Nagao Suzuki

PepT1 mRNA expression is induced by starvation and its level correlates with absorptive transport of cefadroxil longitudinally in the rat intestine.

AbstractPurpose. To establish how closely intestinal transport activity for beta-lactam antibiotics is correlated with PepT1 expression, absolute expression level of PepT1 mRNA and transport activity were determined longitudinally in the small intestine of fed and starved rats. Methods. For evaluation of absolute expression levels of PepT1 mRNA, quantitative RT-PCR by LightCycler® was used. The transport function was determined by quantifying the absorptive transport of cefadroxil across intestinal tissue sheets in a Ussing chamber. Results. PepT1 mRNA expression was highest at the lower region and lowest at the upper region in the fed rats. The value of PepT1 was about 1/5∼1/6 of that of GAPDH. The expression level in the starved rats was increased in all segments, but more profoundly in the upper region. Cefadroxil transport across intestinal tissue was higher in the lower region and lower in the upper region in fed rats, and increased in the upper region in starved rats. An excellent correlation was observed between expression levels and the permeability coefficients (r2= 0.859, p < 0.05). Conclusions. The intestinal transport of cefadroxil is directly proportional to PepT1 expression, suggesting that the PepT1 expression level in the rat small intestine is the major determinant of the absorption of peptide-like compounds.

Involvement of Multidrug Resistance-Associated Protein 2 in Intestinal Secretion of Grepafloxacin in Rats

ABSTRACT We investigated the contribution of multidrug resistance-associated protein 2 (MRP2) to the secretory transport of grepafloxacin and compared its functional role with that of P-glycoprotein (P-gp) by using Sprague-Dawley rats (SDRs) and Eisai hyperbilirubinemic rats (EHBRs), in which MRP2 is hereditarily defective. In intestinal tissue from SDRs mounted in Ussing chambers, the level of transport in the direction from the serosal layer to the mucosal layer was twofold greater than that in the direction from the mucosal layer to the serosal layer. This secretory transport of grepafloxacin was diminished by both probenecid, an MRP2 inhibitor, and cyclosporine, a P-gp inhibitor. In intestinal tissue from EHBRs, the secretory transport of grepafloxacin was lower than that in intestinal tissue from SDRs and was inhibited by cyclosporine but not by probenecid. The absorption of grepafloxacin from intestinal loops in SDRs was in the order of duodenum > jejunum > ileum and was increased by cyclosporine but not by probenecid. The absorption in EHBRs was not higher than that in SDRs. The intestinal secretory clearance in SDRs after intravenous administration of grepafloxacin was shown to be greater for the ileum than for the duodenum, which is in good agreement with the previously reported regional expression profile of MRP2 mRNA. The intestinal secretory clearance was lower in EHBRs than in SDRs. Accordingly, in addition to P-gp, MRP2 might play a role in the secretory transport of grepafloxacin. The function of MRP2 in facilitating grepafloxacin transport in the secretory direction is more pronounced both in vitro and in vivo, while the restriction of entry from the lumen into the cell by MRP2 seems to be negligible, compared with that by P-gp, in the case of grepafloxacin.

Active intestinal secretion of new quinolone antimicrobials and the partial contribution of P-glycoprotein

Transport of quinolone antimicrobials and the contribution of the secretory transporter P‐glycoprotein were studied in‐vivoand in‐vitro. In rat intestinal tissue (Ussing chambers method) and human Caco‐2 cells (Transwell method), grepafloxacin showed secretory‐directed transport. In both experimental systems, the secretory‐directed transport was decreased by ciclosporin A, an inhibitor of P‐glycoprotein, and probenecid, an inhibitor of anion transport systems. This suggested the contribution of P‐glycoprotein and anion‐sensitive transporter(s). The involvement of P‐glycoprotein was investigated by using a P‐glycoprotein over‐expressing cell line, LLC‐GA5‐COL150, and P‐glycoprotein‐gene‐deficient mice (mdr1a(—/—)/1b(—/—) mice). LLC‐GA5‐COL150 cells showed secretory‐directed transport of grepafloxacin, while the parent cell line, LLC‐PK1, did not. The secretory‐directed transport of sparfloxacin and levofloxacin was also detected in LLC‐GA5‐COL150 cells. In the mdr1a(—/—)/1b(—/—) mice, the intestinal secretory clearance was smaller than that in wild‐type mice after intravenous administration of grepafloxacin. Moreover, the absorption from an intestinal loop in mdr1a(—/—)/1b(—/—) mice was larger than that in wild‐type mice. Accordingly, it appears that some quinolones are transported by secretory transporters, including P‐glycoprotein. The involved transporters function in‐vivo not only to transport grepafloxacin from blood to intestine but also to limit its intestinal absorption.

Secretory transport of p-aminohippuric acid across intestinal epithelial cells in Caco-2 cells and isolated intestinal tissue

The intestinal transport of an organic anion, p‐aminohippuric acid (PAH), was studied in Caco‐2 cell monolayers and rat intestinal tissue mounted in Ussing chambers.

Role of nitric oxide in the peristalsis in the isolated guinea-pig ileum

The role of nitric oxide in peristalsis was studied by using NG-nitro-L-arginine, an inhibitor of the biosynthesis of nitric oxide, in the isolated guinea-pig ileum. Constant peristalsis was cyclically induced by distending the ileal wall with intraluminal solution infused at a constant rate. NG-Nitro-L-arginine (10(-6) to 10(-4) M) dose dependently increased the frequency of peristalsis. This effect of NG-nitro-L-arginine was almost hindered by pretreating the ileum with 10(-3) M L-arginine, but not D-arginine. Nitroprusside (5 X 10(-7) M) reversed the frequency increase. In the presence of NG-nitro-L-arginine, peristaltic propulsion occurred at a smaller distension of the ileal wall and the ileum constricted to a smaller diameter at the completion of propulsion. The rate of shortening of longitudinal muscle during distension was raised by NG-nitro-L-arginine, although the peak magnitudes were not changed. Consistent with these effects of NG-nitro-L-arginine on peristalsis, NG-nitro-L-arginine at 10(-5) M increased the contractions of circular muscle in response to electrical field stimulation, but not those of longitudinal muscle. These results suggest that endogenous nitric oxide modulates peristalsis by limiting the contractile activity of the circular muscle of the guinea-pig ileum.

Changes in absorptive function of rat intestine injured by methotrexate.

1. Methotrexate (MTX), an anticancer drug, has been shown to induce acute injury in the small intestine. The present study was designed to investigate the in vivo absorptive function of the small intestine injured by MTX using an amino‐β‐lactam antibiotic cephalexin (CEX). Time‐dependent changes in diamine oxidase (DAO) and alkaline phosphatase (ALP) activity in the small intestine and histopathological findings were also measured in rats treated with MTX (20 mg/kg).

Causative agent of vascular pain among photodegradation products of dacarbazine.

The photodegradation products of the anticancer drug, dacarbazine, cause adverse reactions including local venous pain when injected intravenously. In this study, we attempted to identify which of these products is responsible. We synthesized or purchased five photodegradation products of dacarbazine (dimethylamine, 5‐diazoimidazole‐4‐carboxamide (Diazo‐IC), 4‐carbamoylimidazolium‐5‐olate, 5‐carbamoyl‐2‐(4‐carbamoylimidazol‐5‐ylazo) imidazolium‐5‐olate and 2‐azahypoxanthine) and examined the pain reaction induced by their intraperitoneal administration in mice using an abdominal stretching or constriction assay. Only Diazo‐IC clearly induced pain reaction in mice in a dose‐dependent manner, the other products caused no pain reaction. The threshold concentration for pain reaction in mice was estimated to be about 0.1 mg mL−1. While diclofenac sodium significantly reduced acetic‐acid‐induced pain reaction in mice, it did not influence those induced by Diazo‐IC. This result suggests that the mechanism of Diazo‐IC‐induced pain is different from that of acetic‐acid‐induced inflammatory pain. Dacarbazine itself produced marked relaxation of rat thoracic aorta strips in a concentration‐dependent manner, but there was no difference between the activity of dacarbazine and its photo‐exposed solution, so constriction or relaxation of blood vessels is unlikely to be a factor in the pain reaction. In conclusion, Diazo‐IC generated by photodegradation of dacarbazine solution causes the side‐effect of venous pain. Dacarbazine solution that has turned pink should not be used, because Diazo‐IC is an intermediate in the formation of the reddish product, 5‐carbamoyl‐2‐(4‐carbamoylimidazol‐5‐ylazo) imidazolium‐5‐olate. Drip infusion preparations of dacarbazine should be shielded from light.

Xerostomia-Inducing Drugs Could Increase Chemotherapy-Associated Mucositis in Pediatric Patients

To elucidate the effect of the concomitant usage of other drugs on the occurrence of mucositis during and after chemotherapy, we investigated prescriptions for pediatric patients with hematological cancers at Kanazawa University Hospital, focusing particularly on drugs that have an adverse effect of xerostomia.The patients were divided into two groups consisting of methotrexate-treated (MTX group) and other-anticancer-drug-treated patients (non-MTX group). The occurrence of mucositis in the MTX group was significantly higher than in the non-MTX group.In the MTX-group, the number of concomitantly used drugs during a one-week period after chemotherapy was 6.18 in patients with mucositis and 2.95 in patients without mucositis. In the non-MTX group, there were 3.38 or 3.56 concomitantly used drugs in patients with or without mucositis, respectively.The concomitant drugs were divided into xerostomia-inducing and non-xerostomia-inducing drugs and there-after were classified into pharmacological groups. The occurrence of mucositis was higher among patients using xerostomia-inducing drugs than among those using non-xerostomia-inducing drugs with an exception of diuretics, and this tendency was more prominent in the MTX group than in the non-MTX group. It was recently documented that mucositis was significantly associated with xerostomia during 5-fluorouracil chemotherapy. Therefore, it is assumed that the concomitant usage of xerostomia-inducing drugs reduced the saliva flow, leading to mucositis.We conclude that the concomitant use of xerostomia-inducing drugs is one of the factors that influence the occurrence of mucositis during chemotherapy with MTX and other anticancer agents. It is recommended that the use of xerostomia-inducing drugs should thus be avoided as much as possible to decrease the occurrence of mucositis during chemotherapy.