Development of potent non-acylhydrazone inhibitors of intestinal sodium-dependent phosphate transport protein 2b (NaPi2b).

Inhibition of intestinal sodium-dependent phosphate transport protein 2b (NaPi2b), responsible for intestinal phosphate absorption, is considered to reduce serum phosphate levels, making it a promising therapeutic approach for hyperphosphatemia. Previously, we aimed to identify new drugs for hyperphosphatemia treatment and obtained zwitterionic compound 3 (IC50 = 64 nM) as a potent selective inhibitor of intestinal NaPi2b. This small-molecule compound is gut-restricted owing to its almost membrane-impermeable property. However, when compound 3, containing an acylhydrazone structure, is exposed to plasma, it is easily metabolized and likely produces an acetylhydrazine compound. Clinical studies have shown that acetylhydrazine is a risk factor for hepatic toxicity owing to its microsomal metabolism, wherein toxic reactive intermediates are formed. Therefore, in this study, we aimed to obtain potent NaPi2b inhibitors without an acylhydrazone structure to reduce the risk of hepatic toxicity. We developed compound 18, an anilide compound with zwitterionic property having potent phosphate uptake inhibitory activity in vitro (IC50 = 14 nM) and low bioavailability (FaFg = 5.9%). Oral administration of compound 18 in rats showed a reduction in phosphate absorption comparable to that observed with lanthanum carbonate, a clinically effective phosphate binder used in hyperphosphatemia treatment. Moreover, combined administration of compound 18 and lanthanum carbonate resulted in an additive effect on phosphate absorption inhibition in rats. Our findings suggest that combination therapy with lanthanum carbonate and compound 18 will not only provide better treatment outcomes for hyperphosphatemia but also reduce gastrointestinal side effects in patients.

Discovery and mechanistic study of thiazole-4-acylsulfonamide derivatives as potent and orally active ChemR23 inhibitors with a long-acting effect in cynomolgus monkeys.

Plasmacytoid dendritic cells (pDCs) are a subset of dendritic cells that can secrete large amounts of type I interferon. ChemR23, a G protein-coupled receptor (GPCR) expressed on the surface of pDCs, contributes to the recruitment of pDCs to inflamed tissues through chemotaxis signaling, and is therefore considered an attractive target for the treatment of autoimmune diseases. We previously reported benzoxazole-based compounds that can inhibit ChemR23 signaling through receptor internalization. Although these compounds showed ChemR23 internalization on pDCs in cynomolgus monkeys after oral administration, further improvement of the pharmacokinetics profile was needed for a clinical candidate and we therefore attempted scaffold-hopping from the benzoxazole core structure leading to novel thiazole derivatives. In this report, the design, synthesis, and biological evaluation of new thiazole-based ChemR23 inhibitors were described. Through sequential structure-activity relationship studies regarding (i) the side chain of the N-acylsulfonamide moiety, (ii) the 5-position of the thiazole ring, and (iii) the 1,2,4-oxadiazol-5-one moiety, we have succeeded in finding a potent thiazole-based ChemR23 inhibitor, 14f (IC80 = 12 nM). In addition, the oral administration of 14f at 30 mg/kg to cynomolgus monkeys demonstrated a sustained pharmacological effect of ChemR23 internalization on pDCs until 8 h after dosing, which was considered a longer effect in comparison to previously reported 2-aminobenzoxazole-based ChemR23 inhibitors. This report also shows the synthesis and evaluation of fluorescein-labeled compound 45c for a mechanistic study, and we could confirm the direct binding of our thiazole derivative to ChemR23. We believe that our research on small molecule ChemR23 inhibitors and chemical probe will contribute to the elucidation and analysis of the functions of ChemR23 as well as identifying novel therapeutics for autoimmune diseases.

Discovery of Gut-Restricted Small-Molecule Inhibitors of Intestinal Sodium-Dependent Phosphate Transport Protein 2b (NaPi2b) for the Treatment of Hyperphosphatemia.

NaPi2b is primarily expressed in the small intestine, lungs, and testes and plays an important role in phosphate homeostasis. The inhibition of NaPi2b, responsible for intestinal phosphate absorption, is considered to reduce serum phosphate levels, making it a promising therapeutic approach for hyperphosphatemia. Using a novel phosphate uptake inhibitor 3 (IC50 = 87 nM), identified from an in-house compound collection in human NaPi2b-transfected cells as a prototype compound, we conducted its derivatization based on a Ro5-deviated strategy to develop orally administrable small-molecule NaPi2b inhibitors with nonsystemic exposure. Consequently, compound 15, a zwitterionic compound with a potent in vitro phosphate uptake inhibitory activity (IC50 = 64 nM) and a low membrane permeability (Pe < 0.025 × 10-6 cm/s), was developed. Compound 15 showed a low bioavailability (F = 0.1%) in rats and a reduction in phosphate absorption in the rat intestinal loop assay comparable to sevelamer hydrochloride, a clinically effective phosphate binder for treating hyperphosphatemia.

The design, synthesis and evaluation of 2-aminobenzoxazole analogues as potent and orally efficacious ChemR23 inhibitors.

We previously reported 2-aminobenzoxazole analogue 1 as a potent ChemR23 inhibitor. The compound showed inhibitory activity against chemerin-induced calcium signaling through ChemR23 internalization in CAL-1 cells, which are cell lines of plasmacytoid dendric cells (pDCs). Furthermore, compound 2 inhibited chemotaxis of CAL-1 triggered by chemerin in vitro. However, we noted a difference in the ChemR23 response to our inhibitor between rodents and non-rodents in a previous study. To address this issue, we performed optimization of ChemR23 inhibitors using CAL-1 cells endogenously expressing human ChemR23 and conducted a pharmacokinetics study in cynomolgus monkeys. Various substituents at the 4-position of the benzoxazole ring exhibited potent in vitro bioactivity, while those at the 6-position were not tolerated. Among substituents, a carboxyl group was identified as key for improving the oral bioavailability in cynomolgus monkeys. Compound 38a with the acidic part changed from a tetrazole group to a 1,2,4-oxadiazol-5-one group to improve bioactivity and pharmacokinetic parameters exhibited inhibitory activity against chemerin-induced chemotaxis in vitro. In addition, we confirmed the ChemR23 internalization of pDCs by compound 38a orally administered to cynomolgus monkeys. These 2-aminobenzoxazole-based ChemR23 inhibitors may be useful as novel immunotherapeutic agents capable of suppressing the migration of pDCs, which are known to be major producers of type I interferons in the lesion area of certain autoimmune diseases, such as systemic lupus erythematosus and psoriasis.

Involvement of the active metabolites in the inhibitory activity of K579 on rat plasma dipeptidyl peptidase IV.

K579 ((S)-1-[4-methyl-1-(2-pyrimidinyl)-4-piperidylamino]acetyl-2-pyrrolidinecarbonitrile), which is a long-acting and a slow binding dipeptidyl peptidase IV inhibitor, preserved the endogenously secreted active forms of glucagon-like peptide-1, augmented the insulin response and ameliorated the glucose excursion during oral glucose tolerance test in rats. In this study, we measured plasma concentrations of K579 after oral administration to rats. However, K579 was eliminated rapidly from plasma after oral administration to rats. Therefore, we postulated that there are active metabolites of K579 in rat plasma. We investigated the effect of K579 on plasma dipeptidyl peptidase IV activity using bile duct-cannulated rats. The duration of inhibitory action of plasma dipeptidyl peptidase IV after the administration of K579 in bile duct-cannulated rats was shorter than that in sham-operated rats. Moreover, we investigated the effect of bile obtained from K579-treated rat on plasma dipeptidyl peptidase IV activity in normal rats. The bile collected from K579-treated rats exhibited tardive and potent inhibitory activity of normal rat plasma. These results suggest that K579 sustained the duration of inhibitory action of plasma dipeptidyl peptidase IV by the character as a slow-binding inhibitor and, as well, by the presence of metabolites of K579, which exhibit the inhibitory activity of dipeptidyl peptidase IV.

Sex-dependent pharmacokinetics of S(-)-hydroxyhexamide, a pharmacologically active metabolite of acetohexamide, in rats.

The pharmacokinetic profile of S(-)-hydroxyhexamide (S-HH), a pharmacologically active metabolite of acetohexamide, was examined in male and female rats. S-HH was eliminated more rapidly from plasma in the males than in the females. A significant sex difference was observed in the pharmacokinetic parameters of S-HH in rats. Testectomy caused significant alteration in these parameters of S-HH in male rats, whereas ovariectomy did not in the females. The co-administration of sulfamethazine significantly decreased the plasma clearance (CL(p)) of S-HH in male rats, but had no effect in the females. The plasma concentrations of acetohexamide generated from S-HH showed no sex-related difference. Furthermore, there was no difference in the accumulation of S-HH by renal cortical slices from male and female rats. We propose the possibility that the sex-dependent pharmacokinetics of S-HH in rats is mediated through the male-specific hydroxylation of the cyclohexyl ring catalyzed by a major cytochrome p450 (CYP) isoform (CYP2C11), although the detailed mechanism remains to be elucidated.