Anti-nociceptive action of peripheral mu-opioid receptors by G-beta-gamma protein-mediated inhibition of TRPM3 channels.

Opioids, agonists of µ-opioid receptors (µORs), are the strongest pain killers clinically available. Their action includes a strong central component, which also causes important adverse effects. However, µORs are also found on the peripheral endings of nociceptors and their activation there produces meaningful analgesia. The cellular mechanisms downstream of peripheral µORs are not well understood. Here, we show in neurons of murine dorsal root ganglia that pro-nociceptive TRPM3 channels, present in the peripheral parts of nociceptors, are strongly inhibited by µOR activation, much more than other TRP channels in the same compartment, like TRPV1 and TRPA1. Inhibition of TRPM3 channels occurs via a short signaling cascade involving Gßγ proteins, which form a complex with TRPM3. Accordingly, activation of peripheral µORs in vivo strongly attenuates TRPM3-dependent pain. Our data establish TRPM3 inhibition as important consequence of peripheral µOR activation indicating that pharmacologically antagonizing TRPM3 may be a useful analgesic strategy.

Surface-enhanced infrared absorption studies towards a new optical biosensor.

Reflectometric interference spectroscopy (RIfS), which is well-established in the visual regime, measures the optical thickness change of a sensitive layer caused, e.g., by binding an analyte. When operated in the mid-infrared range the sensor provides additional information via weak absorption spectra (fingerprints). The originally poor spectra are magnified by surface-enhanced infrared absorption (SEIRA). This is demonstrated using the broad complex fluid water band at 3300 cm-1, which is caused by superposition of symmetric, antisymmetric stretching vibration, and the first overtone of the bending vibration under the influence of H-bonds and Fermi resonance effect. The results are compared with a similar experiment performed with an ATR (attenuated total reflectance) set-up.

Transport of treosulfan and temozolomide across an in-vitro blood-brain barrier model.

In vitro, treosulfan (TREO) has shown high effectiveness against malignant gliomas. However, a first clinical trial for newly diagnosed glioblastoma did not show any positive effect. Even though dosing and timing might have been the reasons for this failure, it might also be that TREO does not reach the brain in sufficient amount. Surprisingly, there are no published data on TREO uptake into the brain of patients, despite extensive research on this compound. An in-vitro blood-brain barrier (BBB) model consisting of primary porcine brain capillary endothelial cells was used to determine the transport of TREO across the cell monolayer. Temozolomide (TMZ), the most widely used cytotoxic drug for malignant gliomas, served as a reference. An HPLC-ESI-MS/MS procedure was developed to detect TREO and TMZ in cell culture medium. Parallel to the experimental approach, the permeability of TREO and the reference substance across the in-vitro BBB was estimated on the basis of their physicochemical properties. The detection limit was 30 nmol/l for TREO and 10 nmol/l for TMZ. Drug transport was measured in two directions: influx, apical-to-basolateral (A-to-B), and efflux, basolateral-to-apical (B-to-A). For TREO, the A-to-B permeability was lower (1.6%) than the B-to-A permeability (3.0%). This was in contrast to TMZ, which had higher A-to-B (13.1%) than B-to-A (7.2%) permeability values. The in-vitro BBB model applied simulated the human BBB properly for TMZ. It is, therefore, reasonable to assume that the values for TREO are also meaningful. Considering the lack of noninvasive, significant alternative methods to study transport across the BBB, the porcine brain capillary endothelial cell model was efficient to collect first data for TREO that explain the disappointing clinical results for this drug against cerebral tumors.

Tracking of magnetite labeled nanoparticles in the rat brain using MRI.

This study was performed to explore the feasibility of tracing nanoparticles for drug transport in the healthy rat brain with a clinical MRI scanner. Phantom studies were performed to assess the R1 ( =  1/T1) relaxivity of different magnetically labeled nanoparticle (MLNP) formulations that were based on biodegradable human serum albumin and that were labeled with magnetite of different size. In vivo MRI measurements in 26 rats were done at 3T to study the effect and dynamics of MLNP uptake in the rat brain and body. In the brain, MLNPs induced T1 changes were quantitatively assessed by T1 relaxation time mapping in vivo and compared to post-mortem results from fluorescence imaging. Following intravenous injection of MLNPs, a visible MLNP uptake was seen in the liver and spleen while no visual effect was seen in the brain. However a histogram analysis of T1 changes in the brain demonstrated global and diffuse presence of MLNPs. The magnitude of these T1 changes scaled with post-mortem fluorescence intensity. This study demonstrates the feasibility of tracking even small amounts of magnetite labeled NPs with a sensitive histogram technique in the brain of a living rodent.

Nanoparticulate flurbiprofen reduces amyloid-ß42 generation in an in vitro blood-brain barrier model.

INTRODUCTION: The amyloid-ß42 (Aß42) peptide plays a crucial role in the pathogenesis of Alzheimer's disease (AD), the most common neurodegenerative disorder affecting the elderly. Over the past years, several approaches and compounds developed for the treatment of AD have failed in clinical studies, likely in part due to their low penetration of the blood-brain barrier (BBB). Since nanotechnology-based strategies offer new possibilities for the delivery of drugs to the brain, this technique is studied intensively for the treatment of AD and other neurological disorders. METHODS: The Aß42 lowering drug flurbiprofen was embedded in polylactide (PLA) nanoparticles by emulsification-diffusion technique and their potential as drug carriers in an in vitro BBB model was examined. First, the cytotoxic potential of the PLA-flurbiprofen nanoparticles on endothelial cells and the cellular binding and uptake by endothelial cells was studied. Furthermore, the biological activity of the nanoparticulate flurbiprofen on γ-secretase modulation as well as its in vitro release was examined. Furthermore, the protein corona of the nanoparticles was studied as well as their ability to transport flurbiprofen across an in vitro BBB model. RESULTS: PLA-flurbiprofen nanoparticles were endocytosed by endothelial cells and neither affected the vitality nor barrier function of the endothelial cell monolayer. The exposure of the PLA-flurbiprofen nanoparticles to human plasma occurred in a rapid protein corona formation, resulting in their decoration with bioactive proteins, including apolipoprotein E. Furthermore, luminally administered PLA-flurbiprofen nanoparticles in contrast to free flurbiprofen were able to modulate γ-secretase activity by selectively decreasing Aß42 levels in the abluminal compartment of the BBB model. CONCLUSIONS: In this study, we were able to show that flurbiprofen can be transported by PLA nanoparticles across an in vitro BBB model and most importantly, the transported flurbiprofen modulated γ-secretase activity by selectively decreasing Aß42 levels. These results demonstrate that the modification of drugs via embedding in nanoparticles is a promising tool to facilitate drug delivery to the brain, which enables future development for the treatment of neurodegenerative disorders like AD.

TRPM3 is a nociceptor channel involved in the detection of noxious heat.

Transient receptor potential melastatin-3 (TRPM3) is a broadly expressed Ca(2+)-permeable nonselective cation channel. Previous work has demonstrated robust activation of TRPM3 by the neuroactive steroid pregnenolone sulfate (PS), but its in vivo gating mechanisms and functions remained poorly understood. Here, we provide evidence that TRPM3 functions as a chemo- and thermosensor in the somatosensory system. TRPM3 is molecularly and functionally expressed in a large subset of small-diameter sensory neurons from dorsal root and trigeminal ganglia, and mediates the aversive and nocifensive behavioral responses to PS. Moreover, we demonstrate that TRPM3 is steeply activated by heating and underlies heat sensitivity in a subset of sensory neurons. TRPM3-deficient mice exhibited clear deficits in their avoidance responses to noxious heat and in the development of inflammatory heat hyperalgesia. These experiments reveal an unanticipated role for TRPM3 as a thermosensitive nociceptor channel implicated in the detection of noxious heat.