Lipophilicity in Molecular Modeling

Purpose. The molecular lipophilicity potential (MLP) offers a three-dimensional representation of lipophilicity, a molecular property encoding intermolecular recognition forces and intramolecular interactions. Methods. The interest and applications of the MLP in molecular modeling are varied, as ilustrated here. Results. The MLP is a major tool to assess the dependence of lipophilicity on conformation. As a matter of fact, the MLP combined with an exploration of the conformational space of a solute reveals its "chameleonic behavior, i.e. its capacity to adapt to its molecular environment by hydrophobic collapse or hydrophilic folding. Another successful application of the MLP is its concatenation into 3D-QSAR (Comparative Molecular Field Analysis, CoMFA). Conclusions. Work is in progress to expand the MLP into a docking tool in the modeling of ligand-receptor interactions.     

Advances in LC platforms for drug discovery

Importance of the field: The major application areas of liquid chromatography (LC) in modern drug discovery are the identification and structural characterization of new potential lead compounds from natural and/or synthetic sources and the determination of their physico-chemical and pharmacokinetic properties. Areas covered in this review: Significant advances in terms of LC platforms achieved in the last 5 years are highlighted in this review. Special attention is paid to novel LC strategies used in the discovery of new bioactive molecules and the determination of lipophilicity, pK(a) values, passive drug permeability and in vitro metabolism of new chemical entities. What the reader will gain: Many improvements were achieved in terms of LC instrumentation, columns technology and analytes detection to attain ultra-fast and/or high-resolution chromatographic separations. These advances are particularly beneficial to face the complexity and high number of samples studied in the early phases of the discovery process. Advantages and drawbacks of each strategy are discussed. Take home message: LC and ultra high pressure liquid chromatography coupled with mass spectrometry detection constitute the most promising strategies to achieve high-throughput and/or high-resolution analyses in a drug discovery environment.     

BARD1: an independent predictor of survival in non-small cell lung cancer

BRCA1 mRNA overexpression is correlated with poor survival in NSCLC. However, BRCA1 functions depend on the interaction with BARD1 for its stability, nuclear localization and ubiquitin ligase activity. Expression of alternatively spliced BARD1 isoforms that lack the BRCA1-interaction domain was found upregulated and correlated with poor prognosis in breast and ovarian cancer. These BARD1 isoforms are essential for proliferation of cancer cells in vitro. We investigated whether BARD1 isoforms are expressed in NSCLC. While in lung tissues from healthy controls BARD1 expression was undetectable on the mRNA level and protein level, we found two novel isoforms in addition to previously identified mRNAs expressed in all NSCLC samples tested. Furthermore, the pattern of BARD1 isoform expression was similar in tumor and morphologically normal peri-tumor tissues, and only one novel isoform π was specifically upregulated in tumors. Immunohistochemistry revealed that all 100 NSCLC cases tested expressed isoform-specific BARD1 epitopes, while BARD1 expression was undetectable in biopsies from healthy controls. Statistical analysis showed that the expression of epitopes PVC and WFS, present on isoform π, or epitope WFS alone, expressed on isoforms π, κ and β, were significantly correlated with decreased patient survival. These findings were corroborated in a mouse model of chemically induced lung cancer. Immunostaining of mouse tumors showed that BARD1 epitopes PVC and WFS were specifically upregulated in invasive, but not in confined lung tumors. Thus, BARD1 isoforms might be involved in tumor initiation and invasive progression and might represent a novel prognostic marker for NSCLC.     

Developmental immunotoxicity in male rats after juvenile exposure to di-n-octyltin dichloride (DOTC)

To determine relevant endpoints for evaluating developmental immunotoxicity due to juvenile exposure and optimal age of the animals at assessment, a wide range of immunological parameters were assessed in a juvenile toxicity study. Rats were exposed to di-n-octyltin dichloride (DOTC) by gavage from postnatal day (PND) 10 through PND 21 and via the diet after weaning using a benchmark dose (BMD) approach. Immune assessments were performed in male rats on PNDs 21, 42, and 70 and a subset of animals was used to evaluate the T-cell dependent antibody response (TDAR) to Keyhole limpet hemocyanin. Immune effects were more pronounced on PND 21 and 42 and observed at lower doses than developmental effects. The most sensitive immune parameters affected included TDAR parameters and thymocyte subpopulations with lower confidence limits of the benchmark doses (BMDLs) below the overall no-observed-adverse-effect-level (NOAEL) for DOTC reported so far in literature. These findings illustrate the relative sensitivity of the developing immune system for DOTC, the additional value of assessing functional immune parameters, and underscore the relevance of juvenile immunotoxicity testing in view of the risk assessment of chemicals.     

Greater vasodepressor sensitivity to nicardipine in spontaneously hypertensive rats (SHR) compared to normotensive rats

Summary Differences in the degree of attenuation by the calcium entry blocker, nicardipine, of the pressor responses to -1 (phenylephrine) and -2 (UK 14.304) adrenoceptor agonists was investigated in pentobarbital-anesthetized, normotensive Sprague-Dawley (SD) or Wistar Kyoto (WKY) rats, and spontaneously hypertensive rats (SHR), treated with the ganglionic blocking agent, pentolinium. Following administration of the ganglionic blocking agent, pentolinium, nicardipine produced a significant fall in blood pressure in SHR but not in SD or WKY rats. Nicardipine had no effect on the basal blood pressure of pithed SHR. In rats treated with the ganglionic blocking agent, pentolinium, nicardipine produced parallel shifts to the right in the doseresponse curves for phenylephrine but had no effect on maximal responses to phenylephrine. The decrease in the ED₅₀ of phenylephrine was greater in the SHR than in normotensive rats. Nicardipine produced a decrease in both the ED so and the maximal response to the -2 adrenoceptor agonist, UK 14.304. The decrease in the maximal response was greater in SHR than in WKY normotensive rats but the change in ED₅₀ for UK 14.304 was greater in WKY than in SHR. SD normotensive rats gave intermediate results. We conclude that the inhibition of -adrenoceptor-mediated pressor responses by nicardipine is generally more pronounced in SHR than in normotensive rats. This suggests that hypertension may be accompanied by an increase in the sensitivity of peripheral resistance beds to calcium entry blockers. Send offprint requests to: J. Atkinson     

Blood-to-Brain Transfer of Various Oxicams: Effects of Plasma Binding on Their Brain Delivery

Purpose. The objective of this work was to assess the influence of binding to plasma proteins and to serum on the brain extraction of four antiinflammatory oxicams. Methods. The brain extraction of isoxicam, tenoxicam, meloxicam and piroxicam was investigated in rats using the carotid injection technique. Blood protein binding parameters were determined by equilibrium dialysis using human serum, human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) solutions at various concentrations. Results. All oxicams had low values of brain extraction, between 19% and 39% when dissolved in serum, i.e. under physiological conditions. Brain efflux rate constants calculated from the wash-out curves were the same in the absence or presence of serum. Brain efflux was inversely related to the polarity of the oxicams, such that the higher their H-bonding capacity, the lower their brain efflux. The free dialyzable drug fraction was inversely related to protein concentration. However, rat brain extraction was always higher than expected from in vitro measurements of the dialyzable fraction. Conclusions. Except for piroxicam whose brain extraction was partially decreased in the presence of proteins, the serum unbound and initially bound fractions of oxicams both seem available for transfer into the brain. Modest affinities for AAG rule out any related effect. More surprising is the apparent lack of effect on brain transfer of the high-affinity binding to HSA and serum. The enhanced brain uptake of meloxicam in the presence of AAG could be a result of interactions between this globular protein and the endothelial wall.     

Evidence for the existence of [3H]-trimetazidine binding sites involved in the regulation of the mitochondrial permeability transition pore

1. Trimetazidine is an anti-ischaemic drug effective in different experimental models but its mechanism of action is not fully understood. Data indicate that mitochondria could be the main target of this drug. The aim of this work was to investigate the binding of [³H]-trimetazidine on a purified preparation of rat liver mitochondria.
2. [³H]-trimetazidine binds to two populations of mitochondrial binding sites with K_d values of 0.96 and 84 μM. The total concentration of binding sites is 113 pmol mg⁻¹ protein. Trimetazidine binding sites are differently distributed. The high-affinity ones are located on the outer membranes and represent only a small part (4%) of total binding sites, whereas the low-affinity ones are located on the inner membranes and are more abundant (96%) with a B_max=108 pmol mg⁻¹ protein.
3. Drug displacement studies with pharmacological markers for different mitochondrial targets showed that [³H]-trimetazidine binding sites are different from previously described mitochondrial sites.
4. The possible involvement of [³H]-trimetazidine binding sites in the regulation of the mitochondrial permeability transition pore (MTP), a voltage-dependent channel sensitive to cyclosporin A, was investigated with mitochondrial swelling experiments. Trimetazidine inhibited the mitochondrial swelling induced by Ca²⁺ plus tert-butylhydroperoxide (t-BH). This effect was concentration-dependent with an IC₅₀ value of 200 μM.
5. Assuming that trimetazidine effectiveness may be related to its structure as an amphiphilic cation, we compared it with other compounds exhibiting the same chemical characteristic both for their ability to inhibit MTP opening and to displace [³H]-trimetazidine bound to mitochondria. Selected compounds were drugs known to interact with various biological membranes.
6. A strong correlation between swelling inhibition potency and low-affinity [³H]-trimetazidine binding sites was observed: r=0.907 (n=24; P<0.001).
7. These data suggest that mitochondrial sites labelled with [³H]-trimetazidine may be involved in the MTP inhibiton.

     

Stabilization of the Hydrophilic Sphere of lobitridol,an lodinated Contrast Agent,as Revealed by Experimental and Computational Investigations

Purpose. The regular distribution in space and the stability in time of the hydrophilic sphere surrounding iobitridol were investigated. This is a novel yet important concept in the design of polyiodinated contrast agents since such a sphere is meant to hide their hydrophobic core and thus prevent hydrophobic interactions with biomacromolecules and hence chemotoxicity. Methods. The methods used were experimental (HPLC, ¹H- and ¹³C-NMR spectroscopy) and computational (calculation of conformational behavior and molecular electrostatic potentials). Results. lobitridol exists as a mixture of stereoisomers due to hindered rotation around several bonds. High-temperature molecular dynamics established the existence between 0 and 15 kcal/mol of 238 conformers belonging to 14 classes. Most of these conformers have an inaccessible hydrophobic core, and variable temperature molecular dynamics confirmed that the hydrophilic sphere around iobitridol is stable against external disruption. Conclusions. This study has demonstrated that iobitridol fulfills the physicochemical and structural criteria believed to render a polyiodinated contrast agent inert toward interacting with biomacromolecules.