Design, Synthesis, and Anti-Inflammatory Evaluation of a Novel PPARδ Agonist with a 4-(1-Pyrrolidinyl)piperidine Structure.

Peroxisome proliferator-activated receptor δ (PPARδ) is considered to be a pharmaceutical target to treat metabolic diseases including atherosclerosis, but there is no PPARδ agonist available for clinical use. We have previously reported the discovery of piperidinyl/piperazinyl benzothiazole derivatives as a new series of PPARδ agonists using docking-based virtual screening methods. In the present study, we found that introduction of a pyrrolidine group into the 4-position of their central piperidine rings enhances hPPARδ activity and subtype selectivity. This led to the discovery of 21 having strong PPARδ agonist activity (EC50 = 3.6 nM) with excellent ADME properties. Furthermore, 21 significantly suppressed atherosclerosis progression by 50-60% with reduction of the serum level of MCP-1 in LDLr-KO mice.

Discovery and structure-activity relationship study of 2-piperazinyl-benzothiazole derivatives as potent and selective PPARδ agonists.

Peroxisome proliferator-activated receptor δ (PPARδ) is considered to be a target for treating metabolic syndrome, whereas there is no PPARδ agonist in clinical use. Previously, we have reported the discovery of 2-(1-piperidinyl)-1,3-benzothiazole derivatives as a new series of PPARδ agonists using docking-based virtual screening techniques. In this study, we performed the further optimization study of the lead compound 1 focusing on improvement of hydrophobic interactions in the binding site to enhance agonist efficacy for PPARδ and subtype selectivity, thereby discovering a novel PPARδ agonist 5g which exhibited high in vitro agonist activity (hPPARδ, EC50 = 4.1 nM) and sufficiently high selectivity ratio over PPARα and PPARγ. Moreover, 5g revealed a significant upregulation of high-density lipoprotein cholesterol level in vivo.

Discovery and structure-based design of a new series of potent and selective PPARδ agonists utilizing a virtual screening method.

Novel PPARδ agonists, 2-(1-piperidinyl)-1,3-benzothiazole derivatives were discovered by our proprietary docking-based virtual screening technique. Compound 1 as the initial hit was effectively modified to acquire PPARδ agonist activity, resulting in the discovery of compound 12 with high agonistic potency for PPARδ and selectivity over PPARα and PPARγ. Compound 12 also had good ADME profiles and showed in vivo efficacy as a lead.

Transdermal administration of aqueous pregabalin solution as a potential treatment option for patients with neuropathic pain to avoid central nervous system-mediated side effects.

Pregabalin, (S)-3-isobutyl-γ-aminobutyric acid (GABA), is a widely used adjuvant therapy for patients with neuropathic pain, which is defined as chronic pain caused by lesions or diseases of the somatosensory nervous system. However, dizziness and somnolence (sleepiness) are common dose-limiting side effects, probably due to excessive sedative effects on higher centers of the central nervous system (CNS) which are involved in the anticonvulsant and analgesic actions of pregabalin. We speculated that transdermal delivery would minimize centrally mediated side effects. To test this idea, we evaluated the analgesic effects of pregabalin delivered through the transdermal route in animal models of neuropathic pain. Transdermally administered pregabalin increased the pain thresholds in response to mechanical stimuli in a partial sciatic nerve ligation model in rats and a spinal nerve ligation model in mice, and surprisingly also in normal animals. It is noteworthy that simple transdermal application of an aqueous solution of pregabalin is effective. This could be a useful treatment option to avoid or minimize the CNS-mediated side effects of orally administered pregabalin.

Cooperative therapeutic action of retinoic acid receptor and retinoid x receptor agonists in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative process involving amyloid-ß (Aß) peptide deposition, neuroinflammation, and progressive memory loss. Here, we evaluated whether oral administration of retinoic acid receptor (RAR)α,ß agonist Am80 (tamibarotene) or specific retinoid X receptor (RXR) pan agonist HX630 or their combination could improve deficits in an AD model, 8.5-month-old amyloid-ß protein precursor 23 (AßPP23) mice. Co-administration of Am80 (0.5 mg/kg) and HX630 (5 mg/kg) for 17 days significantly improved memory deficits (Morris water maze) in AßPP23 mice, whereas administration of either agent alone produced no effect. Only co-administration significantly reduced the level of insoluble Aß peptide in the brain. These results thus indicate that effective memory improvement via reduction of insoluble Aß peptide in 8.5-month-old AßPP23 mice requires co-activation of RARα,ß and RXRs. RARα-positive microglia accumulated Aß plaques in the AßPP23 mice. Rat primary microglia co-treated with Am80/HX630 showed increased degradation activity towards 125I-labeled oligomeric Aß1-42 peptide in an insulin-degrading enzyme (IDE)-dependent manner. The co-administration increased mRNA for IDE and membrane-associated IDE protein in vivo, suggesting that IDE contributes to Aß clearance in Am80/HX630-treated AßPP23 mice. Am80/HX630 also increased IL-4Rα expression in microglial MG5 cells. The improvement in memory of Am80/HX630-treated AßPP23 mice was correlated with the levels and signaling of hippocampal interleukin-4 (IL-4). Therefore, Am80/HX630 may promote differentiation of IL-4-responsive M2-like microglia and increase their activity for clearance of oligomeric Aß peptides by restoring impaired IL-4 signaling in AßPP23 mice. Combination treatment with RAR and RXR agonists may be an effective approach for AD therapy.

Silicon-containing GABA derivatives, silagaba compounds, as orally effective agents for treating neuropathic pain without central-nervous-system-related side effects.

Neuropathic pain is a chronic condition resulting from neuronal damage. Pregabalin, the (S)-isomer of 3-isobutyl-γ-aminobutyric acid (GABA), is widely used to treat neuropathic pain, despite the occurrence of central nervous system (CNS)-related side effects such as dizziness and somnolence. Here we describe the pharmacology of novel GABA derivatives containing silicon-carbon bonds, silagaba compounds. Silagaba131, 132, and 161 showed pregabalin-like analgesic activities in animal models of neuropathic pain, but in contrast to pregabalin they did not impair neuromuscular coordination in rotarod tests. Pharmacokinetic studies showed that brain exposure to silagaba compounds was lower than that to pregabalin. Surprisingly, despite their potent analgesic action in vivo, silagaba compounds showed only weak binding to α2-δ protein. These compounds may be useful to study mechanisms of neuropathic pain. Our results also indicate that silagaba132 and 161 are candidates for orally effective treatment of neuropathic pain without CNS-related side effects.

A novel aromatic carboxylic acid inactivates luciferase by acylation of an enzymatically active regulatory lysine residue.

Firefly luciferase (Luc) is widely used as a reporter enzyme in cell-based assays for gene expression. A novel aromatic carboxylic acid, F-53, reported here for the first time, substantially inhibited the enzymatic activity of Luc in a Luc reporter screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (MS/MS) analyses showed that F-53 modifies Luc at lysine-529 via amidation of the F-53 carboxyl group. The lysine-529 residue of Luc, which plays a regulatory catalytic role, can be acetylated. Luc also has a long-chain fatty acyl-CoA synthase activity. An in vitro assay that involved both recombinant Luc and mouse liver microsomes identified F-53-CoA as the reactive form produced from F-53. However, whereas the inhibitory effect of F-53 is observed in Hela cells that transiently expressed Luc, it is not observed in an in vitro assay that involves recombinant Luc alone. Therefore, insights into the activities of certain mammalian transferases can be translated to better understand the acylation by F-53. The insights from this study about the novel inhibitory modification mechanism might help not only to avoid misinterpretation of the results of Luc-based reporter screening assays but also to explain the pharmacological and toxicological effects of carboxylic acid-containing drugs.

Design, synthesis and evaluation of retinoids with novel bulky hydrophobic partial structures.

Many synthetic retinoids contain an aromatic structure with a bulky hydrophobic fragment. In order to obtain retinoids with therapeutic potential that do not bind to or activate retinoic acid X receptors (RXRs), we focused on the introduction of novel hydrophobic moieties, that is, metacyclophane, phenalene and benzoheptalene derivatives. The designed compounds were synthesized and their agonistic activities towards RARs and RXRs were evaluated. Most of the active compounds showed selectivity for RARα and RARß over RARγ, and higher RARß transactivating activity seemed to correlate with higher cell differentiation-inducing activity towards promyelocytic leukemia cell line HL-60. These compounds showed no agonistic activity towards RXRs.

(R)- and (S)-4-Amino-3-(trimethylsilyl)methylbutanoic acids ameliorate neuropathic pain without central nervous system-related side effects.

Neuropathic pain is a chronic pain condition resulting from neuronal damage, and is usually treated with pregabalin or gabapentin, which are structurally related to γ-aminobutyric acid (GABA) and are originally developed as anticonvulsant drugs. Here, we report the synthesis and pharmacology of (R)- and (S)-4-amino-3-(trimethylsilyl)methylbutanoic acids (1a and 1b), which showed analgesic activity as potent as that of pregabalin in the Chung spinal nerve ligation model. However, unlike pregabalin, 1a and 1b do not have antiepileptic effects, and they are therefore promising candidates for selective therapeutic agents to treat neuropathic pain without central nervous system-related side effects.

Tamibarotene: a candidate retinoid drug for Alzheimer's disease.

Tamibarotene (Am80), a synthetic retinoid approved in Japan for treatment of acute promyelocytic leukemia (APL), is a retinoic acid receptor (RAR) agonist with high specificity for RARα and RARß over RARγ. Temporarily and spatially specific expression of RARs suggests their pivotal roles in the adult brain. Am80 is considered to be a promising candidate drug for treatment of Alzheimer's disease (AD) because of its transcriptional controls of multiple target genes involved in etiology and pathology of AD. In APP23 AD model mice, administration of Am80 decreased the deposition of insoluble amyloid-ß(42). In senescence-accelerated mice (SAMP8), Am80 ameliorated the decrease of cortical acetylcholine, as well as reducing anxiety in behavioral tests and improving the sleep deficit. Am80 also effected a significant improvement of memory in the rat scopolamine-induced memory deficit model. Like other retinoids, Am80 also has an immunomodulatory effect and reduces secretion of proinflammatory cytokines and chemokines by astrocytes and microglia surrounding amyloid-ß plaques. In a rat experimental autoimmune encephalomyelitis model, Am80 reduced inflammatory cytokines and showed significant efficacy. Retinoids also promote differentiation of neural stem cells, and Am80 improved the recovery of spinal cord-injured rats. Am80 may also improve vascular factors involved in onset and/or progression of AD. Am80 has been in clinical use for treatment of APL in Japan since 2005, and has been reported to have fewer side effects than other retinoids. We have recently started a clinical study to evaluate the efficacy and safety of Am80 for the treatment of Alzheimer's disease.

Diphenylamine-based retinoid antagonists: regulation of RAR and RXR function depending on the N-substituent.

Based upon the structure-activity relationships of diphenylamine derivatives with retinoid synergistic activity (RXR agonists), novel diphenylamine derivatives with a long alkyl chain (9a and 9b) or a benzyl group (10a-f) as the N-substituent were designed and synthesized. All the synthesized compounds dose-dependently inhibited HL-60 cell differentiation induced by 3.3×10(-10)M Am80. Among them, compound 10f showed the most potent inhibitory activity, and the mechanism was shown, by means of transactivation assay for RARs and RXRs, to involve antagonism against RARs. The N-substituent of the diphenylamine skeleton plays an important role in determining the receptor selectivity for RARs or RXRs, as well as the agonist or antagonist nature of the activity.

Aldosterone-induced kidney injury is mediated by NFκB activation.

BACKGROUND: Aldosterone induces inflammation and fibrosis in the kidney, while nuclear factor κB (NFκB) plays key roles in inflammation mediated by various cytokines. Here, we determined the roles of NFκB activation in aldosterone-induced kidney injury. METHODS: We used unilaterally nephrectomized rats with or without continuous aldosterone infusion and 0.9% saline as drinking water for 3 weeks. IMD-1041, an IKKß inhibitor, and spironolactone were orally administered to inhibit NFκB and mineralocorticoid receptor, respectively. RESULTS: The aldosterone-infused rats exhibited severe kidney injury, hypertension, and increased expression of pro-inflammatory and fibrotic proteins, osteopontin, fibrinogen, collagen type I, and PAI-1. Western blotting confirmed NFκB activation by aldosterone by the increased amount of p65 in the nuclear fraction of the kidney, and oral IMD-1041 prevented the kidney injury and lessened the increase in pro-inflammatory and fibrotic proteins without significant changes in blood pressures. In addition, changes in angiotensin-converting enzyme 2 (ACE2), which has been found to act as a protective factor in various kidney injury models, were examined. Immunofluorescence studies revealed the presence of ACE2 in the brush-border membrane of the proximal convoluted tubules and markedly blunted ACE2 staining in aldosterone-infused rats. The decrease in amount of ACE2 protein was confirmed by Western blotting, and IMD-1041 also prevented the decrease in ACE2. The administration of spironolactone also abolished the effects of aldosterone. CONCLUSION: Our results suggest that aldosterone induces kidney injury via activation of NFκB and mineralocorticoid receptor, and that decreased ACE2 expression may play an important role in aldosterone-induced kidney injury.

[Retinoid therapy for autoimmune diseases].

Retinoid is a collective term for compounds which bind to and activate retinoic acid receptors (RARalpha, beta, gamma and RXRalpha, beta, gamma), members of nuclear hormone receptor superfamily. The most important endogeneous retinoid is all-trans-retinoic acid (ATRA) which is an RARalpha, beta and gamma ligand. ATRA and its mimics have been in clinical use for treatment of acute promyelocytic leukemia (APL) and some skin diseases. Many synthetic retinoids have been developed and attempts to improve their medicinal properties have been made. Among them, tamibarotene (Am80) is an RARalpha- and RARbeta-specific (but RARgamma- and RXRs-nonbinding) synthetic retinoid that is effective in the treatment of psoriasis patients and relapsed APL. Experimentally, this compound is also active in animal models of rheumatoid arthritis and experimental autoimmune encephalomyelitis. On this background, possible application of retinoids for the treatment of autoimmune diseases was discussed. In particular, Th1 dominant autoimmune diseases may be the targets of the retinoids.

Inhibition of IkappaB phosphorylation in cardiomyocytes attenuates myocardial ischemia/reperfusion injury.

OBJECTIVE: Reperfusion injury is related closely to inflammatory reactions such as activation of inflammatory cells and expression of cytotoxic cytokines. We investigated the efficacy of IkappaB phosphorylation blockade in a rat myocardial ischemia/reperfusion injury model. METHODS AND RESULTS: IMD-0354 inhibited phosphorylation of IkappaBalpha and nuclear translocation of nuclear factor-kappa B (NF-kappaB) induced by tumor necrosis factor-alpha (TNF-alpha) in cultured cardiomyocytes. TNF-alpha-induced production of interleukin-1beta and monocyte chemoattractant protein-1 from cultured cardiomyocytes was reduced significantly by IMD-0354. Transient left coronary artery occlusion (30 min) and reperfusion (24 h) were carried out in Sprague-Dawley rats. IMD-0354 (1, 5, 10 mg/kg) was injected intraperitoneally 5 min before the start of reperfusion. Treatment with IMD-0354 resulted in a significant dose-dependent reduction of the infarction area/area at risk ratio (vehicle, 47.0+/-3.4%; 10 mg/kg of IMD-0354, 19.4+/-4.0%; P<0.01) and the preservation of fractional shortening ratio (vehicle, 25.0+/-1.5%; 10 mg/kg of IMD-0354, 42.3+/-1.7%; P<0.01). Histological analysis showed that accumulation of polymorphonuclear neutrophils in the area at risk was decreased significantly. CONCLUSIONS: Inhibition of nuclear translocation of NF-kappaB by IkappaBalpha phosphorylation blockade could provide an effective approach to attenuation of ischemia/reperfusion injury. The cardioprotective effects of IMD-0354 include not only reduction of harmful neutrophil accumulation in myocardium but also inhibition of harmful cytokine and chemokine production by cardiomyocytes.

Determination of endogenous levels of retinoic acid isomers in type II diabetes mellitus patients. Possible correlation with HbA1c values.

A sensitive HPLC method for simultaneous determination of endogenous levels of all-trans-(ATRA), 13-cis-(13cRA), and 9-cis-retinoic acids (9cRA) was applied to serum samples from healthy volunteers and type II diabetes mellitus patients. Levels of 9cRA (around 0.2 ng/ml in both groups) were below the limit of quantification. The concentrations of ATRA and 13cRA were reliably quantified, and the within-day and between-days variances indicated that they were well maintained with little variation. Concentrations of serum ATRA and 13cRA of diabetic patients (ATRA: 1.76 +/- 0.54 ng/ml; 13cRA: 1.77 +/- 0.39ng/ml, n=13) were rather lower than those of healthy subjects (ATRA: 2.08 +/- 0.53 ng/ml; 13cRA: 2.05 +/- 0.26 ng/ml, n = 18), but the differences were not significant, except for the sum of ATRA and 13cRA (p = 0.033). Interestingly, the serum levels of retinoic acids in diabetic patients correlated positively with the hemoglobin A1c (HbA1c) values (ATRA: r = 0.57, p < 0.05; 13cRA: r = 0.62, p < 0.05). The results warrant further studies on the possible involvement of uncontrolled serum retinoic acids levels in the pathogenesis and/or treatment of diabetes mellitus.

Novel retinoid X receptor antagonists: specific inhibition of retinoid synergism in RXR-RAR heterodimer actions.

Several 2-(arylamino)pyrimidine-5-carboxylic acids were designed as novel retinoid X receptor (RXR) antagonists. Compound 6a or 6b alone did not exhibit differentiation-inducing activity toward HL-60 cells and did not affect the activity of a retinoic acid receptor (RAR) agonist, Am80, but did inhibit the synergistic activity of an RXR agonist, PA024 (3), in the presence of Am80. The activity of 6 was ascribed to selective antagonism at the RXR site of RXR-RAR heterodimers.

Potent and selective partial ecdysone agonist activity of chromafenozide in Sf9 cells.

Chromafenozide (ANS-118) is a non-steroidal ecdysone mimic and its insecticidal effect is highly specific to lepidoptera. In order to evaluate the transcription-inducing activity via nuclear ecdysone receptor (EcR) and the mode of action of chromafenozide, ecdysone-responsive reporter gene assay systems were developed in Sf9 and Kc cells. Ponasterone A, a full EcR agonist, induced reporter transcription in a dose-dependent manner in both Sf9 and Kc cells. In contrast, chromafenozide activated reporter transcription with comparable potency to ponasterone A only in Sf9 cells, although its maximum activity was 4-fold lower than that of ponasterone A. When chromafenozide was applied together with ponasterone A to Sf9 cells, it antagonized ponasterone A at nanomolar concentrations. These results suggest that chromafenozide is a potent partial EcR agonist specific to lepidoptera; it appears to bind lepidopteran EcR with comparable affinity to ponasterone A, but may activate the EcR in a different manner.