Lead discovery, chemistry optimization, and biological evaluation studies of novel biamide derivatives as CB2 receptor inverse agonists and osteoclast inhibitors

N,N'-((4-(Dimethylamino)phenyl)methylene)bis(2-phenylacetamide) was discovered by using 3D pharmacophore database searches and was biologically confirmed as a new class of CB(2) inverse agonists. Subsequently, 52 derivatives were designed and synthesized through lead chemistry optimization by modifying the rings A-C and the core structure in further SAR studies. Five compounds were developed and also confirmed as CB(2) inverse agonists with the highest CB(2) binding affinity (CB(2)K(i) of 22-85 nM, EC(50) of 4-28 nM) and best selectivity (CB(1)/CB(2) of 235- to 909-fold). Furthermore, osteoclastogenesis bioassay indicated that PAM compounds showed great inhibition of osteoclast formation. Especially, compound 26 showed 72% inhibition activity even at the low concentration of 0.1 μM. The cytotoxicity assay suggested that the inhibition of PAM compounds on osteoclastogenesis did not result from its cytotoxicity. Therefore, these PAM derivatives could be used as potential leads for the development of a new type of antiosteoporosis agent.

Mitochondrial CB₁ receptors regulate neuronal energy metabolism

The mammalian brain is one of the organs with the highest energy demands, and mitochondria are key determinants of its functions. Here we show that the type-1 cannabinoid receptor (CB(1)) is present at the membranes of mouse neuronal mitochondria (mtCB(1)), where it directly controls cellular respiration and energy production. Through activation of mtCB(1) receptors, exogenous cannabinoids and in situ endocannabinoids decreased cyclic AMP concentration, protein kinase A activity, complex I enzymatic activity and respiration in neuronal mitochondria. In addition, intracellular CB(1) receptors and mitochondrial mechanisms contributed to endocannabinoid-dependent depolarization-induced suppression of inhibition in the hippocampus. Thus, mtCB(1) receptors directly modulate neuronal energy metabolism, revealing a new mechanism of action of G protein-coupled receptor signaling in the brain.

β-Caryophyllene ameliorates cisplatin-induced nephrotoxicity in a cannabinoid 2 receptor-dependent manner

(E)-β-caryophyllene (BCP) is a natural sesquiterpene found in many essential oils of spice (best known for contributing to the spiciness of black pepper) and food plants with recognized anti-inflammatory properties. Recently it was shown that BCP is a natural agonist of endogenous cannabinoid 2 (CB(2)) receptors, which are expressed in immune cells and mediate anti-inflammatory effects. In this study we aimed to test the effects of BCP in a clinically relevant murine model of nephropathy (induced by the widely used antineoplastic drug cisplatin) in which the tubular injury is largely dependent on inflammation and oxidative/nitrative stress. β-caryophyllene dose-dependently ameliorated cisplatin-induced kidney dysfunction, morphological damage, and renal inflammatory response (chemokines MCP-1 and MIP-2, cytokines TNF-α and IL-1β, adhesion molecule ICAM-1, and neutrophil and macrophage infiltration). It also markedly mitigated oxidative/nitrative stress (NOX-2 and NOX-4 expression, 4-HNE and 3-NT content) and cell death. The protective effects of BCP against biochemical and histological markers of nephropathy were absent in CB(2) knockout mice. Thus, BCP may be an excellent therapeutic agent to prevent cisplatin-induced nephrotoxicity through a CB(2) receptor-dependent pathway. Given the excellent safety profile of BCP in humans it has tremendous therapeutic potential in a multitude of diseases associated with inflammation and oxidative stress.

Modulation of L-α-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

The new insight into dynamic crossover in glass forming liquids from the apparent enthalpy analysis

One of the most intriguing phenomena in glass forming systems is the dynamic crossover (T(B)), occurring well above the glass temperature (T(g)). So far, it was estimated mainly from the linearized derivative analysis of the primary relaxation time τ(T) or viscosity η(T) experimental data, originally proposed by Stickel et al. [J. Chem. Phys. 104, 2043 (1996); J. Chem. Phys. 107, 1086 (1997)]. However, this formal procedure is based on the general validity of the Vogel-Fulcher-Tammann equation, which has been strongly questioned recently [T. Hecksher et al. Nature Phys. 4, 737 (2008); P. Lunkenheimer et al. Phys. Rev. E 81, 051504 (2010); J. C. Martinez-Garcia et al. J. Chem. Phys. 134, 024512 (2011)]. We present a qualitatively new way to identify the dynamic crossover based on the apparent enthalpy space (H(a)(') = dlnτ/d(1/T)) analysis via a new plot lnH(a)(') vs. 1∕T supported by the Savitzky-Golay filtering procedure for getting an insight into the noise-distorted high order derivatives. It is shown that depending on the ratio between the "virtual" fragility in the high temperature dynamic domain (m(high)) and the "real" fragility at T(g) (the low temperature dynamic domain, m = m(low)) glass formers can be splitted into two groups related to f < 1 and f > 1, (f = m(high)∕m(low)). The link of this phenomenon to the ratio between the apparent enthalpy and activation energy as well as the behavior of the configurational entropy is indicated.

Angiotensinergic innervation of the kidney: Localization and relationship with catecholaminergic postganglionic and sensory nerve fibers

We describe an angiotensin (Ang) II-containing innervation of the kidney. Cryosections of rat, pig and human kidneys were investigated for the presence of Ang II-containing nerve fibers using a mouse monoclonal antibody against Ang II (4B3). Co-staining was performed with antibodies against synaptophysin, tyrosine 3-hydroxylase, and dopamine beta-hydroxylase to detect catecholaminergic efferent fibers and against calcitonin gene-related peptide to detect sensory fibers. Tagged secondary antibodies and confocal light or laser scanning microscopy were used for immunofluorescence detection. Ang II-containing nerve fibers were densely present in the renal pelvis, the subepithelial layer of the urothelium, the arterial nervous plexus, and the peritubular interstitium of the cortex and outer medulla. They were infrequent in central veins and the renal capsule and absent within glomeruli and the renal papilla. Ang II-positive fibers represented phenotypic subgroups of catecholaminergic postganglionic or sensory fibers with different morphology and intrarenal distribution compared to their Ang II-negative counterparts. The Ang II-positive postganglionic fibers were thicker, produced typically fusiform varicosities and preferentially innervated the outer medulla and periglomerular arterioles. Ang II-negative sensory fibers were highly varicose, prevailing in the pelvis and scarce in the renal periphery compared to the rarely varicose Ang II-positive fibers. Neurons within renal microganglia displayed angiotensinergic, catecholaminergic, or combined phenotypes. Our results suggest that autonomic fibers may be an independent source of intrarenal Ang II acting as a neuropeptide co-transmitter or neuromodulator. The angiotensinergic renal innervation may play a distinct role in the neuronal control of renal sodium reabsorption, vasomotion and renin secretion.