In-column dilution: Improving volume loadability in reverse phase gradient chromatography through the use of a silica pre-column.

Volume overload is a critical limitation in Reversed Phase (RP)-HPLC purification of pharmaceutical compounds. Limited solubility of these materials in most injection solvents leads to large injection volumes in order to maximize throughput. However, peak distortion due to volume overload limits injection amounts, and results in suboptimal use of chromatographic instruments. Volume loading for RP gradient separations was significantly increased by inserting a silica column ahead of the RP separation column. The sole purpose of this column is to dilute the plug of strong injection solvent so that the actual sample constituents are retained when the diluted injection plug arrives at the RP column. This is similar to the concept of a "retention gap" in GC, yet this has never been applied to liquid chromatographic separations. Injection volumes were increased by almost a factor of 3 when using appropriately sized silica columns. A discussion of critical parameters that determine the effectiveness of this approach is provided. The concept is easily applied and does not require any system modifications. It is therefore well suited for open access applications where more instrument intensive approaches, such as "At-Column Dilution", would be less desirable. We will also show that the generic concept which we have titled "In-Column Dilution" can easily be applied to increase the detection sensitivity for analytical application as well by allowing injection of larger sample volumes without peak deterioration for purifications.

Crystal structures of human glycine receptor α3 bound to a novel class of analgesic potentiators.

Current therapies to treat persistent pain and neuropathic pain are limited by poor efficacy, side effects and risk of addiction. Here, we present a novel class of potent selective, central nervous system (CNS)-penetrant potentiators of glycine receptors (GlyRs), ligand-gated ion channels expressed in the CNS. AM-1488 increased the response to exogenous glycine in mouse spinal cord and significantly reversed mechanical allodynia induced by nerve injury in a mouse model of neuropathic pain. We obtained an X-ray crystal structure of human homopentameric GlyRα3 in complex with AM-3607, a potentiator of the same class with increased potency, and the agonist glycine, at 2.6-Å resolution. AM-3607 binds a novel allosteric site between subunits, which is adjacent to the orthosteric site where glycine binds. Our results provide new insights into the potentiation of cysteine-loop receptors by positive allosteric modulators and hold promise in structure-based design of GlyR modulators for the treatment of neuropathic pain.

The Discovery and Hit-to-Lead Optimization of Tricyclic Sulfonamides as Potent and Efficacious Potentiators of Glycine Receptors.

Current pain therapeutics suffer from undesirable psychotropic and sedative side effects, as well as abuse potential. Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels expressed in nerves of the spinal dorsal horn, where their activation is believed to reduce transmission of painful stimuli. Herein, we describe the identification and hit-to-lead optimization of a novel class of tricyclic sulfonamides as allosteric GlyR potentiators. Initial optimization of high-throughput screening (HTS) hit 1 led to the identification of 3, which demonstrated ex vivo potentiation of glycine-activated current in mouse dorsal horn neurons from spinal cord slices. Further improvement of potency and pharmacokinetics produced in vivo proof-of-concept tool molecule 20 (AM-1488), which reversed tactile allodynia in a mouse spared-nerve injury (SNI) model. Additional structural optimization provided highly potent potentiator 32 (AM-3607), which was cocrystallized with human GlyRα3cryst to afford the first described potentiator-bound X-ray cocrystal structure within this class of ligand-gated ion channels (LGICs).