Bromo-substituted indirubins for inhibition of protein kinase-mediated signalling involved in inflammatory mediator release in human monocytes.

Targeting protein kinases that regulate signalling pathways in inflammation is an effective pharmacological approach to alleviate uncontrolled inflammatory diseases. In this context, the natural product indirubin and its 6-bromo-substituted analogue 6-bromoindirubin-3 -glycerol-oxime ether (6BIGOE; 1) were identified as potent inhibitors of glycogen synthase kinase-3ß (GSK-3ß). These inhibitors suppress the release of pro-inflammatory cytokines and prostaglandins (PG) from human monocytes. However, indirubin derivatives target several protein kinases such as cyclin-dependent kinases (CDKs) which has been a major concern for their application in inflammation therapy. Here, we report on a library of 13 5-bromo-substituted indirubin derivatives that have been designed to improve potency and target selectivity. Side-by-side comparison of reference compound 1 (6BIGOE) with 5-bromo derivatives revealed its isomer 2 (5BIGOE), as the most potent derivative able to supress pro-inflammatory cytokine and PG release in lipopolysaccharide-stimulated human monocytes. Analysis of protein kinase inhibition in intact monocytes, supported by our in silico findings, proposed higher selectivity of 1 for GSK-3ß inhibition with lesser potency against CDKs 8 and 9. In contrast, 2 supressed the activity of these CDKs with higher effectiveness than GSK-3ß, representing additional targets of indirubins within the inflammatory response. Encapsulation of 1 and 2 into polymer-based nanoparticles (NP) improved their pharmacological potential. In conclusion, the 5- and 6-brominated indirubins 1 and 2 as dual GSK-3ß and CDK8/9 inhibitors represent a novel concept for intervention with inflammatory disorders.

Drug delivery of 6-bromoindirubin-3'-glycerol-oxime ether employing poly(D,L-lactide-co-glycolide)-based nanoencapsulation techniques with sustainable solvents.

BACKGROUND: Insufficient solubility and stability of bioactive small molecules as well as poor biocompatibility may cause low bioavailability and are common obstacles in drug development. One example of such problematic molecules is 6-bromoindirubin-3'-glycerol-oxime ether (6BIGOE), a hydrophobic indirubin derivative. 6BIGOE potently modulates the release of inflammatory cytokines and lipid mediators from isolated human monocytes through inhibition of glycogen synthase kinase-3 in a favorable fashion. However, 6BIGOE suffers from poor solubility and short half-lives in biological aqueous environment and exerts cytotoxic effects in various mammalian cells. In order to overcome the poor water solubility, instability and cytotoxicity of 6BIGOE, we applied encapsulation into poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles by employing formulation methods using the sustainable solvents Cyrene™ or 400 g/mol poly(ethylene glycol) as suitable technology for efficient drug delivery of 6BIGOE. RESULTS: For all preparation techniques the physicochemical characterization of 6BIGOE-loaded nanoparticles revealed comparable crystallinity, sizes of about 230 nm with low polydispersity, negative zeta potentials around - 15 to - 25 mV, and biphasic release profiles over up to 24 h. Nanoparticles with improved cellular uptake and the ability to mask cytotoxic effects of 6BIGOE were obtained as shown in human monocytes over 48 h as well as in a shell-less hen's egg model. Intriguingly, encapsulation into these nanoparticles fully retains the anti-inflammatory properties of 6BIGOE, that is, favorable modulation of the release of inflammation-relevant cytokines and lipid mediators from human monocytes. CONCLUSIONS: Our formulation method of PLGA-based nanoparticles by applying sustainable, non-toxic solvents is a feasible nanotechnology that circumvents the poor bioavailability and biocompatibility of the cargo 6BIGOE. This technology yields favorable drug delivery systems for efficient interference with inflammatory processes, with improved pharmacotherapeutic potential.