Synthesis and evaluation of novel modified γ-lactam prostanoids as EP4 subtype-selective agonists.

To identify chemically and metabolically stable subtype-selective EP4 agonists, design and synthesis of a series of modified γ-lactam prostanoids has been continued. Prostanoids bearing 2-oxo-1,3-oxazolidine, 2-oxo-1,3-thiazolidine and 5-thioxopyrrolidine as a surrogate for the γ-hydroxycyclopentanone without a troublesome 11-hydroxy group were identified as highly subtype-selective EP4 agonists. Among the tested, several representative compounds demonstrated in vivo efficacy after oral dosing in rats. Their pharmacokinetic and structure-activity relationship studies are presented.

Discovery of orally available 8-aza-5-thiaProstaglandin E1 analogs as highly selective EP4 agonists.

Analogs 8-aza-16-aryl prostaglandin E(1) (PGE(1)) and 8-aza-5-thia-16-arylPGE(1) were synthesized and evaluated with respect to their subtype receptor affinity and EP4 agonist activity for the purposes of identifying subtype-selective EP4 agonists that demonstrate oral efficacy. Using an inhibition assay of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in rats, representative compounds were evaluated for their pharmacokinetic profiles and in vivo efficacy. Structure-activity relationships (SARs) were characterized and presented. Of the compounds tested, several demonstrated better oral exposure and/or in vivo efficacy compared with the previously reported analog 2a.

Endothelium-dependent vascular responses induced by leukotriene B4.

Leukotriene B(4) (LTB(4)) is an inflammatory mediator derived from the 5-lipoxygenase pathway of arachidonic acid metabolism and has recently implicated in the pathogenesis of atherosclerosis. There are two membrane bound receptors for LTB(4): BLT(1) and BLT(2), which represent the high and low affinity receptors, respectively. BLT receptors are expressed on leukocytes, and LTB(4) is a potent chemoattractant for neutrophils, eosinophils, and T lymphocytes. Recent studies have in addition shown that LTB(4) is an indirectly acting vasoconstrictor of isolated vascular preparations. In the guinea pig aorta, the LTB(4)-induced contractions were inhibited by endothelium-denudation. In addition, pre-treatment with the NO synthase inhibitor, L-NOARG, significantly enhanced the contractions induced by LTB(4). The contractile response induced by LTB(4) in the guinea pig aorta was abolished by the selective BLT(1) receptor antagonist U75302 and the expression of BLT(1) receptor mRNA in the guinea pig aorta was established by RT-PCR. Taken together, these results suggest that LTB(4) activates BLT(1) receptors on the endothelium of the guinea pig aorta, associated with the release of both contractile factors and NO.

Effects of a prostaglandin EP4 agonist, ONO-4819, and risedronate on trabecular microstructure and bone strength in mature ovariectomized rats.

The effects of a prostaglandin EP4 agonist, ONO-4819, and risedronate, a representative anti-resorptive drug, on trabecular microarchitecture and biomechanical properties were investigated in mature estrogen-deficient rats; and effects which affected microstructural components that contributed to the improvement of bone strength were also determined. Thirty-three-week-old OVX rats were treated with various doses of ONO-4819, risedronate, or their combination for 11 weeks. Bone mineral density (BMD), trabecular microstructure, and biomechanical strength were determined at the proximal tibia by peripheral quantitative CT, micro CT, and finite element analysis, respectively. Bone histomorphometry was performed at the same site. The results of trabecular structure analysis indicated that whereas risedronate functioned mainly in maintaining trabecular connectivity, ONO-4819 converted the fragile rod-like trabeculae caused by estrogen deficiency to a plate-like structure. In addition, ONO-4819 is one of the few drugs that are capable of increasing trabecular thickness. When the 2 drugs were combined, the beneficial effects of each drug on the trabecular microarchitecture were maintained, resulting in their additive effects on bone strength. The results of bone histomorphometry suggest that ONO-4819 caused an increase in the rate of bone formation by stimulating the differentiation/recruitment of osteoblasts as well as their mineralizing function. ONO-4819 did not stimulate bone resorption, but rather exerted an anti-resorptive function within a certain dose range. ONO-4819 and risedronate increased BMD and improved trabecular structure and biomechanical strength in an additive and independent manner. Thus, EP4 agonist ONO-4819 in combination with risedronate may be an effective treatment for osteoporosis.

Leukotriene B4 is an indirectly acting vasoconstrictor in guinea pig aorta via an inducible type of BLT receptor.

Leukotriene B(4) (LTB(4)) is a potent leukocyte chemoattractant recently implicated in the pathogenesis of atherosclerosis. The aim of this study was to assess the effects of LTB(4) on isolated aortic preparations. Rings of guinea pig aorta were challenged with LTB(4) for recording mechanical responses and measurements of mediator release, and LTB(4) receptor (BLT(1)) expression was assessed by RT-PCR. Single concentrations of LTB(4) induced concentration-dependent contractions that were inhibited by treatment with antihistamines, indomethacin, or the thromboxane receptor antagonist BAYu3405 as well as by denudation of endothelium. In addition, LTB(4) increased the release of histamine and thromboxane in the bath. The contractions induced by LTB(4) were inhibited by either the unselective BLT receptor antagonist ONO-4057 or the selective BLT(1) receptor antagonist U-75302. Pretreatment with all-trans-retinoic acid enhanced the contractions and the release of histamine induced by LTB(4), without affecting either the contractions induced by histamine or the histamine release evoked by calcium ionophore A23187. Analysis by RT-PCR indicated the expression of a BLT(1) receptor in the guinea pig aorta and that BLT(1) receptor mRNA was upregulated after treatment with retinoic acid. These results suggest that LTB(4) contracts the guinea pig aorta via an indirect mechanism involving the release of histamine and thromboxane and that this BLT(1) receptor-mediated response can be upregulated by all-trans-retinoic acid.

The contractile action of leukotriene B4 in the guinea-pig lung involves a vascular component.

Leukotriene B4 (LTB4) is a potent leukocyte chemoattractant, acting on specific receptors, BLT receptors. The aim of this study was to examine the mechanism of action of LTB4 in the guinea-pig lung, using strips of lung parenchyma (GPLP), spirals of trachea (GPT) and bronchus (GPB) and rings of pulmonary artery (GPPA). Mechanical responses were studied in organ baths, and mediator release was assessed using enzyme immuno assay. LTB4 induced similar contractions of GPLP and GPPA, whereas LTB4 had only small contractile effects in GPT and GPB. In addition, the contractile response to LTB4 was reproduced in the human pulmonary artery. In the GPLP, the unselective BLT receptor antagonist ONO-4057 abolished the contractions induced by LTB4, whereas the selective BLT1 receptor antagonist U-75302 only partly inhibited the LTB4-induced contractions. In the GPPA, both antagonists abolished the response to LTB4. The effect of LTB4 in GPPA and GPLP was indirect and mediated by the release of thromboxane A2 and histamine, as supported by selective pharmacologic interventions and measurements of thromboxane B2 and histamine in the organ baths. In conclusion, the results indicate a new biological function of LTB4, namely to constrict isolated pulmonary arteries. Moreover, the findings suggest that the LTB4-induced contractions of GPPA were mediated by a BLT1 receptor, whereas BLT2 receptor activation accounted for a major part of the contraction of GPLP, making the latter preparation a suitable assay for BLT2 receptors. British Journal of Pharmacology (2004) 141, 449-456. doi:10.1038/sj.bjp.0705641

Design and synthesis of a selective EP4-receptor agonist. Part 4: practical synthesis and biological evaluation of a novel highly selective EP4-receptor agonist.

A practical method of synthesizing a highly selective EP4-receptor agonist 1 using Corey lactone 2 as a key intermediate was developed. Selective methanesulfonylation of the primary alcohol of the diol 8 under the newly devised conditions followed by the protection of the remaining secondary alcohol are key reactions in this new method. Further biological evaluation of 1a-b is also reported.

Receptor preferences of cysteinyl-leukotrienes in the guinea pig lung parenchyma.

Two cysteinyl-leukotriene receptors, CysLT(1) and CysLT(2) receptors, have been cloned, but the contractions to cysteinyl-leukotrienes in the guinea pig lung parenchyma have been reported to be resistant to CysLT(2) receptor antagonism and to be only partially inhibited by CysLT(1) receptor antagonism. The receptor preferences of the individual cysteinyl-leukotrienes (leukotriene C(4), D(4) and E(4)) in the guinea pig lung parenchyma were studied in organ baths. CysLT(1) receptor antagonists competitively inhibited the contraction to leukotriene E(4), but exhibited only weak antagonism of contractions to leukotriene C(4) and D(4). In the presence of the cyclooxygenese inhibitor indomethacin and the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG), the CysLT(1) receptor antagonists did not further inhibit the leukotriene D(4)-induced contraction. These results suggest that leukotriene E(4) solely activates a CysLT(1) receptor, and that the CysLT(1) receptor antagonist-resistant contraction to leukotriene D(4) and C(4) is mediated via another CysLT receptor.