Discovery of Selective Tertiary Amide Inhibitors of Cyclin-Dependent Kinase 2 (CDK2).

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and are frequently altered in cancer cells, thereby leading to uncontrolled proliferation. In this context, CDK2 has emerged as an appealing target for anticancer drug development. Herein, we describe the discovery of a series of selective small molecule inhibitors of CDK2 beginning with historical compounds from our ERK2 program (e.g., compound 6). Structure-based drug design led to the potent and selective tool compound 32, where excellent selectivity against ERK2 and CDK4 was achieved by filling the lipophilic DFG-1 pocket and targeting interactions with CDK2-specific lower hinge binding residues, respectively. Compound 32 demonstrated 112% tumor growth inhibition in mice bearing OVCAR3 tumors with 50 mg/kg bis in die (BID) oral dosing.

Medicinal chemistry strategies to extend duration of action of inhaled drugs for intracellular targets.

Prolonging duration of action of inhaled drugs is a challenging endeavor and guidance for medicinal chemistry teams is very limited, particularly if the site of action is intracellular. Herein, we identified recent literature reports of newly designed inhaled compounds with intracellular targets and summarized learnings from different approaches and strategies undertaken by medicinal chemistry teams. We highlight key properties that have shown to lead to longer duration of action and provide guidance on the best strategy to follow while designing a new inhaled drug with an intracellular target.

Reversible inhibition of efflux transporters by hydrogel microdevices.

Oral drug delivery is a preferred administration route due to its low cost, high patient compliance and fewer adverse events compared to intravenous administration. However, many pharmaceuticals suffer from poor solubility and low oral bioavailability. One major factor that contributes to low bioavailability are efflux transporters which prevent drug absorption through intestinal epithelial cells. P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are two important efflux transporters in the intestine functioning to prevent toxic materials from entering systemic circulation. However, due to its broad substrate specificity, P-gp limits the absorption of many therapeutics, including chemotherapeutics and antibacterial agents. Methods to inhibit P-gp with competitive inhibitors have not been clinically successful. Here, we show that micron scale devices (microdevices) made from a commonly used biomaterial, polyethylene glycol (PEG), inhibit P-gp through a biosimilar mucus in Caco-2 cells and that transporter function is restored when the microdevices are removed. Microdevices were shown to inhibit P-gp mediated transport of calcein AM, doxorubicin, and rhodamine 123 (R123) and BCRP mediated transport of BODIPY-FL-prazosin. When in contact with Caco-2 cells, microdevices decrease the cell surface amount of P-gp without affecting the passive transport. Moreover, there was an increase in mucosal to serosal transport of R123 with microdevices in an ex-vivo mouse model and increased absorption in vivo. This biomaterial-based approach to inhibit efflux transporters can be applied to a range of drug delivery systems and allows for a nonpharmacologic method to increase intestinal drug absorption while limiting toxic effects.

Human intestinal spheroids cultured using Sacrificial Micromolding as a model system for studying drug transport.

In vitro models of the small intestine are crucial tools for the prediction of drug absorption. The Caco-2 monolayer transwell model has been widely employed to assess drug absorption across the intestine. However, it is now well-established that 3D in vitro models capture tissue-specific architecture and interactions with the extracellular matrix and therefore better recapitulate the complex in vivo environment. However, these models need to be characterized for barrier properties and changes in gene expression and transporter function. Here, we report that geometrically controlled self-assembling multicellular intestinal Caco-2 spheroids cultured using Sacrificial Micromolding display reproducible intestinal features and functions that are more representative of the in vivo small intestine than the widely used 2D transwell model. We show that Caco-2 cell maturation and differentiation into the intestinal epithelial phenotype occur faster in spheroids and that they are viable for a longer period of time. Finally, we were able to invert the polarity of the spheroids by culturing them around Matrigel beads allowing superficial access to the apical membrane and making the model more physiological. This robust and reproducible in vitro intestinal model could serve as a valuable system to expedite drug screening as well as to study intestinal transporter function.

Recent advances in intraocular sustained-release drug delivery devices.

Topical eye-drop administration and intravitreal injections are the current standard for ocular drug delivery. However, patient adherence to the drug regimen and insufficient administration frequency are well-documented challenges to this field. In this review, we describe recent advances in intraocular implants designed to deliver therapeutics for months to years, to obviate the issues of patient adherence. We highlight recent advances in monolithic ocular implants in the literature, the commercialization pipeline, and approved for the market. We also describe design considerations based on material selection, active pharmaceutical ingredient, and implantation site.

Co-Delivery of Timolol and Brimonidine with a Polymer Thin-Film Intraocular Device.

PURPOSE: We developed a polycaprolactone (PCL) co-delivery implant that achieves zero-order release of 2 ocular hypotensive agents, timolol maleate and brimonidine tartrate. We also demonstrate intraocular pressure (IOP)-lowering effects of the implant for 3 months in vivo. METHODS: Two PCL thin-film compartments were attached to form a V-shaped co-delivery device using film thicknesses of ∼40 and 20 µm for timolol and brimonidine compartments, respectively. In vitro release kinetics were measured in pH- and temperature-controlled fluid chambers. Empty or drug-loaded devices were implanted intracamerally in normotensive rabbits for up to 13 weeks with weekly measurements of IOP. For ocular concentrations, rabbits were euthanized at 4, 8, or 13 weeks, aqueous fluid was collected, and ocular tissues were dissected. Drug concentrations were measured by liquid chromatography-tandem mass spectrometry. RESULTS: In vitro studies show zero-order release kinetics for both timolol (1.75 µg/day) and brimonidine (0.48 µg/day) for up to 60 days. In rabbit eyes, the device achieved an average aqueous fluid concentration of 98.1 ± 68.3 ng/mL for timolol and 5.5 ± 3.6 ng/mL for brimonidine. Over 13 weeks, the drug-loaded co-delivery device resulted in a statistically significant cumulative reduction in IOP compared to untreated eyes (P < 0.05) and empty-device eyes (P < 0.05). CONCLUSIONS: The co-delivery device demonstrated a zero-order release profile in vitro for 2 hypotensive agents over 60 days. In vivo, the device led to significant cumulative IOP reduction of 3.4 ± 1.6 mmHg over 13 weeks. Acceptable ocular tolerance was seen, and systemic drug levels were unmeasurable.

Probing the luminal microenvironment of reconstituted epithelial microtissues.

Polymeric microparticles can serve as carriers or sensors to instruct or characterize tissue biology. However, incorporating microparticles into tissues for in vitro assays remains a challenge. We exploit three-dimensional cell-patterning technologies and directed epithelial self-organization to deliver microparticles to the lumen of reconstituted human intestinal microtissues. We also develop a novel pH-sensitive microsensor that can measure the luminal pH of reconstituted epithelial microtissues. These studies offer a novel approach for investigating luminal microenvironments and drug-delivery across epithelial barriers.