SummaryONO-4641 is a next-generation sphingosine 1-phosphate (S1P) receptor agonist selective for S1P receptors 1 and 5. The objective of the study was to characterize the immunomodulatory effects of ONO-4641 using preclinical data. ONO-4641 was tested in both in-vitro pharmacological studies as well as in-vivo models of transient or relapsing-remitting experimental autoimmune encephalomyelitis (EAE). In vitro, ONO-4641 showed highly potent agonistic activities versus S1P receptors 1 and 5 [half maximal effective concentration (EC50) values of 0·0273 and 0·334 nM, respectively], and had profound S1P receptor 1 down-regulating effects on the cell membrane. ONO-4641 decreased peripheral blood lymphocyte counts in rats by inhibiting lymphocyte egress from secondary lymphoid tissues. In a rat experimental autoimmune encephalomyelitis (EAE) model, ONO-4641 suppressed the onset of disease and inhibited lymphocyte infiltration into the spinal cord in a dose-dependent manner at doses of 0·03 and 0·1 mg/kg. Furthermore, ONO-4641 prevented relapse of disease in a non-obese diabetic mouse model of relapsing-remitting EAE. These observations suggest that ONO-4641 may provide therapeutic benefits in the treatment of multiple sclerosis.

Key words: temperature-sensitive simian virus 40 large T-antigen/transgenic rat/gastric epithelial cell lines/cathepsin E/ prostaglandin E2

Temocapril is a novel ACE inhibitor that is cleared via dual excretion routes in humans. Borderline or mildly hypertensive patients with normal renal function [group 1, creatinine clearance (CLCR) > 70 ml/min (4.2 L/h), n = 12], moderate renal impairment [group 2, CLCR 30 to 70 ml/min (1.8 to 4.2 L/h), n = 12] or severe renal impairment [group 3, CLCR < 30 ml/min (1.8 L/h), n = 12] received a single oral dose of either temocapril 1 mg (n = 6, each group) or enalapril 5mg (n = 6, each group). These 2 drugs gave similar values for the area under the plasma concentration-time curve (AUC) of the active diacids. The maximum plasma concentration of enalapril diacid was increased 2- and 6-fold in moderate and severe renal impairment, respectively, whereas that of temocapril diacid was not altered. The AUC of enalapril diacid increased 13-fold at CLCR values < 30 ml/min, but that of temocapril diacid increased only 2-fold. The duration of plasma ACE inhibition due to enalapril was greatly prolonged by the impairment of renal function, whereas that due to temocapril was affected very little. Urinary recovery of temocapril diacid was decreased markedly in patients with severe renal dysfunction, most probably because the diacid was excreted through the biliary route. On the other hand, urinary recovery of enalapril diacid remained fairly high even in patients with severe renal impairment, because of extremely high plasma diacid concentrations resulting from the lack of biliary excretion. These observations suggest that temocapril is beneficial in the treatment of hypertension in patients with severely impaired renal function.

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Aim: To develop conditionally immortalized gastric mucosal cell lines that show distinct types of cell differentiation from transgenic mice harboring temperature-sensitive simian virus 40 (tsSV40) large T antigen. Methods: Gastric mucosal cells from the transgenic mice were cultured at a permissive temperature (33°C), and proliferative cells were then cloned by colony formation. Results: Eight gastric cell lines showed epithelial-like morphology and grew at 33°C. Three different types of the cell lines have been established: (1) MGE12-1, MGE3-2, and MGE509 cells expressing mRNAs for pit cell markers (gastric mucin and cathepsin E); (2) MGE02, MGE503, and MGE511 cells expressing mRNAs for pit and zymogenic (pepsinogen F) cell markers, and (3) MGE507 and MGE727 cells expressing mRNAs for pit, zymogenic, and parietal (H,K-ATPase α-subunit) cell markers. Moreover, the TaqMan assay showed that mRNA levels of mucin, H,K-ATPase α-subunit, and pepsinogen F were influenced by nonpermissive temperature (39°C) in MGE503 and MGE727 cells. Conclusion: These gastric epithelial cell lines seem to reflect different stages of development of gastric mucosal cells.

The method for the simultaneous determination of angiotensin-converting enzyme (ACE) inhibitor enalapril and its active metabolite enalaprilat in plasma and urine was developed by gas chromatography/mass spectrometry. Enalapril and enalaprilat in plasma and urine were extracted and cleaned up by using Sep-Pak C18 and silica cartridges. Derivatization was carried out using diazomethane and trifluoroacetic anhydride. Detection by selected ion monitoring was selected to m/z 288 (enalaprilat) and 302 (enalapril). The detection limit of enalapril and enalaprilat was 200 pg/mL in plasma and 2 ng/mL in urine. This method was applied to the pharmacokinetic analysis of enalapril and enalaprilat in body fluids.

Despite the usefulness of angiotensin converting enzyme (ACE; EC 3.4.15.1) inhibitors for patients with renal insufficiency, some hesitation has been exercised in applying ACE inhibitors to the treatment of such patients because most ACE inhibitors are excreted mainly into the urine. In this context, development of an ACE inhibitor which is excreted into the bile has been sought. The pharmacokinetic properties of the novel ACE inhibitor, temocapril hydrochloride (temocapril HCl; CS-622), were investigated in six healthy volunteers. This drug is excreted mainly into the bile in animal studies. Temocapril HCl was given in a single dose of 0.5, 1.0, and 2.0 mg, and 36, 44, and 38 per cent of the administered drug was excreted in the feces and 17, 19, and 24 per cent in the urine as the de-esterified active diacid form (the diacid metabolite) within 48 h, respectively. The plasma ACE activity was markedly inhibited. No abnormal clinical findings suggestive of side-effects were observed. Thus, from the pharmacokinetic standpoint, temocapril HCl is expected to be a useful drug for patients with renal dysfunction.

The discovery of 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid 13n (ceralifimod, ONO-4641), a sphingosine-1-phosphate (S1P) receptor agonist selective for S1P and S1P, is described. While it has been revealed that the modulation of the S1P receptor is an effective way to treat autoimmune diseases such as relapsing-remitting multiple sclerosis (RRMS), it was also reported that activation of the S1P receptor is implicated in some undesirable effects. We carried out a structure-activity relationship (SAR) study of hit compound 6 with an amino acid moiety in the hydrophilic head region. Following identification of a lead compound with a dihydronaphthalene central core by inducing conformational constraint, optimization of the lipophilic tail region led to the discovery of 13n as a clinical candidate that exhibited >30 000-fold selectivity for S1P over S1P and was potent in a peripheral lymphocyte lowering (PLL) test in mice (ED = 0.029 mg/kg, 24 h after oral dosing).