Aqueous Two-Phase Systems (ATPSs) have been extensively studied for their ability to simultaneously separate and purify active pharmaceutical ingredients (APIs) and key intermediates with high yields and high purity. Depending on the ATPS composition, it can be adapted for the separation and purification of cells, nucleic acids, proteins, antibodies, and small molecules. This method has been shown to be scalable, allowing it to be used in the milliliter scale for early drug development to thousands of liters in manufacture for commercial supply. The benefits of ATPS in pharmaceutical separations is increasingly being recognized and investigated by larger pharmaceutical companies. ATPSs use identical instrumentation and similar methodology, therefore a change from traditional methods has a theoretical low barrier of adoption. The cost of typical components used to form an ATPS at large scale, particularly that of polymer-polymer systems, is the primary challenge to widespread use across industry. However, there are a few polymer-salt examples where the increase in yield at commercial scale justifies the cost of using ATPSs for macromolecule purification. More recently, Ionic Liquids (ILs) have been used for ATPS separations that is more sustainable as a solvent, and more economical than polymers often used in ATPSs for small molecule applications. Such IL-ATPSs still retain much of the attractive characteristics such as customizable chemical and physical properties, stability, safety, and most importantly, can provide higher yield separations of organic compounds, and efficient solvent recycling to lower financial and environmental costs of large scale manufacturing.

Liquid forms of pharmaceuticals (ionic liquids and deep eutectic solvents) offer a number of potential advantages over solid-state drugs; a key question is the role of intermolecular hydrogen bonding interactions in enabling membrane transport. Characterization is challenging since high sample viscosities, typical of liquid pharmaceutical formulations, hamper the use of conventional solution NMR at ambient temperature. Here, we report the application of magic-angle spinning (MAS) NMR spectroscopy to the deep eutectic pharmaceutical, lidocaine ibuprofen. Using variable temperature MAS NMR, the neat system, at a fixed molar ratio, can be studied over a wide range of temperatures, characterized by changing mobility, using a single experimental setup. Specific intermolecular hydrogen bonding interactions are identified by two-dimensional 1 H− 1 H NOESY and ROESY MAS NMR experiments. Hydrogen-bonding dynamics are quantitatively determined by following the chemical exchange process between the labile protons by means of line-width analysis of variable temperature 1 H MAS NMR spectra.

The structure and molecular order in the thermotropic ionic liquid crystal (ILC), [choline][geranate(H)octanoate], an analogue of Choline And GEranate (CAGE), which has potential for use as a broad-spectrum antimicrobial and transdermal and oral delivery agent, were investigated by magic-angle spinning (MAS) nuclear magnetic resonance (NMR), polarizing optical microscopy, small-angle X-ray scattering (SAXS), and mass spectrometry. Mass spectrometry and the 1 H NMR chemical shift reveal that CAGE-oct is a dynamic system, with metathesis (the exchange of interacting ions) and hydrogen exchange occurring between hydrogen-bonded/ionic complexes such as [(choline)(geranate)(H)(octanoate)], [(choline)(octanoate) 2 (H)], and [(choline)-(geranate) 2 (H)]. These clusters, which are shown by mass spectrometry to be significantly more stable than expected for typical electrostatic ion clusters, involve hydrogen bonding between the carboxylic acid, carboxylate, and hydroxyl groups, with rapid hydrogen bond breaking and re-formation observed to average the 1 H chemical shifts. The formation of a partial bilayer liquid crystal (LC) phase was identified by SAXS and polarizing optical microscopy at temperatures below ∼293 K. The occurrence of this transition close to room temperature could be utilized as a potential temperature-induced "switch" of the anisotropic properties for particular applications. The presence of an isotropic component of approximately 23% was observed to coexist with the LC phase, as detected by polarizing optical microscopy and quantified by both 1 H− 13 C dipolar-chemical shift correlation (DIPSHIFT) and 1 H double-quantum (DQ) MAS NMR experiments. At temperatures above the LC-to-isotropic transition, intermediate-range order (clustering of polar and nonpolar domains), a feature of many ILs, persists. Site-specific order parameters for the LC phase of CAGE-oct were obtained from the MAS NMR measurement of the partially averaged 13 C− 1 H dipolar couplings (D CH ) by cross-polarization (CP) build-up curves and DIPSHIFT experiments, and 1 H− 1 H dipolar couplings (D HH ) by double-quantum (DQ) build-up curves. The corresponding order parameters, S CH and S HH , are in the range 0−0.2 and are lower compared to those for smectic (i.e., layered) phases of conventional nonionic liquid crystals, resembling those of lamellar phases formed by lyotropic surfactant−solvent systems.

The properties of aqueous solutions of the CAGE deep eutectic solvent are predicted with the SAFT-γ Mie approach.

Appropriate control of cell death is a fundamental biological process which is frequently dysregulated during tumor development and therapeutic resistance. Apoptosis is a form of regulated cell death initiated by either the extracellular environment (extrinsic) or following internal cellular damage (intrinsic). It is controlled by the BCL-2 family which includes anti-apoptotic regulators like BCL-2, BCL-XL and MCL-1 that bind and sequester various pro-apoptotic BH3-only proteins (BIM, BAD, BID, NOXA, PUMA, etc.), and the pro-apoptotic effectors (BAK, BAX, etc.) responsible for mitochondrial pore formation and MOMP (mitochondrial outer membrane permeabilization). MOMP results in intermembrane space protein release, leading to caspase activation in an irreversible path to programmed cell death. Of the anti-apoptotic regulators, MCL-1 is one of the most frequently and highly amplified genes in human cancers such as myeloid leukemia making it a compelling therapeutic target. Since BCL-2 proteins interact through protein-protein interactions, they have long been elusive targets. The success of selective BCL-2 protein inhibitor Venetoclax in the treatment of various hematological cancers, however spurred interest in MCL-1 as an oncology target. Using structure-based drug design, major breakthroughs were made in the development of MCL-1 inhibitors, with several candidates entering clinical studies in the past five years. JNJ-4355, a highly potent 1,4-indolyl macrocycle (MCL-1 Ki = 18 pM, Cell (MOLP8) AC50 = 8.7 nM) was optimized to address shortcomings from first generation MCL-1 inhibitors: it has improved physicochemical properties (CHI LogD7.4 = 2.35, EPSA = 151 Å2), resulting in greatly improved equilibrium solubility (3.14 mM in buffer pH 7) and reduced protein binding (99.93%). JNJ-4355 showed promising in vitro potency data in cancer cell lines and AML patient-derived samples (cell killing AC50 0.29-75 nM in 25/27 evaluable samples). In vivo MCL-1:BAK complex disruption was confirmed in a mouse MOLM13 (AML) xenograft. Efficacy was demonstrated in a mouse MOLP8 (multiple myeloma) xenograft resulting in complete tumor regression after a single IV dose of JNJ-4355. Citation Format: Frederik J. Rombouts, Lento William, Ingrid Velter, Ann Vos, Aldo Peschiulli, Reuillon Tristan, Maria Dominguez Blanco, Matthieu Jouffroy, Lisa McQueen, Helena Steyvers, Mariette Bekkers, Cristina Altrocchi, Beth Pietrak, Seong Joo Koo, Lawrence Szewczuk, David Walker, Kathryn Packman, Ruud Bueters, Petra Vinken, Amy Johnson, Ricardo Attar, Ulrike Philippar. In pursuit of MCL-1 inhibitors with improved therapeutic window for the treatment of hematological malignancies: Discovery of JNJ-4355 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2133.