CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3′-end processing and associated splicing factors.

This review project, including the publication of this article, was funded by Takeda Pharmaceutical Company Limited.

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The present study evaluated the specific intermolecular interactions between carbamazepine (CBZ) and substituents of hypromellose acetate succinate (HPMC-AS), as well as the mechanism of inhibition of recrystallization of solid dispersions (SDs) using Fourier-transform infrared (FTIR) and solid-state nuclear magnetic resonance (NMR) spectroscopy. CBZ and HPMC derivatives, including HPMC, hypromellose acetate (HPMC-A), and hypromellose succinate (HPMC-S), were spray-dried to prepare CBZ/polymer spray-dried samples (SPDs). CBZ/HPMC SPD and CBZ/HPMC-A SPD recrystallized within 10 days at 60 °C and 0% relative humidity, whereas CBZ/HPMC-S SPD maintained its amorphous state for a longer period. FTIR and solid-state NMR measurements using 13 C cross polarization (CP), 1 H single-pulse, and 1 H− 15 N CP-based heteronuclear single quantum correlation filter experiment with very fast magic angle spinning (MAS) at 70 kHz identified molecular interactions in CBZ/polymer SPDs. Although the HPMC backbone and substituents did not interact notably with CBZ and disrupt CBZ−CBZ intermolecular interactions (formed in the amorphous CBZ), acetate and succinate substituents on HPMC-A and HPMC-S disrupted CBZ−CBZ intermolecular interactions through formation of CBZ/polymer interactions. The acetate substituent formed a hydrogen bond with the NH 2 group of CBZ, whereas the succinate substituent formed molecular interactions with both the CO and NH 2 groups of CBZ. Formation of relatively strong molecular interactions between CBZ and the succinate substituent followed by disruption of CBZ−CBZ intermolecular interactions effectively stabilized the amorphous state of CBZ in CBZ/HPMC-S SPD. The correlation between CBZ−polymer interactions and ability of polymers to effectively inhibit CBZ recrystallization is reflected in various commercial HPMC-AS. For example, HPMC-AS LF grade, containing higher amounts of the succinate group, was found to effectively inhibit the recrystallization of CBZ through strong molecular interactions as compared with the HPMC-AS HF grade. The present study demonstrated that a detailed investigation of molecular interactions between the drug and the polymer using FTIR and solid-state NMR continued...

Recently, choline and geranic acid (CAGE), an ionic liquid (IL), has been recognized to be a superior biocompatible material for oral and transdermal drug delivery systems (DDS). When CAGE is administered, CAGE would be exposed to various types of physiological fluids, such as intestinal and intradermal fluids. However, the effect of physiological fluids on the structure of CAGE remains unclear. In the present study, molecular structures of CAGE with different ratios of water were investigated using small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR). The SAXS pattern of CAGE showed an IL-specific broad peak derived from nanoscale aggregation until 17 vol % water. Meanwhile, narrow peaks were observed in samples with 25−50 vol % water, showing a transition to the lamellar phase. With more than 67 vol % water, CAGE was found to exist as micelles in water. The 1 H NMR spectra indicated that protons of H 2 O, OH in choline (CH), and COOH in geranic acid (GA) were observed as only one peak up to 17 vol % water. This peak shifted to a high magnetic field, and the integral values increased with the water content, speculating that water is localized close to the COOH and OH groups to allow proton exchange. The 13 C NMR spectra showed that peaks related to the carboxyl group shifted with adding water. Moreover, only GA peaks were observed in the lamellar phase through 13 C cross-polarization magic-angle spinning NMR, suggesting that the main rigid component of the lamellar phase was GA. Taken together, this study suggested that CAGE still maintained its IL structure up to 17 vol % water, then transitioned to the lamellar phase with 25−50 vol % water, and finally changed to the micellar phase with more than 67 vol % water. This information would be useful in the formulation and development of DDS using CAGE.

To judge the developability and analyze functional mechanism of co-amorphouses, we investigated the physicochemical properties of co-amorphouses and compare the properties with the co-crystals having the same drug and counters. Co-amorphous compounds are a novel approach to improve the physicochemical properties of drugs. A co-amorphous is in an amorphous solid state allowing non-ionic interactions between drug molecules and counter molecules. The co-amorphous compounds composed of itraconazole (ITZ) with the organic carboxyl acid, fumaric acid (FA) or L-tartaric acid (TA), were prepared by mechanical grinding. Potential interactions within ITZ-FA co-amorphous were assessed by Raman spectroscopy. ITZ-FA coamorphous was not crystallized as the co-crystal or as a single ITZ crystal, suggesting that the amorphous state, like the amorphous solid dispersion, was physically stable and that ITZ-FA co-amorphous was also chemically stable. In contrast, no clear interactions were observed within ITZ-TA co-amorphous, and the coamorphous was physically stable but chemically unstable. The solubility of the co-amorphous state was much higher than those of ITZ crystal and the co-crystals and was almost identical to that of amorphous ITZ. A co-amorphous compound like ITZ-FA co-amorphous might be feasible to implement in the development of solid drug products and bring some merits compared to the co-crystals, and the function is governed by the interaction between a drug and a counter. The co-amorphous approach may be an effective strategy for drug development and can contribute to the production of novel drugs with improved functions.Key words co-amorphous; co-crystal; stability; solubilization; interaction; itraconazole Solubility improvement is one of the most important challenges for drug development of poorly soluble drugs. Amorphization of drugs is a major effective strategy to prepare solubilized formulations, because the crystal packing effect on a drug substance significantly decreases its solubility. 1-3)An amorphous compound is in a higher energy state and has a higher mobility than a crystalline compound, improving its solubility and dissolution properties. However, due to the higher energy state, the physical and chemical stability of amorphous compounds are relatively lower than those of a crystal. 4,5) In many cases, an amorphous drug is formulated by solid dispersion with hydrophilic polymers to improve the amorphous drug stability. [6][7][8] The complex formed with polymers can prevent the drug's crystallization and maintain the physical stability of the amorphous state. Generally, weak interactions cannot sufficiently stabilize amorphous drugs. 9)The prediction and design leading to the interactions between the drug and the polymer are still difficult to accomplish. Therefore, solid dispersions combined with several kinds and amounts of polymers need to be tested.As another approach to enhance a drug's solubility, pharmaceutical co-crystals have been investigated and adapted to the drug development processes in...

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Low-frequency Raman spectroscopy has advantage to discriminate and quantify polymorphs where common 2D superstructures are piled up with different mode.