Mechanisms of osteoclastogenesis inhibition by a novel class of biphenyl-type cannabinoid CB(2) receptor inverse agonists

The cannabinoid CB(2) receptor is known to modulate osteoclast function by poorly understood mechanisms. Here, we report that the natural biphenyl neolignan 4'-O-methylhonokiol (MH) is a CB(2) receptor-selective antiosteoclastogenic lead structure (K(i) < 50 nM). Intriguingly, MH triggers a simultaneous G(i) inverse agonist response and a strong CB(2) receptor-dependent increase in intracellular calcium. The most active inverse agonists from a library of MH derivatives inhibited osteoclastogenesis in RANK ligand-stimulated RAW264.7 cells and primary human macrophages. Moreover, these ligands potently inhibited the osteoclastogenic action of endocannabinoids. Our data show that CB(2) receptor-mediated cAMP formation, but not intracellular calcium, is crucially involved in the regulation of osteoclastogenesis, primarily by inhibiting macrophage chemotaxis and TNF-α expression. MH is an easily accessible CB(2) receptor-selective scaffold that exhibits a novel type of functional heterogeneity.

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.

Continuous, Passive Liquid-Liquid Extraction and Emulsion Separation Within Microfluidic and Millifluidic Devices

Liquid-liquid extraction (LLE) is a method used to separate compounds based on their relative activity in two immiscible phases. By significantly reducing the scale of liquid-liquid extraction to the micro- and milli-fluidic levels, this separation process can bemade suitable for low volume, high value materials.

Efficient Liquid Liquid Extraction By Emulsion Formation and Separation in Robust Milli-Fluidic Devices

Conventional liquid liquid extraction (LLE) methods require large volumes of fluids to achieve the desired mass transfer of a solute, which is unsuitable for systems dealing with a low volume or high value product. An alternative to these methods is to scale down the process. Millifluidic devices share many of the benefits of microfluidic systems, including low fluid volumes, increased interfacial area-to-volume ratio, and predictability. A robust millifluidic device was created from acrylic, glass, and aluminum. The channel is lined with a hydrogel cured in the bottom half of the device channel. This hydrogel stabilizes co-current laminar flow of immiscible organic and aqueous phases. Mass transfer of the solute occurs across the interface of these contacting phases. Using a y-junction, an aqueous emulsion is created in an organic phase. The emulsion travels through a length of tubing and then enters the co-current laminar flow device, where the emulsion is broken and each phase can be collected separately. The inclusion of this emulsion formation and separation increases the contact area between the organic and aqueous phases, therefore increasing the area over which mass transfer can occur. Using this design, 95% extraction efficiency was obtained, where 100% is represented by equilibrium. By continuing to explore this LLE process, the process can be optimized and with better understanding may be more accurately modeled. This system has the potential to scale up to the industrial level and provide the efficient extraction required with low fluid volumes and a well-behaved system.

Numerical solutions of elliptic inverse problems via the equation error method

To estimate a parameter in an elliptic boundary value problem, the method of equation error chooses the value that minimizes the error in the PDE and boundary condition (the solution of the BVP having been replaced by a measurement). The estimated parameter converges to the exact value as the measured data converge to the exact value, provided Tikhonov regularization is used to control the instability inherent in the problem. The error in the estimated solution can be bounded in an appropriate quotient norm; estimates can be derived for both the underlying (infinite-dimensional) problem and a finite-element discretization that can be implemented in a practical algorithm. Numerical experiments demonstrate the efficacy and limitations of the method.