Shuichi Ohuchida

Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation

Bone remodeling, comprising resorption of existing bone and de novo bone formation, is required for the maintenance of a constant bone mass. Prostaglandin (PG)E2 promotes both bone resorption and bone formation. By infusing PGE2 to mice lacking each of four PGE receptor (EP) subtypes, we have identified EP4 as the receptor that mediates bone formation in response to this agent. Consistently, bone formation was induced in wild-type mice by infusion of an EP4-selective agonist and not agonists specific for other EP subtypes. In culture of bone marrow cells from wild-type mice, PGE2 induced expression of core-binding factor α1 (Runx2/Cbfa1) and enhanced formation of mineralized nodules, both of which were absent in the culture of cells from EP4-deficient mice. Furthermore, administration of the EP4 agonist restored bone mass and strength normally lost in rats subjected to ovariectomy or immobilization. Histomorphometric analysis revealed that the EP4 agonist induced significant increases in the volume of cancellous bone, osteoid formation, and the number of osteoblasts in the affected bone of immobilized rats, indicating that activation of EP4 induces de novo bone formation. In addition, osteoclasts were found on the increased bone surface at a density comparable to that found in the bone of control animals. These results suggest that activation of EP4 induces bone remodeling in vivo and that EP4-selective drugs may be beneficial in humans with osteoporosis.

Regulation of TNFα and interleukin-10 production by prostaglandins I2 and E2: studies with prostaglandin receptor-deficient mice and prostaglandin E-receptor subtype-selective synthetic agonists

To know which receptors of prostaglandins are involved in the regulation of TNFalpha and interleukin 10 (IL-10) production, we examined the production of these cytokines in murine peritoneal macrophages stimulated with zymosan. The presence of PGE(2) or the PGI(2) analog carbacyclin in the medium reduced the TNFalpha production to one-half, whereas IL-10 production increased several fold; and indomethacin caused the reverse effects, suggesting that endogenous prostaglandins may have a regulatory effect on the cytokine production. Among prostaglandin E (EP) receptor-selective synthetic agonists, EP2 and EP4 agonists caused down-regulation of the zymosan-induced TNFalpha production, but up-regulation on the IL-10 production; while EP1 and EP3 agonists showed no effect. Macrophages harvested from prostaglandin I (IP) receptor-deficient mice showed the up- and down-regulatory effects on the cytokine production by the EP2 and EP4 agonists or PGE(2), but no effect was obtained by carbacyclin. On the contrary, macrophages from EP2-deficient mice showed the effect by PGE(2), carbacyclin, and the EP4 agonist, but not by the EP2 agonist; and the cells from EP4-deficient mice showed the effect by PGE(2), carbacyclin, and EP2 agonist, but not by the EP4 agonist. These functional effects of prostaglandins well accorded with the mRNA expression of TNFalpha and IL-10 when such expression was examined by the RT-PCR method. The peritoneal macrophages from normal mice expressed IP, EP2, and EP4 receptors, but not EP1 and EP3, when examined by RT-PCR. Thus the results suggest that PGI(2) and PGE(2) generated simultaneously with cytokines by macrophages treated with zymosan may influence the cytokine production through IP, EP2, and EP4 receptors.

Inhibitory effect of a prostaglandin E receptor subtype EP1 selective antagonist, ONO-8713, on development of azoxymethane-induced aberrant crypt foci in mice

We previously reported that prostaglandin E(2) contributes to colon carcinogenesis through its binding to the prostaglandin E receptor subtype EP(1) using a genetic approach in EP(1)-knockout mice and a pharmacological approach with the EP(1) selective antagonist, ONO-8711. In the present study, we examined the effects of another more selective EP(1) receptor antagonist, ONO-8713, on development of azoxymethane (AOM)-induced aberrant crypt foci (ACFs) in male C57BL/6J mice treated i.p. with 10mg/kg body weight AOM once a week for 3weeks. Administration of ONO-8713 at doses of 250, 500 and 1000ppm in diet during and post-AOM treatment for 5weeks resulted in a dose-dependent reduction of ACF formation, being 15, 30 and 36% inhibition of the control value, respectively. The level of inhibition was similar to that with ONO-8711. Moreover, ONO-8713 suppressed the development of ACF when administered at post-AOM, as in the case of ONO-8711. The data confirm EP(1) receptor involvement in colon carcinogenesis.

Prevention of Diabetic Nephropathy in Rats by Prostaglandin E Receptor EP1-Selective Antagonist

Local production of prostaglandins (PGs) in the kidney is increased in clinical and experimental diabetic nephropathy, but the role of PGs in the pathogenesis and progression of diabetic nephropathy has remained unclear. It is here shown that an orally active antagonist selective for the PGE receptor EP1 subtype potently prevents the progression of nephropathy in streptozotocin-induced diabetic rats. The effects are shown by ameliorated renal and glomerular hypertrophy, decreased mesangial expansion, inhibited transcriptional activation of transforming growth factor-beta (TGF-beta) and fibronectin, and complete suppression of proteinuria. In vitro, this agent completely inhibits TGF-beta and fibronectin upregulation in mesangial cells cultured under high-glucose conditions. These data indicate that the PGE2-EP1 system plays a crucial role in the development of diabetic renal injury in rats. It is further shown that both the EP1 antagonist and aspirin, a nonselective PG synthase inhibitor, markedly attenuate mesangial expansion, whereas only the EP1 antagonist inhibits glomerular hypertrophy and proteinuria, which suggests that these changes are caused by different mechanisms. This study reveals a potential usefulness of selective EP1 blockade as a novel therapeutic strategy for diabetic nephropathy and also brings a new insight into our understanding of this disease.

Role of Prostaglandin E Receptor EP1 Subtype in the Development of Renal Injury in Genetically Hypertensive Rats

Abstract—One of the major causes of end-stage renal diseases is hypertensive renal disease, in which enhanced renal prostaglandin (PG) E2 production has been shown. PGE2, a major arachidonic acid metabolite produced in the kidney, acts on 4 receptor subtypes, EP1 through EP4, but the pathophysiological importance of the PGE2/EP subtypes in the development of hypertensive renal injury remains to be elucidated. In this study, we investigated whether an orally active EP1-selective antagonist (EP1A) prevents the progression of renal damage in stroke-prone spontaneously hypertensive rats (SHRSP), a model of human malignant hypertension. Ten-week-old SHRSP, with established hypertension but with minimal renal damage, were given EP1A or vehicle for 5 weeks. After the treatment period, vehicle-treated SHRSP showed prominent proliferative lesions in arterioles, characterized by decreased &agr;-smooth muscle actin expression in multilayered vascular smooth muscle cells. Upregulation of transforming growth factor-&bgr; expression and tubulointerstitial fibrosis were also observed in vehicle-treated SHRSP. All these changes were dramatically attenuated in EP1A-treated SHRSP. Moreover, EP1A treatment significantly inhibited both increase in urinary protein excretion and decrease in creatinine clearance but had little effect on systemic blood pressure. These findings indicate that the PGE2/EP1 signaling pathway plays a crucial role in the development of renal injury in SHRSP. This study opens a novel therapeutic potential of selective blockade of EP1 for the treatment of hypertensive renal disease.

Combined effects of prostaglandin E receptor subtype EP1 and subtype EP4 antagonists on intestinal tumorigenesis in adenomatous polyposis coli gene knockout mice

Previous studies have shown that prostaglandin E2 (PGE2) is involved in intestinal carcinogenesis through its binding to the PGE2 receptor subtypes EP1 and EP4 and activation of downstream pathways. ONO‐8711 and ONO‐AE2–227, prostaglandin E receptor subtype EP1‐ and EP4‐selective antagonists, respectively, are known to suppress formation of intestinal polyps in adenomatous polyposis coli gene‐deficient mice. The present study was designed to investigate the combined effects of EP1 and EP4 antagonists on spontaneous polyp formation in APC1309 mice in order to determine the contribution of each receptor to intestinal tumorigenesis. APC1309 mice were treated with 400 ppm of ONO‐8711 alone, 400 ppm of ONO‐AE2–227 alone or both in combination in the diet for 6 weeks. The mean area of polyps found in the intestine, calculated as the longer diameter × the shorter diameter ×π, was reduced by 12%, 43% (P<0.01) and 56% (P<0.01) of the mean control value (8.8 mm2) in the ONO‐8711 alone, ONO‐AE2–227 alone and combination treatment groups, respectively, suggesting clear additive effects of the combination. The same additive tendency for suppression was also observed with respect to the numbers of polyps in the intestine. Polyp size reduction was more remarkable with the EP4 antagonist, while the number reduction was more pronounced with the EP1 antagonist. Our results indicate that EP1 and EP4 may have separate intrinsic roles and, to some extent, contribute to polyp formation independently. Thus, combination treatment has potential for the chemoprevention of colon carcinogenesis.

Design and synthesis of a highly selective EP2-receptor agonist

EP2-receptor selective agonist 3 was identified by the structural hybridization of butaprost 1a and PGE(2) 2a. Based on this information, a chemically more stabilized 4 was discovered as another highly selective EP2-receptor agonist, iv administration of which to anesthetized rats suppressed uterine motility, while PGE(2) 2a stimulated uterine motility.

Design and synthesis of a selective EP4-receptor agonist. Part 3: 16-phenyl-5-thiaPGE1 and 9-β-halo derivatives with improved stability

To identify a new selective EP4-agonist with improved chemical stability, further chemical modification of those reported previously was continued. We focused our attention on chemical modification of the alpha chain of 3,7-dithiaPGE(1) and selected 5-thiaPGE(1) as a new chemical lead. Introduction of an optimized omega chain to the 5-thiaPG skeleton afforded m-methoxymethyl derivative 33a, which showed the most potent EP4-receptor agonist activity and good subtype-selectivity both in vitro and in vivo. 9beta-HaloPGF derivatives were also synthesized and biologically evaluated in an attempt to block self-degradation of the beta-hydroxyketone moiety. Among these series, and 39b showed potent agonist activity and good subtype-selectivity. Structure-activity relationships (SARs) are also discussed.

Suppression of azoxymethane-induced colon cancer development in rats by a prostaglandin E receptor EP1-selective antagonist

Prostaglandin E2 is involved in colon carcinogenesis through its binding to the PGE2 receptor subtypes EP1, EP2, EP3 and EP4. We have demonstrated that administration of ONO‐8711, an EP1‐selective antagonist, suppresses development of AOM‐induced ACF in C57BL/6 mice and F344 rats. ONO‐8711 also reduced the numbers of intestinal polyps in Min mice. In the present study, we investigated the long‐term effects of ONO‐8711 on colon cancer development in rats treated with AOM. Male F344 rats were injected subcutaneously with AOM (15 mg/kg body weight) once a week for the first 2 weeks to develop colon cancer. Administration of 400 or 800 p.p.m. ONO‐8711 in their diets for 32 weeks reduced the incidence, multiplicity and volume of colon carcinomas. The incidence of colon adenocarcinomas in AOM‐treated rats was 97, 83 and 76% (P < 0.05) in the 0, 400 and 800 p.p.m. of ONO‐8711 groups, respectively. The multiplicity of adenocarcinomas was also decreased significantly, being 3.31 ± 0.33, 2.34 ± 0.27 (P < 0.05) and 2.06 ± 0.34 (P < 0.01) with 0, 400 and 800 p.p.m. of ONO‐8711, respectively. Moreover, treatment with 800 p.p.m. ONO‐8711 reduced the mean volume of adenocarcinomas to 49% (P < 0.05) of the value for the AOM treatment alone. Furthermore, the BrdU labeling index was decreased significantly in colon cancer cells by 800 p.p.m. ONO‐8711. These results confirm that EP1 is involved in colon carcinogenesis and that EP1‐selective antagonists might be promising candidates for colon cancer chemopreventive agents. (Cancer Sci 2005; 96: 260 –264)

Design and synthesis of a highly selective EP4-receptor agonist. Part 1: 3,7-dithiaPG derivatives with high selectivity.

To identify new highly selective EP4-agonists, further modification of the 16-phenyl moiety of 1 was continued. 16-(3-methoxymethyl)phenyl derivatives 13-(6q) and 16-(3-ethoxymethyl)phenyl derivatives 13-(7e) showed more selectivity and potent agonist activity than 1. 16-(3-methyl-4-hydroxy)phenyl derivative 18-(14e) demonstrated excellent subtype selectivity, while both its receptor affinity and agonist activity were less potent than those of 13-(6q). Structure-activity relationships (SARs) are also discussed.