ME3183, a novel phosphodiesterase-4 inhibitor, exhibits potent anti-inflammatory effects and is well tolerated in a non-clinical study.

Phosphodiesterase 4 (PDE4) inhibitors are expected to exhibit efficacy against inflammatory diseases due to their broad pharmacological activity. The launched PDE4 inhibitors apremilast, crisaborole, and roflumilast have not exhibited sufficient inhibitory potential due to poor margins of effectiveness and tolerability. In this report, we describe the non-clinical efficacy, brain translocation, and vomit-inducing effects of ME3183 compared with apremilast. ME3183 showed extensive cytokine suppression in vitro studies using human peripheral blood mononuclear cells and T cells. ME3183 also significantly suppressed skin inflammation in a chronic oxazolone-induced dermatitis model and showed antipruritic effects in a substance P-induced mouse pruritus model. In these in vitro and in vivo studies, ME3183 also significantly suppressed cytokines, and focusing on tumor necrosis factor-α as a psoriasis-related cytokine and interleukin-4 as an atopic dermatitis-related cytokine, ME3183 potently inhibited both cytokines. ME3183 showed in vivo efficacy at lower doses than apremilast. The brain distribution of ME3183 was sufficiently low in mice and rats. The effective dose of ME3183 for emesis was similar to that of apremilast in ferrets. Given its high-potency inhibitory effects, ME3183 would have a wide margin of efficacy and tolerability. These wide margins demonstrate the effectiveness of ME3183 in treating many inflammatory diseases, such as psoriasis and atopic dermatitis. An on-going phase 2 trial is expected to further demonstrate the efficacy and safety of ME3183.

Exploring how plasma- and muscle-related parameters affect trout hemolysis as a route to prevent hemoglobin-mediated lipid oxidation of fish muscle.

Hemoglobin (Hb) is a powerful promoter of lipid oxidation, particularly in muscle of small pelagic fish species and fish by-products, both having high Hb-levels and highly unsaturated lipids. As Hb is located within the red blood cells (RBCs) it is here hypothesized that the perishable polyunsaturated fatty acids (PUFAs) can be protected from oxidation by limiting hemolysis during early fish processing. Using a model system consisting of washed-resuspended trout (Oncorhynchus mykiss) RBCs (wr-RBCs), the aim of this study was to evaluate how RBC lysis under cold storage was affected by selected parameters linked to blood or muscle: bacterial growth, energy status, pH, RBC membrane lipid oxidation and colloidal osmotic pressure (COP). The results indicated that bacterial growth had a modest effect on hemolysis while pH-values typical for post mortem fish muscle (6.4-6.8), and absence of glucose or albumin stimulated hemolysis. The rapid hemolysis observed at pH 6.4-6.8 correlated with lipid oxidation of the RBC membrane, while the lower hemolysis at pH 7.2-8.0 occurred with low, or without any RBC membrane lipid oxidation. When hemin was added to the RBCs at pH 6.8 hemolysis was induced without parallel RBC membrane oxidation, pointing at Hb-autoxidation and hemin-release per se as important events triggering lysis in fish muscle. Altogether, the study provided valuable findings which ultimately can aid development of new tools to combat lipid oxidation in post mortem fish muscle by limiting hemolysis.

The phosphodiesterase 4 inhibitor AA6216 suppresses activity of fibrosis-specific macrophages.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a form of chronic, progressive fibrosing interstitial pneumonia of unknown cause, with a poor prognosis. We previously showed the antifibrotic effects of a novel phosphodiesterase 4 (PDE4) inhibitor, AA6216. In this study, we examined the effect of AA6216 on the pulmonary accumulation of segregated-nucleus-containing atypical monocytes (SatMs), which produce tumor necrosis factor (TNF)-α and are involved in murine lung fibrosis. METHODS: Mice were treated with bleomycin intratracheally at day 0 and either 10 mg/kg AA6216, 100 mg/kg nintedanib, or vehicle orally once daily from day 0 to 8. On day 9, we isolated the bronchoalveolar lavage fluid and analyzed the SatM ratio. In addition, we evaluated the effect of AA6216 on TNF-α production from SatMs isolated from murine bone marrow. RESULTS: AA6216, and not the antifibrotic agent nintedanib, significantly suppressed the pulmonary accumulation of SatMs (AA6216: 68.3 ± 5.4%, Nintedanib: 129.8 ± 19.7%). Furthermore, AA6216 dose-dependently inhibited the production of TNF-α by SatMs. CONCLUSIONS: AA6216 suppresses pathogenic SatMs in the lung, which contributes to its antifibrotic effects.

Anti-inflammatory effects of a novel phosphodiesterase-4 inhibitor, AA6216, in mouse dermatitis models.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that is commonly treated with corticosteroids. However, these drugs have long-term adverse effects, representing an unmet need for new treatments. AD is associated with dysregulation of phosphodiesterase 4 (PDE4) activity in inflammatory cells and the topical PDE4 inhibitor, crisaborole, is approved by the US FDA for mild-to-moderate AD. In this study, we compared the effects of a novel PDE4 inhibitor, AA6216, with those of crisaborole on skin inflammation. We found that AA6216 is a more potent inhibitor of PDE4 and of cytokine production (TNF-α, IL-12/23p40, IL-4, IL-13, and IFN-γ) by human peripheral blood mononuclear cells (PBMCs) stimulated by phytohemagglutinin (PHA) or anti-CD3 antibodies, with IC50 values ranging from 5.9 to 47 nM. AA6216 also significantly suppressed skin inflammation in three mouse models of dermatitis. In acute and chronic oxazolone-induced dermatitis models, topical AA6216 exhibited stronger inhibitory effects on ear inflammation and cytokine production (TNFα, IL-1ß, and IL-4) in skin lesions compared with crisaborole. In a Dermatophagoides farinae-induced dermatitis model, AA6216 significantly reduced the dermatitis score, based on the development of erythema/hemorrhage, scarring/dryness, edema, and excoriation/erosion, compared with a clinically used topical AD drug, tacrolimus. These results suggest the possibility that AA6216 is a novel and effective topical therapeutic agent for the treatment of dermatitis including AD.

Entropy-Driven Supramolecular Ring-Opening Polymerization of a Cyclic Hemoglobin Monomer for Constructing a Hemoglobin-PEG Alternating Polymer with Structural Regularity.

Our earlier report described that a cyclic hemoglobin (Hb) monomer with two ß subunits of a Hb molecule (α2ß2) bound through a flexible polyethylene glycol (PEG) chain undergoes reversible supramolecular ring-opening polymerization (S-ROP) to produce a supramolecular Hb polymer with a Hb-PEG alternating structure. In this work, we polymerized cyclic Hb monomers with different ring sizes (2, 5, 10, or 20 kDa PEG) to evaluate the thermodynamics of S-ROP equilibrium. Quantification of the produced supramolecular Hb polymers and the remaining cyclic Hb monomers in the equilibrium state revealed a negligibly small enthalpy change in S-ROP (ΔHp ≤ 1 kJ·mol-1) and a markedly positive entropy change increasing with the ring size (ΔSp = 26.8-33.2 J·mol-1·K-1). The results suggest an entropy-driven mechanism in S-ROP: a cyclic Hb monomer with the larger ring size prefers to form a supramolecular Hb polymer. The S-ROP used for this study has the potential to construct submicrometer-sized Hb-PEG alternating polymers having structural regularity.

A novel phosphodiesterase 4 inhibitor, AA6216, reduces macrophage activity and fibrosis in the lung.

Idiopathic pulmonary fibrosis (IPF) is an intractable disease with poor prognosis, and therapeutic options are limited. While the pathogenic mechanism is unknown, cytokines, such as transforming growth factor (TGF)-ß, and immune cells, such as monocytes and macrophages, that produce them, seem to be involved in fibrosis. Some phosphodiesterase 4 (PDE4) inhibitors reportedly have anti-fibrotic potential by acting on these disease-related factors. Therefore, we evaluated the effect of a novel PDE4 inhibitor, AA6216, on nonclinical IPF-related models and samples from IPF patients. First, we examined the inhibitory effect of AA6216 on the production of TGF-ß1 from a human monocytic cell line, THP-1. Second, we analyzed the impact of AA6216 on TNF-α production by human alveolar macrophages collected from patients with IPF. Finally, we investigated the anti-fibrotic potency of AA6216 on bleomycin-induced lung fibrosis in mice. We found that AA6216 significantly inhibited TGF-ß1 production by THP-1 cells. It also significantly suppressed TNF-α production by alveolar macrophages from patients with IPF. In the mouse model of bleomycin-induced pulmonary fibrosis, therapeutic administration of AA6216 significantly reduced fibrosis scores, collagen-stained areas, and TGF-ß1 in bronchoalveolar lavage fluid. AA6216 may represent a new agent for the treatment of IPF with a distinct mechanism of action from that of conventional anti-fibrotic agents.

Antioxidative Pseudoenzymatic Mechanism of NAD(P)H Coexisting with Oxyhemoglobin for Suppressed Methemoglobin Formation.

Oxyhemoglobin (HbO2) coexisting with equimolar NADH retards autoxidation and oxidant-induced metHb formation based on the pseudocatalase (CAT) and pseudosuperoxide dismutase (SOD) activities. In this work, we compared the effects of NADH with those of NADPH and estimated the binding site of NAD(P)H to HbO2 to elucidate the antioxidative mechanisms. The results clarified that pseudo-CAT and pseudo-SOD activities of HbO2 coexisting with NADPH were similar to activities obtained with NADH. Prompt MetHb formation (<40 min) facilitated by oxidants (H2O2, NO, and NaNO2) was hindered by NADPH. These effects were similar to those of NADH. However, we found that NADPH is thermally unstable compared to NADH and that NADPH cannot sustain antioxidative effects for a long period of autoxidation to metHb such as 24 h. Lineweaver-Burk plots clarified that the Michaelis constants of these pseudoenzymatic activities are in the millimolar range. Addition of inositol hexaphosphate (IHP) and 2,3-diphosphoglycerate (DPG), which are known to bind not only with deoxyHb but also weakly with HbO2, showed competitive inhibition of pseudoenzymatic activities. These results suggest that the binding site of NADH and NADPH on HbO2 is the same as those of IHP and DPG. 31P nuclear magnetic resonance definitively showed 1:1 stoichiometric binding of NADH to HbO2. High-performance liquid chromatography analysis showed that NADH preferentially inhibited autoxidation of α-subunit heme. Docking simulations also predicted that the binding site of relaxed-state HbO2 with NAD(P)H is the same as those with IHP and DPG. Collectively, the pseudoenzymatic activities of HbO2 coexisting with NAD(P)H are induced by the 1:1 stoichiometric binding of NAD(P)H to HbO2.

Ring-Opening Polymerization of Hemoglobin.

Hemoglobin (Hb), an oxygen-carrying protein, has an α2ß2 tetrameric structure that dissociates reversibly into two αß dimers (α2ß2 ⇄ 2αß). We synthesized a cyclic Hb-ring monomer with two ß subunits bound through a 10 kDa polyethylene glycol (PEG) chain. The monomer induced ring-opening polymerization to produce a supramolecular polymer via intersubunit interaction of αß dimers of an Hb molecule at the PEG terminals. Both the ring-closed monomer and the ring-opened supramolecular polymer were then fixed covalently by intramolecular cross-linking of two ß subunits. Quantification of fixed products at various monomer concentrations revealed the equilibrium constant ( K), a ratio of propagation and depropagation rate constants, as 5.68 mM-1. The average degree of polymerization ([Formula: see text]) increased proportionally, concomitantly with the initial monomer concentration. Hb polymer with [Formula: see text] = 13.2 ( Mn = ca. 1 MDa) was obtained by cross-linking at 2.33 mM. Our novel strategy of ring-opening polymerization of Hb will eventually realize a highly aligned and efficiently polymerized Hb for creating artificial oxygen carriers for a clinical use.

Analysis of Dimeric αß Subunit Exchange between PEGylated and Native Hemoglobins (α2ß2 Tetramer) in an Equilibrated State by Intramolecular ßß-Cross-Linking.

Various chemical modifications of hemoglobin (Hb) including PEGylation have been investigated to produce red blood cell substitutes. Some of those modifications are designed on the premise that the α2ß2 tetrameric structure of Hb is fundamentally stable and that it rarely dissociates into two αß dimers in a physiological condition. However, in the present work using the "clipping" method we detected and quantitatively analyzed the considerable degree of exchange reaction of αß subunits between ß93Cys-bis-PEGylated and native Hbs through dissociation into αß dimers and restructuring to α2ß2 tetramer in a physiological condition. The equilibrium constant ( Keq) of subunit exchange reactions increased from 0.82 to 2.86 with increasing molecular weight of PEG from 2 to 40 kDa, indicating that longer PEG chains enhanced such exchange reaction. The results suggest that the exchange might occur for other modified Hbs even at a practically high concentration for use as a red blood cell substitute.

Evaluation of four antiseptics using a novel murine norovirus.

We isolated a novel murine norovirus (MNV), MT30-2 strain, from feces of conventional mice in Japan to evaluate the virucidal activity of four antiseptics. The MNV MT30-2 strain was inactivated by as little as 0.2% (w/v) povidone-iodine (PVP-I) and 0.1% (w/v) sodium hypochlorite (NaOCl) treatment as determined by a novel plaque assay. Importantly, PVP-I reduced the MNV titer by 4 log(10) within 15 s of exposure. The other two antiseptics, benzethonium chloride (BEC) and chlorhexidine gluconate (CHG), did not reduce the MNV titer even when treatment lasted for 60 s. When the virus titer was reduced by PVP-I or NaOCl treatment, the amount of MNV RNA was not reduced, indicating that the presence of viral RNA was not related to the virucidal activity of the antiseptics. PVP-I and NaOCl will be useful in controlling the spread of MNV, which is a common problem in mice colonies. In this study, we isolated a novel MNV and newly revealed that two antiseptics (PVP-I and NaOCl) were able to inactivate MNV at low concentrations and in a short contact time.

ME3738 enhances the effect of interferon and inhibits hepatitis C virus replication both in vitro and in vivo.

BACKGROUND & AIMS: ME3738 (22ß-methoxyolean-12-ene-3ß, 24-diol), a derivative of soyasapogenol B, attenuates liver disease in several animal models of acute and chronic liver injury. ME3738 is thought to inhibit replication of hepatitis C virus (HCV) by enhancing interferon (IFN)-ß production, as determined using the HCV full-length binary expression system. We examined the effect of ME3738 combined with IFN-α on HCV replication using the genotype 1b subgenomic replicon system and an in vivo mouse HCV model. METHODS: HCV replicon cells (ORN/3-5B/KE cells and Con1 cells) were incubated with ME3738 and/or IFN-α, and then intracellular IFN-stimulated genes (ISGs) and HCV RNA replication were analyzed by reverse-transcription-real time polymerase chain reaction and luciferase reporter assay. HCV-infected human hepatocyte chimeric mice were also treated with ME3738 and/or IFN-α for 4 weeks. Mouse serum HCV RNA titer, HCV core antigen, and ISGs expression in the liver were measured. RESULTS: ME3738 induced gene expression of oligoadenylate synthetase 1 and inhibited HCV replication in both HCV replicon cells. The drug enhanced the effect of IFN to significantly increase ISG expression levels, inhibit HCV replication in replicon cells, and reduce mouse serum HCV RNA and core antigen levels in mouse livers. The combination treatment was not hepatotoxic as evident histologically and did not reduce human serum albumin in mice. CONCLUSIONS: ME3738 inhibited HCV replication, enhancing the effect of IFN-α to increase ISG expression both in vitro and in vivo, suggesting that the combination of ME3738 and IFN might be useful therapeutically for patients with chronic hepatitis C.

Initiation of DNA replication after fertilization is regulated by p90Rsk at pre-RC/pre-IC transition in starfish eggs.

Initiation of DNA replication in eukaryotic cells is controlled through an ordered assembly of protein complexes at replication origins. The molecules involved in this process are well conserved but diversely regulated. Typically, initiation of DNA replication is regulated in response to developmental events in multicellular organisms. Here, we elucidate the regulation of the first S phase of the embryonic cell cycle after fertilization. Unless fertilization occurs, the Mos-MAPK-p90Rsk pathway causes the G1-phase arrest after completion of meiosis in starfish eggs. Fertilization shuts down this pathway, leading to the first S phase with no requirement of new protein synthesis. However, how and in which stage the initiation complex for DNA replication is arrested by p90Rsk remains unclear. We find that in G1-arrested eggs, chromatin is loaded with the Mcm complex to form the prereplicative complex (pre-RC). Inactivation of p90Rsk is necessary and sufficient for further loading of Cdc45 onto chromatin to form the preinitiation complex (pre-IC) and the subsequent initiation of DNA replication. However, cyclin A-, B-, and E-Cdk's activity and Cdc7 accumulation are dispensable for these processes. These observations define the stage of G1 arrest in unfertilized eggs at transition point from pre-RC to pre-IC, and reveal a unique role of p90Rsk for a negative regulator of this transition. Thus, initiation of DNA replication in the meiosis-to-mitosis transition is regulated at the pre-RC stage as like in the G1 checkpoint, but in a manner different from the checkpoint.

Novel photosystem involving protonation and deprotonation processes modelled on a PYP photocycle.

The synthesis of novel ortho-coumaric acid derivatives, with an amide group linked with an olefin moiety, which introduced photoinduced switching of the intramolecular hydrogen bonds is presented. An intramolecular OH...O=C hydrogen bond formed in a Z-phenol compound was switched to an intramolecular NH...O hydrogen bond in Z phenolate state via deprotonation. The pK(a) value of the Z-phenol derivative was lower than that of E-phenol, and a novel photocycle system involving protonation and deprotonation processes was achieved.

Manipulation of an intramolecular NH...O hydrogen bond by photoswitching between stable E/Z isomers of the cinnamate framework.

Novel carboxylic acid derivatives were synthesized, which allowed switching of the intramolecular distance between amide group and carboxylic oxygen atoms using E to Z photoisomerization of the cinnamate framework. An intramolecular NH...O hydrogen bond was formed in the Z carboxylate compound not only in solution but also in the solid state. The pK(a) value of the carboxylic acid was lowered as a consequence of the E/Z photoisomerization.

Photoinduced switching of intramolecular hydrogen bond between amide NH and carboxyl oxygen.

In this study, we synthesized two novel carboxylic acid and carboxylate compounds, both of which had an amide group linked with an azomethine moiety to introduce photoinduced switching of the intramolecular NH...O hydrogen bond. We suggest that the cis-carboxylate compound forms a stronger intramolecular NH...O hydrogen bond than the cis-carboxylic acid compound.