Mitsuko Miyamoto

TRA-418, a novel compound having both thromboxane A2 receptor antagonistic and prostaglandin I2 receptor agonistic activities: its antiplatelet effects in human and animal platelets

Summary.  TRA‐418 is a novel compound that has been found in our screening for compounds having both thromboxane A2 (TP) receptor antagonistic and prostaglandin I2 (IP) receptor agonistic activities. In the binding assays, TRA‐418 showed a 10‐fold higher affinity to TP‐receptors than IP‐receptors. TRA‐418 inhibited platelet aggregation induced by the TP‐receptor agonist, U‐46619 and by arachidonic acid at concentrations lower than those required for inhibition of ADP‐induced aggregations. Furthermore, TRA‐418 inhibited not only platelet aggregation induced by ADP alone, but also that induced by ADP in the presence of the TP‐receptor antagonist, SQ‐29548. When the IC50 values of TRA‐418 for platelet aggregation were estimated in platelet preparations from monkeys, dogs, cats, and rats using ADP and arachidonic acid as the platelet stimulating agents, it was found that the values estimated in monkey platelets were quite similar to those estimated in human platelets. In ex vivo platelet aggregation in monkeys, TRA‐418 exhibited significant inhibitory effects on arachidonic acid‐induced aggregation in platelet preparations from monkeys treated at 3 µg kg min−1 or higher doses, where neither a significant decrease in blood pressure nor a significant increase in heart rate was observed. These results are consistent with the fact that TRA‐418 has a relatively potent TP‐receptor antagonistic activity together with a relatively weak IP‐receptor agonistic activity.

Beraprost sodium improves survival rates in anti-glomerular basement membrane glomerulonephritis and 5/6 nephrectomized chronic kidney disease rats.

Beraprost sodium, a stable prostacyclin analog, was showed to improve survival rates in two different rat models, anti-glomerular basement membrane (GBM) glomerulonephritis (GN) and 5/6 nephrectomized (Nx) chronic kidney disease (CKD) rats. In the anti-GBM rat, beraprost sodium (0.2 and 0.6 mg/kg/day) improved survival rate (hazard ratio for beraprost sodium 0.6 mg/kg/day group, 0.10; 95% confidence interval, 0.01 to 0.68). Subsequently, in the 5/6 Nx CKD rat, beraprost sodium (0.6 mg/kg/day) improved survival rate (hazard ratio for beraprost sodium, 0.46; 95% confidence interval, 0.23 to 0.92), serum creatinine doubling time and the slope of the reciprocal of serum creatinine. In the anti-GBM GN rats, beraprost sodium suppressed the serum accumulation of representative uremic toxins such as indoxyl sulfate. Furthermore, beraprost sodium inhibited human aortic endothelial cell (HAEC) injury induced by indoxyl sulfate, indicating that beraprost sodium might have a protective effect against cardiovascular damage due to CKD. These results show that beraprost sodium can improve the survival rates in two rat models of anti-GBM GN and 5/6 Nx CKD rats by protecting endothelial cells and thereby ameliorating decreased renal function. Therefore, clinical studies are needed in patients with chronic kidney failure to determine whether beraprost sodium will become a useful medication in CKD.

Effects of TRA-418, a novel TP-receptor antagonist, and IP-receptor agonist, on human platelet activation and aggregation.

{4‐[2‐(1,1‐Diphenylethylsulfanyl)‐ethyl]‐3,4‐dihydro‐2H‐benzo[1,4]oxazin‐8‐yloxy}‐acetic acid N‐Methyl‐D‐glucamine salt (TRA‐418) has both thromboxane A2 (TP)‐receptor antagonist and prostacyclin (IP)‐receptor agonist properties. The present study examined the advantageous effects of TRA‐418 based on the dual activities, over an agent having either activity alone and also the difference in the effects of TRA‐418 and a glycoprotein αIIb/β3 integrin (GPIIb/IIIa) inhibitor. TRA‐418 inhibited platelet GPIIb/IIIa activation as well as P‐selectin expression induced by adenosine 5′‐diphosphate, thrombin receptor agonist peptide 1–6 (Ser‐Phe‐Leu‐Leu‐Arg‐Asn‐NH2), and U‐46619 in the presence of epinephrine (U‐46619+ epinephrine). TRA‐418 also inhibited platelet aggregation induced by those platelet‐stimulants in Ca2+ chelating anticoagulant, citrate and in nonchelating anticoagulant, D‐phenylalanyl‐L‐prolyl‐L‐arginyl‐chloromethyl ketone (PPACK). The TP‐receptor antagonist SQ‐29548 inhibited only U‐46619+epinephrine‐induced GPIIb/IIIa activation, P‐selectin expression, and platelet aggregation. The IP‐receptor agonist beraprost sodium inhibited platelet activation. Beraprost also inhibited platelet aggregation induced by platelet stimulants we tested in citrate and in PPACK. The GPIIb/IIIa inhibitor abciximab blocked GPIIb/IIIa activation and platelet aggregation. However, abciximab showed slight inhibitory effects on P‐selectin expression. TRA‐418 is more advantageous as an antiplatelet agent than TP‐receptor antagonists or IP‐receptor agonists separately used. TRA‐418 showed a different inhibitory profile from abciximab in the effects on P‐selectin expression.

Increased leukocyte aggregates are associated with atherosclerosis in patients with hemodialysis.

Little data are available on the role of blood rheology in atherosclerosis in hemodialysis (HD) patients. This study sought to assess the relationship between leukocytes conjugated with platelets (leukocyte aggregates [LA]) and atherosclerosis in patients with HD. The present study included 118 patients on HD. As surrogate markers of atherosclerosis, aortic stiffness measured by brachial‐ankle pulse wave velocity, and carotid intima‐media thickness (IMT) were measured. As an assessment of LA, a method, microchannel array flow analyzer, which makes it possible to directly observe the flow of blood cell elements through the microchannel, was used. We measured a number of LA during 50 μL flow of whole blood through microchannels. In 12 age‐matched healthy individuals, a number of LA during 50 μL flow of whole blood was 25.7±5.4, whereas in HD patients it was significantly increased up to 48.2±16.4 (P<0.001). Flow cytometry demonstrated that LA were predominantly monocytes. Leukocyte aggregates were positively associated with plasma levels of fibrinogen (P<0.01), or serum high‐sensitive C‐reactive protein (P<0.01). Moreover, LA had highly significant associations with brachial‐ankle pulse wave velocity (P<0.001) and IMT (P<0.001). In conclusion, we demonstrated hemorheologically that monocyte‐platelet conjugates play an important role in aortic stiffness and IMT in HD patients.

Effect of fatty acids on the phosphate binding of TRK-390, a novel, highly selective phosphate-binding polymer

Phosphate binders are used for the treatment of hyperphosphatemia in hemodialysis patients with chronic kidney disease. Sevelamer, a phosphate-binding polymer, has been reported to bind bile acids or fatty acids and thereby decrease its phosphate-binding capacity. The novel phosphate binder TRK-390 is a poly (allylamine) polymer that has been shown to have enhanced phosphate selectivity, with low bile-acid-binding. In this study we evaluated the effect of fatty acids on the phosphate-binding capacity of TRK-390. In the absence of fatty acids and bile acids, the phosphate-binding capacity of TRK-390 was similar to that of sevelamer. In the presence of fatty acids and bile acids, the phosphate-binding capacity of TRK-390 was reduced to 83%; in contrast, that of sevelamer was reduced to 35%. TRK-390 and sevelamer showed a similar effect in lowering urinary phosphate excretion in normal rats fed a normal diet. However, urinary phosphate excretion of rats treated with TRK-390 was reduced by about one half of that obtained with sevelamer, when given with a high-fat diet that had a fat content similar to the diet of hemodialysis patients. TRK-390 was superior in terms of phosphate selectivity in the presence of fatty acids and bile acids in vitro, and the phosphate-binding capacity of TRK-390 in vivo was shown to be less affected by fat in comparison with that of sevelamer. Thus, TRK-390 is expected to be useful as a novel highly selective phosphate binder.

TRA-418, a thromboxane A2 receptor antagonist and prostacyclin receptor agonist, inhibits platelet-leukocyte interaction in human whole blood

TRA-418, a compound with both thromboxane A2 receptor (TP receptor) antagonistic and prostacyclin receptor (IP receptor) agonistic activities, was synthesised in our laboratory as a new antithrombotic agent. In this study, we examined the effects of TRA-418 on platelet-leukocyte interactions in human whole blood. Platelet-leukocyte interactions were induced by U-46619 in the presence of epinephrine (U-46619 + epinephrine) or with thrombin receptor agonist peptide 1-6 (TRAP). Platelet-leukocyte interactions were assessed by flow cytometry, with examination of both platelet-neutrophil and platelet-monocyte complexes. In a control experiment, the TP receptor antagonist SQ-29548 significantly inhibited the induction of platelet-leukocyte complexes by the combination of U-46619 and epinephrine, but not TRAP-induced formation of platelet-leukocyte complexes. Conversely, the IP receptor agonist beraprost sodium inhibited platelet-leukocyte complex formation induced by both methods, although the IC50 values of beraprost sodium for U-46619 + epinephrine were at least 10-fold greater than for TRAP. Under such conditions, TRA-418 inhibited both U-46619 + epinephrine-induced and TRAP-induced platelet-leukocyte complex formation in a concentration-dependent manner, in a similar range. These results suggest that TRA-418 exerts its inhibitory effects on platelet-leukocyte interactions by acting as a TP receptor antagonist as well as an IP receptor agonist in an additive or synergistic manner. These inhibitory effects of TRA-418 on formation of platelet-leukocyte complexes suggest the compound is beneficial effects as an antithrombotic agent.

A prostacyclin analog prevents the regression of renal microvascular network by inhibiting mitochondria-dependent apoptosis in the kidney of rat progressive glomerulonephritis.

We have previously demonstrated that renoprotective effects of a prostacyclin analog, beraprost sodium, on the kidney of anti-glomerular basement membrane glomerulonephritis (GN) rats. The aim of this study is to address the renoprotection mechanism of beraprost sodium, especially in the terminal stage of GN. Beraprost sodium was orally administrated from 2 to 7 weeks after induction of GN, and renal function, morphology, protein and mRNA levels were analyzed. We found the beraprost sodium treatment suppressed the structural regression of renal microvascular network and decline of renal blood flow occurred in the kidney of GN rats. To address the mechanism of the structural maintenance, we focused on apoptosis because the increased number of apoptotic renal microvascular endothelial cells and tubular epithelial cells was observed in the kidneys of GN rats as compared with normal and beraprost sodium treated rats. Protein and mRNA analyses demonstrated that mitochondria-dependent apoptotic pathway was activated in the kidneys of GN rats, and beraprost sodium suppressed the activation by modulating the expression patterns of pro- and anti-apoptotic factors. These results suggest that inhibition of mitochondria-dependent apoptosis of renal cells in GN kidney and consequent maintenance of renal functional structures, including microvascular network might contribute to the renoprotective effect of beraprost sodium in GN.

Piperidine derivatives as nonprostanoid IP receptor agonists

We searched for a strong and selective nonprostanoid IP agonist bearing piperidine and benzanilide moieties. Through optimization of substituents on the benzanilide moiety, the crucial part of the agonist, 43 (2-((1-(2-(N-(4-tolyl)benzo[d][1,3]dioxole-5-carboxamido)ethyl)piperidin-4-yl)oxy)acetic acid monohydrate monohydrochloride) was discovered and exhibited strong platelet aggregation inhibition (IC50=21nM) and 100-fold selectivity for IP receptor over other PG receptors. The systemic exposure level and bioavailability after oral administration of 43 were also good in dog.

Synthesis of Novel IP Agonists via N-Aminoethyl Cyclic Amines Prepared by Decarboxylative Ring-Opening Reactions

An efficient synthesis of a highly potent and selective IP (PGI2 receptor) agonist that is not structurally analogous to PGI2 is described. This synthesis is accomplished through the following key steps: Nucleophilic ring-opening of 3-(4-chlorophenyl)-oxazolidin-2-one prepared by a one-pot procedure with 4-piperidinol and selective O-alkylation of 1-(2-(4-chlorophenylamino)ethyl)piperidin-4-ol. The obtained compound is a potent and selective IP agonist displaying a long duration of action.