resonance technology (BIAcore) and solid phase binding (SPB) for determination of kinetic parameters shows that SPB provides not only a cost-effective approach for determination of realtime kinetic parameters of macromolecular ligand-ligate interaction but also a method with several advantages over the BIAcore system in investigating the mechanism of antigen-antibody interaction.

Mutations in beta ` subunit of Escherichia coli RNA polymerase perturb the activator polymerase functional interaction required for promoter clearance

P>Transcription activator C employs a unique mechanism to activate mom gene of bacteriophage Mu. The activation process involves, facilitating the recruitment of RNA polymerase (RNAP) by altering the topology of the promoter and enhancing the promoter clearance by reducing the abortive transcription. To understand the basis of this multi-step activation mechanism, we investigated the nature of the physical interaction between C and RNAP during the process. A variety of assays revealed that only DNA-bound C contacts the beta' subunit of RNAP. Consistent to these results, we have also isolated RNAP mutants having mutations in the beta' subunit which were compromised in C-mediated activation. Mutant RNAPs show reduced productive transcription and increased abortive initiation specifically at the C-dependent mom promoter. Positive control (pc) mutants of C, defective in interaction with RNAP, retained the property of recruiting RNAP to the promoter but were unable to enhance promoter clearance. These results strongly suggest that the recruitment of RNAP to the mom promoter does not require physical interaction with C, whereas a contact between the beta' subunit and the activator, and the subsequent allosteric changes in the active site of the enzyme are essential for the enhancement of promoter clearance.

Mutagenicity associated with O6-methylguanine-DNA damage and mechanism of nucleotide flipping by AGT during repair

Methylated guanine damage at O6 position (i.e. O6MG) is dangerous due to its mutagenic and carcinogenic character that often gives rise to G:C-A:T mutation. However, the reason for this mutagenicity is not known precisely and has been a matter of controversy. Further, although it is known that O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6MG paired with cytosine in DNA, the complete mechanism of target recognition and repair is not known completely. All these aspects of DNA damage and repair have been addressed here by employing high level density functional theory in gas phase and aqueous medium. It is found that the actual cause of O6MG mediated mutation may arise due to the fact that DNA polymerases incorporate thymine opposite to O6MG, misreading the resulting O6MG:T complex as an A:T base pair due to their analogous binding energies and structural alignments. It is further revealed that AGT mediated nucleotide flipping occurs in two successive steps. The intercalation of the finger residue Arg 128 into the DNA double helix and its interaction with the O6MG: C base pair followed by rotation of the O6MG nucleotide are found to be crucial for the damage recognition and nucleotide flipping.

Interaction anisotropy and shear instability of aspirin polymorphs established by nanoindentation

Nanoindentation is applied to the two polymorphs of aspirin to examine and differentiate their interaction anisotropy and shear instability. Aspirin provides an excellent test system for the technique because: (i) polymorphs I and II exhibit structural similarity in two dimensions, thereby facilitating clear examination of the differences in mechanical response in relation to well-defined differences between the two crystal structures; (ii) single crystals of the metastable polymorph II have only recently become accessible; (iii) shear instability has been proposed for II. Different elastic moduli and hardness values determined for the two polymorphs are correlated with their crystal structures, and the interpretation is supported by measured thermal expansion coefficients. The stress-induced transformation of the metastable polymorph II to the stable polymorph I can be brought about rapidly by mechanical milling, and proceeds via a slip mechanism. This work establishes that nanoindentation provides ``signature'' responses for the two aspirin polymorphs, despite their very similar crystal structures. It also demonstrates the value of the technique to quantify stability relationships and phase transformations in molecular crystals, enabling a deeper understanding of polymorphism in the context of crystal engineering.

Nonspecific Interaction between DNA and Protein allows for Cooperativity: A Case Study with Mycobacterium DNA Binding Protein

Different DNA-binding proteins have different interaction modes with DNA. Sequence-specific DNA protein interaction has been mostly associated with regulatory processes inside a cell, and as such extensive studies have been made. Adequate data is also available on nonspecific DNA protein interaction, as an intermediate to protein's search for its cognate partner. Multidomain nonspecific DNA protein interaction involving physical sequestering of DNA has often been implicated to regulate gene expression indirectly. However, data available on this type of interaction is limited. One such interaction is the binding of DNA with mycobacterium DNA binding proteins. We have used the Langmuir-Blodgett technique to evaluate for the first time the kinetics and thermodynamics of Mycobacterium smegmatis Dps 1 binding to DNA. By immobilizing one of the interacting partners, we have shown that, when a kinetic bottleneck is applied, the binding mechanism showed cooperative binding (n = 2.72) at lower temperatures, but the degree of cooperativity gradually reduces (n = 1.38) as the temperature was increased We have also compared the kinetics and thermodynamics of sequence-specific and nonspecific DNA protein interactions under the same set of conditions.

ZnO/Ag nanohybrid: synthesis, characterization, synergistic antibacterial activity and its mechanism

A highly homogeneous ZnO/Ag nanohybrid has been synthesized by a novel route, employing chitosan as mediator by purely electrostatic interaction. By employing various techniques such as powder XRD, UV-visible, IR spectroscopy and electron (SEM, TEM) microscopy, the formation of the nanohybrid has been established. The synergistic antibacterial effect of ZnO/Ag nanohybrid on Gram-positive and Gram-negative bacteria is found to be more effective, compared to the individual components (ZnO and Ag). Cytotoxicity experiments are carried out and the results are correlated to the solubility of the nanohybrid. A possible mechanism has been proposed for the antibacterial activity of ZnO/Ag nanohybrid, based on TEM studies on bacteria, carried out by employing the microtome technique and by EPR measurements on the hybrid.

Sensitivity of nucleation phenomena on range of interaction potential

Theoretical and computational investigations of nucleation have been plagued by the sensitivity of the phase diagram to the range of the interaction potential. As the surface tension depends strongly on the range of interaction potential and as the classical nucleation theory (CNT) predicts the free energy barrier to be directly proportional to the cube of the surface tension, one expects a strong sensitivity of nucleation barrier to the range of the potential; however, CNT leaves many aspects unexplored. We find for gas-liquid nucleation in Lennard-Jones system that on increasing the range of interaction the kinetic spinodal (KS) (where the mechanism of nucleation changes from activated to barrierless) shifts deeper into the metastable region. Therefore the system remains metastable for larger value of supersaturation and this allows one to explore the high metastable region without encountering the KS. On increasing the range of interaction, both the critical cluster size and pre-critical minima in the free energy surface of kth largest cluster, at respective kinetic spinodals, shift towards smaller cluster size. In order to separate surface tension contribution to the increase in the barrier from other non-trivial factors, we introduce a new scaling form for surface tension and use it to capture both the temperature and the interaction range dependence of surface tension. Surprisingly, we find only a weak non-trivial contribution from other factors to the free energy barrier of nucleation. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.3685835]

Ribosome-RNA interaction: a potential target for developing antiviral against hepatitis C virus

Hepatitis C virus (HCV), a member of Flaviviridae, encoding a positive-sense single-stranded RNA translates by cap-independent mechanism using the internal ribosome entry site (IRES) present in the 5' UTR of the virus. The IRES has complex stem loop structures and is capable of recruiting the 40S ribosomal subunit in a factor-independent fashion. As the IRES sequence is highly conserved throughout the HCV genotypes and the translation is the first obligatory step of the HCV life cycle, the IRE'S-mediated translation, or more specifically, the ribosome HCV RNA interaction is an attractive target to design effective antivirals. This article will focus on the mechanism of the HCV IRES translation and the various ways in which the interaction of ribosome and IRES has been targeted.

Interaction of nucleic acids with carbon nanotubes and dendrimers

Nucleic acid interaction with nanoscale objects like carbon nanotubes (CNTs) and dendrimers is of fundamental interest because of their potential application in CNT separation, gene therapy and antisense therapy. Combining nucleic acids with CNTs and dendrimers also opens the door towards controllable self-assembly to generate various supra-molecular and nano-structures with desired morphologies. The interaction between these nanoscale objects also serve as a model system for studying DNA compaction, which is a fundamental process in chromatin organization. By using fully atomistic simulations, here we report various aspects of the interactions and binding modes of DNA and small interfering RNA (siRNA) with CNTs, graphene and dendrimers. Our results give a microscopic picture and mechanism of the adsorption of single- and double-strand DNA (ssDNA and dsDNA) on CNT and graphene. The nucleic acid-CNT interaction is dominated by the dispersive van der Waals (vdW) interaction. In contrast, the complexation of DNA (both ssDNA and dsDNA) and siRNA with various generations of poly-amido-amine (PAMAM) dendrimers is governed by electrostatic interactions. Our results reveal that both the DNA and siRNA form stable complex with the PAMAM dendrimer at a physiological pH when the dendrimer is positively charged due to the protonation of the primary amines. The size and binding energy of the complex increase with increase in dendrimer generation. We also give a summary of the current status in these fields and discuss future prospects.

Mechanism for the Compressive Strain Induced Oscillations in the Conductance of Carbon Nanotubes

We report on the monotonic increase and the oscillation of electrical conductance in multiwalled carbon nanotubes with compressive strain. Combined experimental and theoretical analyses confirm that the conductance variation with strain is because of the transition from sp(2) to configurations that are promoted by the interaction of walls in the nanotubes. The intrawall interaction is the reason for the monotonic increase in the conduction, while the oscillations are attributable to interwall interactions. This explains the observed electromechanical oscillation in multiwalled nanotubes and its absence in single-walled nanotubes, thereby resolving a long-standing debate on the interpretation of these results. Moreover, the current carrying capability of nanotubes can be enhanced significantly by controlling applied strains. DOI: 10.1103/PhysRevLett.110.095504