E7386, a Selective Inhibitor of the Interaction between ß-Catenin and CBP, Exerts Antitumor Activity in Tumor Models with Activated Canonical Wnt Signaling.

The Wnt/ß-catenin signaling pathway plays crucial roles in embryonic development and the development of multiple types of cancer, and its aberrant activation provides cancer cells with escape mechanisms from immune checkpoint inhibitors. E7386, an orally active selective inhibitor of the interaction between ß-catenin and CREB binding protein, which is part of the Wnt/ß-catenin signaling pathway, disrupts the Wnt/ß-catenin signaling pathway in HEK293 and adenomatous polyposis coli (APC)-mutated human gastric cancer ECC10 cells. It also inhibited tumor growth in an ECC10 xenograft model and suppressed polyp formation in the intestinal tract of ApcMin /+ mice, in which mutation of Apc activates the Wnt/ß-catenin signaling pathway. E7386 demonstrated antitumor activity against mouse mammary tumors developed in mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice. Gene expression profiling using RNA sequencing data of MMTV-Wnt1 tumor tissue from mice treated with E7386 showed that E7386 downregulated genes in the hypoxia signaling pathway and immune responses related to the CCL2, and IHC analysis showed that E7386 induced infiltration of CD8+ cells into tumor tissues. Furthermore, E7386 showed synergistic antitumor activity against MMTV-Wnt1 tumor in combination with anti-PD-1 antibody. In conclusion, E7386 demonstrates clear antitumor activity via modulation of the Wnt/ß-catenin signaling pathway and alteration of the tumor and immune microenvironments, and its antitumor activity can be enhanced in combination with anti-PD-1 antibody. SIGNIFICANCE: These findings demonstrate that the novel anticancer agent, E7386, modulates Wnt/ß-catenin signaling, altering the tumor immune microenvironment and exhibiting synergistic antitumor activity in combination with anti-PD-1 antibody.

Design, synthesis, and structure-activity relationships of a series of novel N-aryl-2-phenylcyclopropanecarboxamide that are potent and orally active orexin receptor antagonists.

Herein we describe the design, synthesis, and structure-activity relationships (SARs) of a novel phenylcyclopropane series represented by 7 and 33 b as antagonists of orexin 1 and orexin 2 receptors. With 4 serving as the initial lead for the development of orexin antagonists, exploration of SAR resulted in improved binding affinity for orexin 1 and orexin 2 receptors. Among the synthesized compounds, 33 b ((-)-N-(5-cyanopyridin-2-yl)-2-[(3,4-dimethoxyphenyl)oxymethyl]-2-phenylcyclopropanecarboxamide) exhibited potent in vitro activity and oral efficacy in animal sleep measurement experiments. The results of our study suggest that compound 33 b may serve as a valuable template for the development of new orexin receptor antagonists.

Crystal structure and dehydration behavior of E6070, a novel IKKß protein kinase inhibitor for the treatment of rheumatoid arthritis.

This study is focused on solving the crystal structure of E6070, a novel IKKß protein kinase inhibitor, and characterizing its solid state. E6070 exists as a stable crystalline trihydrate (hydrate I) that undergoes two dehydration events on heating. Neither crystal form nor water content changed under the relative humidity range from 11% to 93% at 25 °C. Crystal transformation to hydrate II occurred in the first dehydration process beginning at ~70 °C followed by loss of almost all remaining crystal lattice water as heating continues up to 160 °C. The observed weight loss of the first step was significantly higher than two equivalents of water. The X-ray powder diffraction proved that the anhydrate is an amorphous form that reversibly reforms the original trihydrate when exposed to atmospheric moisture. Hydrate II more readily forms hydrate I than from the anhydrate. The activation energy of the second step accompanied with amorphism was approximately 1.5 times greater than that of the first. In addition, a gradual decline of activation energy depending on the extent of dehydration was observed, implying reversible conversion in both steps. Therefore, the trihydrate was considered suitable for further development as its solid state is controllable during the manufacturing processes and the shelf life.

Inhibition of crystal nucleation and growth by water-soluble polymers and its impact on the supersaturation profiles of amorphous drugs.

The impact of water-soluble polymers on drug supersaturation behavior was investigated to elucidate the role of water-soluble polymers in enhancing the supersaturation levels of amorphous pharmaceuticals. Hydroxypropyl methylcellulose (HPMC), polyvinylpyrrolidone (PVP), and Eudragit L-100 (Eudragit) were used as representative polymers, and griseofulvin and danazol were used as model drugs. Supersaturation profiles of amorphous drugs were measured in biorelevant dissolution tests. Crystal growth rate was measured from the decrease in dissolved drug concentration in the presence of seed crystals. Nucleation kinetics was evaluated by measuring the induction time for nucleation. All experiments were performed in the presence and absence of polymers. The degree of supersaturation of the amorphous model drugs increased with an increase in the inhibitory efficiency of polymers against crystal nucleation and growth (HPMC > PVP > Eudragit). In the presence of HPMC, the addition of seed crystals diminished the supersaturation ratio dramatically for griseofulvin and moderately for danazol. The results demonstrated that the polymers contributed to drug supersaturation by inhibiting both nucleation and growth. The effect of the polymers was drug dependent. The detailed characterization of polymers would allow selection of appropriate crystallization inhibitors and a planned quality control strategy for the development of supersaturable formulations.

Investigation for the amorphous state of ER-34122, a dual 5-lipoxygenase/cyclooxygenase inhibitor with poor aqueous solubility, in HPMC solid dispersion prepared by the solvent evaporation method.

ER-34122, a poorly water-soluble dual 5-lipoxygenase/cyclooxygenase inhibitor, exists as a crystalline form. According to an Oak Ridge thermal ellipsoid plot drawing, carbonyl oxygen O (5) makes an intermolecular hydrogen bond with the hydrogen bonded to N (3) in the crystal structure. The FTIR and the solid-state ¹³C NMR spectra suggest that the network is spread out in the amorphous state and the hydrogen bonding gets weaker than that in the crystalline phase, because the carbonyl signals significantly shift in both spectra. When amorphous ER-34122 was heated, crystallization occurred at around 140°C. Similar crystallization happened in the solid dispersion; however, the degree of crystallization was much lower than that observed in the pure amorphous material. Also, the DSC thermogram of the solid dispersion did not show any exothermic peaks implying crystallization. The heat of fusion (ΔHf) determined in the pure amorphous material was nearly equal to that for the crystalline form, whereas the ΔHf value obtained in the solid dispersion was less than a third of them. These data prove that crystallization of the amorphous form is dramatically restrained in the solid dispersion system. The carbonyl wavenumber shifts in the FTIR spectra indicate that the average hydrogen bond in the solid dispersion is lower than that in the pure amorphous material. Therefore, HPMC will suppress formation of the intermolecular network observed in ER-34122 crystal and preserve the amorphous state, which is thermodynamically less stable, in the solid dispersed system.

Evaluation of drug supersaturation by thermodynamic and kinetic approaches for the prediction of oral absorbability in amorphous pharmaceuticals.

Supersaturation behavior of model drugs, danazol, griseofulvin, itraconazole, vemurafenib, and ER-34122, was analyzed by both thermodynamic and kinetic approaches to better understand the absorption characteristics of amorphous pharmaceuticals. For each amorphous drug, the extent of supersaturation during in vitro dissolution was proved to be similar to that in vivo, which was estimated from relative bioavailability data. The theoretical limit of supersaturation was thermodynamically calculated from several thermal properties and water sorption isotherms of amorphous solids. in vitro and in vivo supersaturation of amorphous vemurafenib was thermodynamically controlled and was in good agreement with the theoretical limit. On the contrary, the supersaturation ratio of the other four drugs was highly overestimated by the thermodynamic calculation. However, it was satisfactorily explained by considering supersaturation stability, which indicated how long supersaturation can be maintained without crystal nucleation. Supersaturation stability was evaluated by measuring the induction time for crystal nucleation kinetically. Concomitant use of thermodynamic and kinetic approaches is, therefore, invaluable in evaluating supersaturation behavior of amorphous materials and assessing development potential of poorly water-soluble drugs.

Quantitative crystallinity determination for E1010, a novel carbapenem antibiotic, using differential scanning calorimetry.

OBJECTIVES: The objective of this study was to develop a quantitative crystallinity analysis method for the bulk drug of E1010 ((+)-(4R,5S,6S)-6-[(R)-1-hydroxyethyl]-3-[(2S,4S)-2-[(R)-1-hydroxy-1-[(R)-pyrrolidin-3 -yl]methyl]pyrrolidin-4-yl]thio-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid monohydrochloride), a novel carbapenem antibiotic. METHODS: X-ray analyses, thermal analyses and hygroscopicity measurements were used to elucidate the crystal structure and the solid state properties. To develop a quantitative method for the crystallinity of E1010 bulk drug, the relationship between enthalpy change obtained by differential scanning calorimetry (DSC) and crystalline form ratio was investigated. KEY FINDINGS: E1010 bulk drug was found to exist in a crystalline trihydrate formed in two layers, i.e. a layer of E1010 free form, and a layer consisting of chloride ions and water molecules. The thermal analysis showed an endothermic peak derived from dehydration with the loss of crystal lattices at around 100°C as an onset. The enthalpy change value for the endothermic peak correlated well with crystalline content in binary physical mixtures of the crystalline trihydrate and the amorphous form. In addition, for nine lots of the bulk drug, a positive correlation between the enthalpy change and chemical stability in the solid state was observed. CONCLUSIONS: This quantitative analysis of crystallinity using DSC could be applicable for the quality control of the bulk drug to detect variability among manufacturing batches and to estimate the chemical stability of partially amorphous samples.

Supersaturation-nucleation behavior of poorly soluble drugs and its impact on the oral absorption of drugs in thermodynamically high-energy forms.

In order to better understand the oral absorption behavior of poorly water-soluble drugs, their supersaturation-nucleation behavior was characterized in fasted state simulated intestinal fluid. The induction time (t(ind)) for nucleation was measured for four model drugs: itraconazole, erlotinib, troglitazone, and PLX4032. Supersaturated solutions were prepared by solvent shift method, and nucleation initiation was monitored by ultraviolet detection. The relationship between t(ind) and degree of supersaturation was analyzed in terms of classical nucleation theory. The defined supersaturation stability proved to be compound specific. Clinical data on oral absorption were investigated for drugs in thermodynamically high-energy forms such as amorphous forms and salts and was compared with in vitro supersaturation-nucleation characteristics. Solubility-limited maximum absorbable dose was proportionate to intestinal effective drug concentrations, which are related to supersaturation stability and thermodynamic solubility. Supersaturation stability was shown to be an important factor in determining the effect of high-energy forms. The characterization of supersaturation-nucleation behavior by the presented method is, therefore, valuable for assessing the potential absorbability of poorly water-soluble drugs.

Solvent shift method for anti-precipitant screening of poorly soluble drugs using biorelevant medium and dimethyl sulfoxide.

96-well plate based anti-precipitant screening using bio-relevant medium FaSSIF (fasted-state simulated small intestinal fluid) is a useful technique for discovering anti-precipitants that maintain supersaturation of poorly soluble drugs. In a previous report, two disadvantages of the solvent evaporation method (solvent casting method) were mentioned: precipitation during the evaporation process and the use of volatile solvents to dissolve compounds. In this report, we propose a solvent shift method using DMSO (dimethyl sulfoxide). Initially, the drug substance was dissolved in DMSO at a high concentration and diluted with FaSSIF that contained anti-precipitants. To evaluate the validity of the method, itraconazole (ITZ) was used as the poorly soluble model drug. The solvent shift method resolved the disadvantages of the evaporation method, and AQOAT (HPMC-AS) was found as the most appropriate anti-precipitant for ITZ in a facile and expeditious manner when compared with the solvent evaporation method. In the large scale JP paddle method, AQOAT-based solid dispersion maintained a higher concentration than Tc-5Ew (HPMC)-based formulation; this result corresponded well with the small scale of the solvent shift method.

Frozen water phase method for logD measurement using a 96-well plate.

In this study, a frozen water phase method for logD measurement using a 96-well plate was developed. In the case of logD measurement of compounds, the problem of octanol contamination often occurs; in lipophilic compounds, the concentration of the octanol phase is much higher than that of the water phase. When the water phase is separated from the octanol phase, a small amount of octanol phase contamination could strongly influence the concentration of the water phase. To avoid this problem, the frozen water phase method was developed. The water phase was frozen in liquid nitrogen and then the unfrozen octanol phase was removed. To remove the portion of the octanol remaining on the frozen water phase, the surface of the frozen water phase was washed with octanol and water/ethanol (50/50, v/v). The validity of the method was confirmed by results of commercially available drugs at the logD range from 0 to 4. Further, it was found that this method had the ability to evaluate the pH-logD profile of compounds in the range from pH 2 to pH 12. As a result, we developed the convenient and accurate method that is effective in preventing contamination with a wide dynamic range.

Reliable on-line sample preparation of basic compounds from plasma using a reversed phase restricted access media in column-switching LC.

We investigated on-line sample preparation of basic compounds from plasma using a methylcellulose-immobilized reversed-phase restricted-access media in column-switching liquid chromatography (LC). Dilution of the plasma sample with phosphate buffered saline prevented or delayed the formation of fibrin clots at 4 degrees C and resulted in reproducible on-line sample preparation over a 30-h period. The use of an ion-pair reagent in the extraction LC enhanced recoveries of hydrophilic basic compounds. The ability of the methods to quantify compounds in plasma were validated and the method was successfully applied to the pharmacokinetic study of a hydrophilic basic compound injected into the bloodstream of rats.

Effective on-line purification for cationic compounds in rat bile using a column-switching LC technique.

An on-line purification method for cationic compounds and their metabolites in rat bile was investigated using a column-switching technique. 8-Hydroxyquinoline and its glucuronide were used as test compounds. Bile samples were injected directly into the system and successful on-line extraction with high purification efficiency for analytes was achieved using two-dimensional extraction LC; that is, reversed-phase chromatography followed by cation-exchange chromatography. After removal of the endogenous component by extraction LC, chromatographic separation of the target analyte was performed on an analytical ODS column, followed by identification using UV detection. The quantitative ability of the method was evaluated on the basis of injection repeatability, linearity and accuracy. This novel method was also applied to LC/MS analysis in order to characterise the pharmacokinetics of propranolol in rats, and the metabolites were successfully identified.

Solid state characterization of E2101, a novel antispastic drug.

E2101, a novel antispastic drug, was found to exist in at least two polymorphs that were confirmed by X-ray powder diffraction (XRD). These two species are designated forms I and II. The physicochemical and thermodynamic properties of these polymorphs were characterized by variable temperature XRD, thermal analysis, hygroscopicity measurements, and dissolution studies. The transition temperature was also estimated from the solubilities determined at various temperatures. The E2101 polymorphs were anhydrous and adsorbed little moisture under high humidity conditions. The melting onsets and heats of fusion for form I were 148.1 +/- 0.2 degrees C and 38.2 +/- 1.0 kJ/mol, respectively, and for form II were 139.8 +/- 0.4 degrees C and 35.2 +/- 0.5 kJ/mol, respectively. The intrinsic dissolution rate of form II in JP 2 medium was 1.5-fold faster than that of form I, corresponding to the rank order of the aqueous solubility and the enthalpy of fusion. Accordingly, form I was thought to be thermodynamically more stable than form II and thus suitable for further development. According to the thermal analysis and variable temperature XRD results, the recrystallization of form I occurred at approximately 145 degrees C after form II melted, however, no crystal transition behavior was observed below the lower melting point. The DSC thermograms at various heating rates and van't Hoff plots from the solubility studies indicated that the polymorphic pair would be monotropic.

Improvement of dissolution and oral absorption of ER-34122, a poorly water-soluble dual 5-lipoxygenase/cyclooxygenase inhibitor with anti-inflammatory activity by preparing solid dispersion.

Several formulation approaches were attempted to improve the dissolution and the oral absorption of ER-34122, which is a novel dual 5-lipoxygenase/cyclooxygenase inhibitor with potent anti-inflammatory activity. The solid dispersion of ER-34122 with hydroxypropylmethylcellulose (TC-5RW), which is an inert solid carrier, resulted in a significant improvement in the dissolution rate of ER-34122. The solid dispersion was prepared by a solvent evaporation method using ethanol and water. The solid-state characteristics of the solid dispersion, the corresponding physical mixture, and ER-34122 alone were investigated by X-ray powder diffraction, Fourier transform infrared spectroscopy (FTIR), and an automated controlled-atmosphere microbalance. The X-ray powder diffraction patterns suggest that the solid dispersion exists in a totally amorphous state and the others exist in a crystalline state. The FTIR spectra results suggest that ER-34122 can interact with TC-5RW through intermolecular hydrogen bonding in the solid dispersion. This interaction may cause a stabilization of ER-34122 in the higher-energy, faster-dissolving amorphous state. The dissolution rate of ER-34122 from the solid dispersion was significantly greater than that from the physical mixture or the pure drug. Furthermore, when orally administrated to beagle dogs, ER-34122 showed about a 100-fold increase in both maximum concentration (C(max)) and area under the curve of concentration versus time (AUC) compared with the pure drug. Consequently, it was determined that the solid dispersion technique with TC-5RW provides a promising way to increase the dissolution rate and the oral absorption of poorly water-soluble drugs such as ER-34122.