Mizagliflozin, a novel selective SGLT1 inhibitor, exhibits potential in the amelioration of chronic constipation.

Chronic constipation is a highly common functional gastrointestinal disorder that adversely affects patient quality of life. At present, limited therapeutic options are available for the treatment of chronic constipation, which indicates the need for new therapeutic agents. Herein, we report the potential of mizagliflozin, a novel selective sodium glucose co-transporter 1 (SGLT1) inhibitor, for the amelioration of chronic constipation. Mizagliflozin's inhibitory activity against SGLTs was evaluated by an in vitro assay of cells transiently expressing SGLTs. The safety profile of an initial single dose (2-160mg, orally) and multiple doses (2-20mg, orally, once daily immediately prior to breakfast on Days 1 and 13, and three times daily immediately prior to every meal on Days 3-12) of mizagliflozin was determined by performing a phase I study in healthy male subjects. In addition, the effect of mizagliflozin and lubiprostone on fecal wet weight was compared using a dog model of loperamide-induced constipation and rat model of low-fiber-diet-induced constipation. Mizagliflozin potently inhibited human SGLT1 in a highly selective manner. The results of the phase I study showed mizagliflozin increased stool frequency and loosened stool consistency; these effects increased progressively with an increase in the dosage and the number of doses of mizagliflozin. In addition, the oral administration of mizagliflozin increased fecal wet weight in a dog model of loperamide-induced constipation and a rat model of low-fiber-diet-induced constipation, similar to lubiprostone. These results suggest the potential use of a novel selective SGLT1 inhibitor, mizagliflozin, for the amelioration of chronic constipation.

Cloning of the dog bile salt export pump (BSEP; ABCB11) and functional comparison with the human and rat proteins.

The dog bile salt export pump (BSEP; ABCB11) was cloned and expressed in a Sf9 insect cell system. The deduced amino acid sequence encodes a 1325-amino-acid protein, which shows 89.4% and 80.2% homology with human BSEP and rat Bsep, respectively. The transcript of the dog Bsep gene was detected at a high level in liver, but not other tissues, by quantitative RT-PCR. The BSEP-expressing membrane vesicles isolated from Sf9 cells exhibited saturable uptake of [(3)H]taurocholic acid with Michaelis constants (K(m)) of 33.7, 22.2 and 19.9 microM for the dog, rat and human transporters, respectively. The uptake of [(3)H]taurocholic acid by all three transporters was significantly inhibited by troglitazone, glibenclamide, and other several inhibitors, while pravastatin inhibited dog Bsep and human BSEP, but not rat Bsep at 100 microM. The IC(50) of troglitazone for dog Bsep, human BSEP, and rat Bsep were 32, 20, and 60 microM, and those of pravastatin were 441, 240 and >1,000 microM, respectively. In conclusion, while dog Bsep shows similar ATP-dependent bile acid transport characteristics to human BSEP and rat Bsep, there is a species difference in affinity for drugs such as pravastatin and troglitazone.

Molecular cloning and functional characterization of cynomolgus monkey multidrug resistance-associated protein 2 (MRP2).

The monkey is an important experimental model in the pharmacological evaluation of new drugs. We isolated monkey multidrug resistance-associated protein 2 (MRP2) cDNA to examine expression profiles among various tissues and measured ATPase activity to assess substrate specificity. The amino acid sequence encoded by monkey MRP2 cDNA was very similar (96% identity) to the reported human MRP2 cDNA (GenBank accession no. NM_000392). The tissue distribution of MRP2 in monkeys was partially different from that in humans. We found relatively high expression of MRP2 in the monkey kidney and small intestine using Northern blotting. Substrate specificity was compared between human and monkey MRP2. The affinity of 17beta-estradiol 17-(beta-d-glucuronide), methotrexate, vinblastine, and probenecid to monkey MRP2 was higher than that to human MRP2. Functional and expression differences between human and monkey MRP2 should be incorporated into the evaluation of candidate drugs.

BCRP/ABCG2 levels account for the resistance to topoisomerase I inhibitors and reversal effects by gefitinib in non-small cell lung cancer.

PURPOSE: Breast cancer resistance protein (BCRP) confers resistance against topoisomerase I inhibitors in cancer cells. Very recently, we reported that gefitinib reverses BCRP-mediated drug resistance by direct inhibition. However, it remains undetermined how much BCRP contributes to the resistance to topoisomerase I inhibitors in non-small cell lung cancer (NSCLC). The present study was designed to examine whether BCRP levels in NSCLC cells are correlated with the resistance to topoisomerase I inhibitors and the reversal effect by gefitinib. METHODS: BCRP levels and its function were evaluated by Western blotting and flowcytometry, respectively. Gefitinib-insensitive NSCLC cells expressed various levels of BCRP, which were closely correlated not only with the IC50 values of SN-38 (r=0.874, P<0.05) and those of topotecan (r=0.968, P<0.001), but also with the reversal effects of 1 microM gefitinib on SN-38 resistance (r=0.956, P<0.001) and topotecan resistance (r=0.977, P=0.0001). RESULTS: BCRP levels accounted for between 80 and 90% of the variation in the resistance to topoisomerase I inhibitors and the reversal effects by gefitinib. Also, gefitinib increased intracellular topotecan accumulation in proportion to the BCRP levels. CONCLUSIONS: These findings suggest that BCRP is the most important molecule responsible for topoisomerase I inhibitor resistance, and that the development of BCRP inhibitors is an effective approach for overcoming this resistance. In addition, the examination of BCRP levels in NSCLC tissues may identify an optimal patient population for treatment with topoisomerase I inhibitors alone or in combination with BCRP inhibitors.

Gefitinib, an EGFR tyrosine kinase inhibitor, directly inhibits the function of P-glycoprotein in multidrug resistant cancer cells.

Gefitinib (Iressa) is a selective epidermal growth factor receptor tyrosine kinase inhibitor and is used for the treatment of lung cancer. Recently, we discovered that it inhibits the breast cancer resistance protein, which is an ATP-binding cassette transporter. P-glycoprotein (Pgp) also pumps multiple types of drugs out of the cell using energy generated from ATP, and confers multidrug resistance on cancer cells. This study was designed to examine whether gefitinib inhibits the function of Pgp. We used multidrug resistant PC-6/PTX lung cancer and MCF-7/Adr breast cancer cells which overexpress Pgp and measured their drug sensitivity and drug-efflux function by tetrazolium assay and flowcytometry, respectively. In addition, the drug-stimulated ATPase activity of Pgp was measured using insect membranes that express human Pgp. Epidermal growth factor receptor was expressed in MCF-7/Adr, but not in PC-6/PTX cells, and the overexpression of Pgp did not confer resistance to gefitinib to both cell types. However, clinically achievable levels of gefitinib moderately reversed the Pgp-mediated resistance to paclitaxel and docetaxel in Pgp overexpressing cells. In addition, gefitinib increased the intracellular accumulation of the Pgp substrate rhodamine-123 in resistant cells, and activated ATPase in a preparation of pure Pgp-expressing membrane. These findings suggest that gefitinib directly interacts with Pgp and inhibits its function. Gefitinib may clinically inhibit the excretion of Pgp substrate drugs including anticancer agents, and its drug-interaction should therefore be considered.

Role of radial fibers in controlling the onset of myelination.

Recent in vitro study showed that astrocytes induce oligodendrocyte processes to adhere to axons. However, the role of astrocytes in myelination in vivo remains unknown. We have, therefore, conducted a study to clarify the possible involvement of astrocytes during the initial myelination process. In newborn mice, the expression of glial fibrillary acidic protein (GFAP), a marker for astrocytes, was restricted to a few fibrous architectures in the subventricular zone (SVZ), but we did not observe any GFAP-positive astrocytes. Prior to the onset of myelination, GFAP became transiently expressed in the cells with radial fibers elongating from the SVZ to the pia of cerebral cortex, and myelin-associated glycoprotein (MAG)-positive premyelinating oligodendrocytes appeared as neighbors to them, with the processes attaching to radial fibers, but not to axons. These GFAP-positive "radial" cells lost their fibrous architecture and became typical GFAP-positive astrocytes at about 10 days postnatally, when myelination set in, indicating that the disappearance of radial fibers coordinates with the initiation of myelination. From these results, we propose that premyelinating oligodendrocytes are in contact with radial fibers rather than axons and that the cytoarchitectural transformation of radial fibers into astrocytes is involved substantially in controlling the onset of initial myelination. Our proposal was further confirmed by GFAP-deficient mice, in which the disappearance of these radial fibers and the initiation of myelination were delayed in parallel. Our findings together suggest that myelination in vivo is in concert with astrocytic differentiation, involving radial fibers therein, rather than being a mere axon-oligodendrocyte interaction.

Protective role of phosphorylation in turnover of glial fibrillary acidic protein in mice.

Glial fibrillary acidic protein (GFAP), the principal intermediate filament (IF) protein of mature astrocytes in the CNS, plays specific roles in astrocyte functions. GFAP has multiple phosphorylation sites at its N-terminal head domain. To examine the role of phosphorylation at these sites, we generated a series of substitution mutant mice in which phosphorylation sites (Ser/Thr) were replaced by Ala, in different combinations. Gfap(hm3/hm3) mice carrying substitutions at all five phosphorylation sites showed extensive decrease in both filament formation and amounts of GFAP. Gfap(hm1/hm1) and Gfap(hm2/hm2) mice, which carry substitutions at three of five sites and in different combinations, showed differential phenotypes. Although Gfap(hm3/hm3) mice retained GFAP filaments in Bergmann glia in the cerebellum, the (Gfap(hm3/hm3):Vim(-/-)) mice lacked GFAP filaments. Pulse-chase experiments of cultured astrocytes indicated that the Hm3-GFAP encoded by Gfap(hm3) was unstable particularly in the absence of vimentin, another IF protein. These results revealed the role of phosphorylation in turnover of GFAP and a synergistic role of GFAP and vimentin in the dynamics of glial filaments. The data further suggest that each of the phosphorylated sites has a distinct impact on the dynamics of GFAP.

Normal development of serotonergic neurons in mice lacking S100B.

S100B, a glia-derived calcium binding protein, exhibits strong neurite extension activity in cultured serotonergic neurons. Using S100B-knockout mice, we examined whether this protein possesses in vivo serotonergic trophic activity. The distribution of serotonergic fibers, determined by immunohistochemistry, in the brains of S100B-knockout mice was quite similar to that of wild-type mice. Furthermore, the content of serotonin and its metabolite 5-hydroxyindole-3-acetic acid in knockout mice was also indistinguishable from those of wild-type mice. Our findings argue against the hypothesis that S100B has a crucial role in neurite extension of serotonergic neurons.

Distribution of phosphorylated glial fibrillary acidic protein in the mouse central nervous system.

BACKGROUND: Glial fibrillary acidic protein (GFAP) is the principal component of intermediate filaments (IFs) in mature astrocytes in the central nervous system (CNS). Like other IF proteins, GFAP has multiple phosphorylation sites in the N-terminal head domain. The distribution of phospho-GFAP in vivo has not been elucidated. RESULTS: We generated Gfap(hwt) knock-in mice, in which the coding region for the head domain of GFAP is replaced with the corresponding human sequence. In combination with a series of monoclonal antibodies (mAbs) reactive to human phospho-GFAP, we visualized the distribution of phospho-GFAP in vivo in mice. GFAP phosphorylated at Thr7, Ser8 and/or Ser13 increased postnatally in the CNS of these mice. Limited populations of GFAP-positive astrocytes were labelled with anti-phospho-GFAP mAbs in most brain areas, whereas almost all the astrocytes in the optic nerve and spinal cord were labelled. Astrocytes in the subventricular zone and rostral migratory stream preferentially contained phospho-GFAP. In a cold injury model of the cerebral cortex, we detected phospho-GFAP in reactive astrocytes at 2-3 weeks after the injury. CONCLUSIONS: Phospho-GFAP provides a molecular marker indicating the heterogeneity of astrocytes, and Gfap(hwt) knock-in mice will aid in monitoring intracellular conditions of astrocytes, under various conditions. Our results suggest that the phosphorylation of GFAP plays a role in non-dividing astrocytes in vivo.