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 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.

Direct binding of ligandin to uridine 5'-diphosphate glucuronosyltransferase 1A1.

AIM: Bilirubin, a final degradation product of heme produced mainly in the spleen, is carried to the liver through its binding to albumin in the blood circulation. After its transport to hepatocytes, ligandin (glutathione S-transferase; GST) carries bilirubin to the endoplasmic reticulum (ER). uridine 5'-diphosphate-glucuronosyltransferase 1A1 (UGT1A1) glucuronidates bilirubin for solubilization in the ER. METHODS: By GST pull-down and co-immunoprecipitation assays, GSTA2, a member of the alpha-class of GST, was observed to directly bind to UGT1A1 through the region present inside the ER. RESULTS: GSTA2 was detected in the microsomal fraction together with the cytosolic fraction after hepatocyte fractionation. CONCLUSION: These results strongly suggest that bilirubin is directly delivered to UGT1A1 from ligandin for glucuronidation.

UDP-glucuronosyltransferase1A1 directly binds to albumin.

UDP-glucuronosyltransferase1A1 (UGT1A1) catalyses glucuronidation of bilirubin (the final break down product of heme which is produced mainly in the spleen and liver) and is located on the lumen of the endoplasmic reticulum (ER). To identify partner UGTs that form hetero-oligomers with UGT1A1, or other proteins that bind directly to UGT1A1, yeast two-hybrid screening was performed using UGT1A1 as bait. From these studies, cDNA clones specific for human serum albumin (HSA) were unexpectedly isolated. The direct interaction between UGT1A1 and albumin was confirmed in vitro by a pull-down assay. FITC-albumin uptake into HepG2 and Huh7 cells was observed only when bilirubin are present in the culture medium. Furthermore, the endocytosis inhibitor phenylarsine oxide (PAO) prevented albumin uptake into the cells, suggesting that the albumin/bilirubin complex is internalized through receptor-mediated endocytosis. From these studies, it would appear that production of large amounts of toxic bilirubin might use different uptake pathways for entry into hepatocytes.