Identification of the Uptake Transporter Responsible for Distribution of Acotiamide into Stomach Tissue.

The acetylcholinesterase inhibitor, acotiamide, improves gastric motility and is clinically used to treat functional dyspepsia. The present study aimed to identify the transporters involved in the distribution of acotiamide in stomach tissue. Acotiamide uptake by the gastric cancer-derived model cell line, Hs746 T, was Na+- and pH-independent. The initial uptake velocity of acotiamide was saturable with increasing concentrations of acotiamide and was inhibited by selective serotonin reuptake inhibitors, which are potent inhibitors of the plasma membrane monoamine transporter (PMAT). The uptake of acotiamide by PMAT gene-transfected HEK293 cells was saturable, with similar Km (197.9 µM) values to those of uptake by Hs 746T cells (106 µM). Moreover, immunoreactivity of PMAT was found in the gastric smooth muscle and vascular endothelial cells. These results suggest that PMAT contributes to the distribution of acotiamide in the stomach, where it exerts its pharmacological effects.

Pharmacokinetics and Pharmacodynamics of Azeloprazole Sodium, a Novel Proton Pump Inhibitor, in Healthy Japanese Volunteers.

The pharmacokinetics (PK) and pharmacodynamics (PD) of proton pump inhibitors differ among cytochrome P450 (CYP) 2C19 genotypes. Therefore, we developed azeloprazole sodium (Z-215), a novel proton pump inhibitor, whose metabolism is not affected by CYP2C19 activity in vitro. However, the PK and PD of azeloprazole sodium have not been evaluated in Japanese subjects. We conducted an open-label, crossover study in healthy Japanese male volunteers to evaluate the plasma concentration and intragastric pH with respect to CYP2C19 genotype after repeated administration of 10, 20, and 40 mg azeloprazole sodium and 10 and 20 mg rabeprazole sodium (rabeprazole). The plasma concentration profile of azeloprazole sodium was similar among genotypes, whereas that of rabeprazole differed. The 24-hour intragastric pH ≥ 4 holding time ratio (pH ≥ 4 HTR) of azeloprazole sodium was similar among genotypes. The pH ≥ 4 HTR was 52.5%-60.3%, 55.1%-65.8%, and 69.4%-77.1% after administration of 10, 20, and 40 mg azeloprazole sodium, respectively, and 59.2%-72.3% and 64.4%-91.2% after administration of 10 and 20 mg rabeprazole, respectively, on the fifth day of dosing. The maximum plasma concentration (Cmax ), area under the plasma concentration-time curve (AUC), and pH ≥ 4 HTR of azeloprazole sodium were proportional to dose. The Cmax , AUC, and pH ≥ 4 HTR on day 5 were slightly higher following administration of 20 mg azeloprazole sodium before comparison with after a meal. No serious adverse events were observed. These results suggest that azeloprazole sodium is useful for treating gastroesophageal reflux disease in all CYP2C19 genotypes.

Mass balance and metabolism of Z-215, a novel proton pump inhibitor, in healthy volunteers.

The human mass balance of [14 C]Z-215, a novel proton pump inhibitor, was characterised in six healthy male volunteers following single oral administration of [14 C]Z-215 (20 mg, 3.7 MBq) to determine the elimination pathway of Z-215 and the distribution of its metabolites in plasma, urine, and faeces (NCT02618629). [14 C]Z-215 was rapidly absorbed, with a Cmax of 434 ng/mL at 0.38 h for Z-215 and 732 ng eq./mL at 0.5 h for total radioactivity. Means of 59.61% and 31.36% of the administered radioactive dose were excreted in urine and faeces, respectively, within 168 h post-dose. The majority of the dose was recovered within 24 h in urine and 96 h in faeces. Unchanged Z-215 was excreted in urine at trace levels but was not detected in faeces. The main components in plasma were Z-215 and Z-215 sulphone, accounting for 29.8% and 13.3% of the total circulating radioactivity, respectively. Additionally, Z-215 was metabolised through oxidation, reduction and conjugation. Our in vitro Z-215 metabolism study showed that the major isozyme contributing to the oxidation of Z-215, including the formation of Z-215 sulphone, was CYP3A4. In conclusion, Z-215 is well absorbed in humans and primarily eliminated via metabolism, where CYP3A4 plays an important role.

Physiologically-Based Pharmacokinetic and Pharmacodynamic Modeling for the Inhibition of Acetylcholinesterase by Acotiamide, A Novel Gastroprokinetic Agent for the Treatment of Functional Dyspepsia, in Rat Stomach.

PURPOSE: Acotiamide, a gastroprokinetic agent used to treat functional dyspepsia, is transported to at least two compartments in rat stomach. However, the role of these stomach compartments in pharmacokinetics and pharmacodynamics of acotiamide remains unclear. Thus, the purpose of this study was to elucidate the relationship of the blood and stomach concentration of acotiamide with its inhibitory effect on acetylcholinesterase (AChE). METHODS: Concentration profiles of acotiamide and acetylcholine (ACh) were determined after intravenous administration to rats and analyzed by physiologically-based pharmacokinetic and pharmacodynamic (PBPK/PD) model containing vascular space, precursor pool and deep pool of stomach. RESULTS: Acotiamide was eliminated from the blood and stomach in a biexponential manner. Our PBPK/PD model estimated that acotiamide concentration in the precursor pool exceeded 2 µM at approximately 2 h after administration. Acotiamide inhibited AChE activity in vitro with a 50% inhibitory concentration of 1.79 µM. ACh reached the maximum concentration at 2 h after administration. CONCLUSIONS: Our PBPK model well described the profile of acotiamide and ACh concentration in the stomach in the assumption that acotiamide was distributed by carrier mediated process and inhibited AChE in the precursor pool of stomach. Thus, Acotiamide in the precursor pool plays an important role for producing the pharmacological action.

Distribution of acotiamide, an orally active acetylcholinesterase inhibitor, into the myenteric plexus of rat and dog stomachs.

AIMS: Acotiamide is the first-in-class drug for the treatment of functional dyspepsia. Although pharmacological and therapeutic actions of acotiamide are thought to be derived from its inhibitory effects on acetylcholinesterase (AChE), whether the concentration of acotiamide at the site of action is sufficient to inhibit AChE remains unclear. Since major site of acotiamide action is thought to be the cholinergic nerve terminals in gastric myenteric plexus, we studied the distribution of [(14)C]acotiamide into gastric myenteric plexus. MAIN METHODS: Distribution of [(14)C]acotiamide was evaluated using macro- and micro-autoradiography in rats and dogs. KEY FINDINGS: The results of macro-autoradiography showed the concentration of radioactivity was 27.9µM in rat stomach, which was 12 times higher than IC50 of acotiamide for rat AChE. Being different from rats, the distribution of radioactivity in the muscular layer was distinguishable from that in the mucosal layer in dog stomach. The concentration of radioactivity in the muscular layer of dog stomach (1.41µM) was approximately two-times lower than those in the mucosal layer, however, it was approximately 1.2 times higher than IC50 of acotiamide for dog AChE. The results of micro-autoradiography also showed the radioactivity distributed homogenously in the muscular layer of rat stomach, suggesting the concentration of radioactivity around the ganglion of myenteric plexus is similar to that in the muscular layer of stomach. SIGNIFICANCE: These findings suggest acotiamide distributes to the myenteric plexus of stomach, a putative site of acotiamide action, with adequate concentrations to inhibit AChE, in both of rat and dog stomachs.

Mechanism for distribution of acotiamide, a novel gastroprokinetic agent for the treatment of functional dyspepsia, in rat stomach.

The novel gastroprokinetic agent acotiamide improves gastric motility by inhibiting acetylcholinesterase activity in stomach; however, the mechanism of distribution of acotiamide from blood to stomach has not been clarified. Here, the tissue distribution of acotiamide was investigated in rats. The tissue-to-plasma concentration ratio (K(p,app,in vivo)) for stomach decreased from 4.1 to 2.4 mL/g of tissue at steady state with increasing plasma concentrations, whereas the K(p,app,in vivo) for skeletal muscle was much lower and constant, regardless of the concentration of acotiamide in plasma. In vitro binding to stomach tissue protein exhibited a linear profile, with a predicted K(p,app,in vitro) of 2.2 from free fractions under linear conditions. Therefore, protein binding to stomach tissue might only play a limited role in the stomach distribution of acotiamide. The influx permeability (f (u,b) × PS(inf,app)) in the stomach exhibited dose-dependent saturation at the lowest range of examined blood unbound concentrations of acotiamide, whereas that in skeletal muscle exhibited only minimal dose dependence. In addition, the unbound concentration ratio of stomach to plasma (2.8) at steady state was markedly higher than unity. Taken together, these results suggest that carrier-mediated concentrative uptake processes play an important role in the distribution of acotiamide to the stomach but not skeletal muscle.

Acotiamide hydrochloride (Z-338) enhances gastric motility and emptying by inhibiting acetylcholinesterase activity in rats.

In clinical trials, acotiamide hydrochloride (acotiamide: Z-338) has been reported to be useful in the treatment of functional dyspepsia. Here, we investigated the effects of acotiamide on gastric contraction and emptying activities in rats in comparison with itopride hydrochloride (itopride) and mosapride citrate (mosapride). We also examined in vitro the compound's inhibitory effect on acetylcholinesterase (AChE) activity derived from rat stomach. In in vivo studies, acotiamide (30 and 100mg/kg s.c.) and itopride (100mg/kg s.c.) markedly enhanced normal gastric antral motility in rats. In gastric motility dysfunction models, acotiamide (100mg/kg s.c.) and itopride (100mg/kg s.c.) improved both gastric antral hypomotility and the delayed gastric emptying induced by clonidine, an α(2)-adrenoceptor agonist. In contrast, mosapride (10mg/kg s.c.) had no effect on these models. Like the AChE inhibitors itopride (30 mg/kg s.c.) and neostigmine (10 µg/kg s.c.), acotiamide (10mg/kg s.c.) also clearly enhanced gastric body contractions induced by electrical stimulation of the vagus, which were abolished by atropine and hexamethonium, whereas mosapride (3 and 10mg/kg s.c.) did not. In in vitro studies, acotiamide concentration-dependently inhibited rat stomach-derived AChE activity (IC(50)=2.3 µmol/l). In addition, stomach tissue concentrations of acotiamide after administration at 10mg/kg s.c. were sufficient to produce inhibition of AChE activity in rat stomach. These results suggest that acotiamide stimulates gastric motility and improves gastric motility dysfunction in rats by inhibiting AChE activity, and may suggest a role for acotiamide in improving gastric motility dysfunction in patients with functional dyspepsia.

Concentration dependence of 5-aminosalicylic acid pharmacological actions in intestinal mucosa after oral administration of a pH-dependent formulation.

Asacol, a medication that delivers delayed release 5-aminosalicylic acid (5-ASA), is a useful therapeutic agent for inflammatory bowel disease (IBD), but the relationship between its pharmacological actions and intestinal concentrations has not been studied in detail. Therefore, our aim was to assess 5-ASA's pharmacological actions as a function of its concentration at its target site. We first evaluated 5-ASA's release profiles in vitro by the paddle method and found that Asacol starts to release 5-ASA at pH ≥ 7. Orally administered Asacol pharmacokinetic parameters were evaluated in dogs. Asacol's T(max) was much longer than that of the time-dependent release 5-ASA formulation. We also determined 5-ASA's distribution in the intestinal mucosa and found that it is effectively delivered there by Asacol. These results indicated that Asacol released 5-ASA in a pH-dependent manner, resulting in efficient delivery to the large intestine. We also compared the mucosal 5-ASA concentrations with the IC(50) values for scavenging free radicals or suppressing LTB(4) production. The 5-ASA concentration in the large intestine was higher than IC(50) values necessary to suppress inflammatory processes. We also report the release characteristics of Asacol and the targeted delivery of 5-ASA to affected sites in IBD patients.

Mechanism of the regulation of organic cation/carnitine transporter 1 (SLC22A4) by rheumatoid arthritis-associated transcriptional factor RUNX1 and inflammatory cytokines.

Recently, it was reported that the organic cation/carnitine transporter 1 (OCTN1, SLC22A4) is associated with chronic inflammatory diseases, such as rheumatoid arthritis (RA) and Crohn's disease. OCTN1 in humans is expressed in synovial tissues of individuals with rheumatoid arthritis. Furthermore octn1 in mice is expressed in inflamed joints with collagen-induced arthritis, a model of human arthritis, but not in the joints of normal mice. OCTN1 should be involved in the inflammatory disease and in the present study, the regulatory mechanism of OCTN1 expression was characterized using the human fibroblast-like synoviocyte cell line MH7A, derived from RA patients. A luciferase-reporter gene assay and gel shift assay demonstrated that RUNX1, which is an essential hematopoietic transcription factor associated with acute myeloid leukemia and is related to RA and Sp1, is involved in the regulation of OCTN1 promoter activity. Inflammatory cytokines such as interleukin-1beta and tumor necrosis factor-alpha increased the expression of OCTN1 mRNA. Furthermore, overexpression of nuclear factor-kappaB (NF-kappaB) activated promoter activity of OCTN1. These results clearly demonstrate that expression of OCTN1 is regulated by various factors, including RUNX1, inflammatory cytokines, and NF-kappaB, all of which are also related to the pathogenesis of RA. Further studies on the physiological substrate(s) of OCTN1 should be done to clarify the roles of OCTN1 in these diseases.

Characterization of human OATP2B1 (SLCO2B1) gene promoter regulation.

PURPOSE: We investigated transcriptional regulation of organic anion transporter OATP2B1 (SLCO2B1) that is expressed in multiple tissues such as liver, small intestine, and others and compared it with that of liver-specific OATPs. METHODS: The promoter activity was examined by luciferase assay. Specific bindings of transcription factors to the promoter region were examined by gel mobility shift assay using native and mutated nucleotides of the promoter region of OATP2B1. RESULTS: Deletion-mutation study of the promoter region of OATP2B1 showed that the -59 region that included the Sp1 binding site had basal promoter activity, whereas promoter activities of the further upper region were different between intestine-derived Caco-2 cells and liver-derived HepG2 cells. The association of Sp1 to the promoter region was confirmed by gel shift assay and overexpression of Sp1 in cultured cells. Although the promoter of OATP2B1 has a putative HNF1alpha binding site, overexpression of HNF1alpha did not induce the expression of OATP2B1. CONCLUSION: Sp1, a transcription factor, was required for constitutive expression of OATP2B1 in liver and small intestine, whereas HNF1alpha, which is involved in the expression of liver-specific OATPs, did not seem to play a role in OATP2B1 expression. Accordingly, it was suggested that the tissue expression profile of OATP2B1 was different from that of other liver-specific OATPs.

Involvement of uric acid transporter in increased renal clearance of the xanthine oxidase inhibitor oxypurinol induced by a uricosuric agent, benzbromarone.

Benzbromarone has been reported to increase the renal clearance of oxypurinol, an active metabolite of allopurinol. We examined the renal transport of oxypurinol to determine whether such a change in renal clearance could be explained by altered transporter-mediated reabsorption. Since the first step of reabsorption takes place at the renal epithelial apical membrane, we focused on membrane transporters. Benzbromarone is an inhibitor of reabsorption of uric acid mediated by the uric acid transporter (URAT) URAT1 (SLC22A12), which is expressed at the apical membrane of proximal tubular cells in humans. Uptake of oxypurinol by Xenopus oocytes injected with complementary RNA of URAT1 was significantly higher than that by water-injected oocytes, and the uptake was saturable, with a K(m) of about 800 microM. Moreover, benzbromarone inhibited the oxypurinol uptake by URAT1 at concentrations as low as 0.01 microM. The uptake of oxypurinol by another organic anion transporter (OAT), OAT4 (SLC22A11), which is also expressed at the apical membrane of proximal tubular epithelial cells, was negligible, whereas the uptake of [3H]estrone-3-sulfate by OAT4 was significantly inhibited by oxypurinol. Furthermore, neither the transport activity of organic cation/carnitine transporter (OCTN) 1 nor OCTN2 was affected by oxypurinol or benzbromarone. These results indicate that URAT1 is involved in renal reabsorption of oxypurinol, and the increment of renal clearance of oxypurinol upon concomitant administration of benzbromarone could be due to drug interaction at URAT1.

Regulation of testis-specific carnitine transporter (octn3) gene by proximal cis-acting elements Sp1 in mice.

The mouse octn transporter family consists of three genes, octn1, octn2 and octn3. The gene products octn2 and octn3, which transport carnitine with high affinity, are both expressed in testis, where carnitine is required to maintain sperm cell motility. Here, we focused on the regulatory mechanism of the expression of octn3 in an attempt to determine whether the differential tissue expression profiles of the octn2 and octn3 genes reflect distinct physiological roles of octn2 and octn3. The promoter activity of the octn3 gene was examined by luciferase assay and gel mobility shift assay using the mouse Sertoli cell line TM4 as host cells. Deletion-mutant assay demonstrated that a gene segment of the 5'-untranslated region located at about -500 bp relative to the transcription start site is required for constitutive octn3 transcription. Deletion of the Sp1-binding site within the region resulted in loss of transcriptional activity. In addition, overexpression of Sp1 in TM4 cells led to a further increase of transcription of octn3. These results demonstrated that Sp1-binding sites are necessary and sufficient for constitutive octn3 gene transcription. Furthermore, the expressions of both of octn2 and octn3 genes in TM4 cells were up-regulated by palmitic acid, whereas carnitine increased only the expression of octn2 without any change in octn3 expression. Accordingly, the expressions of octn2 and 3 are regulated by distinct mechanisms, suggesting distinct roles of octn2 and octn3 in carnitine transport.