Induction of multidrug resistance-associated protein 2 in liver, intestine and kidney of streptozotocin-induced diabetic rats.

Many studies have demonstrated that Mrp2 is highly regulated in some physiopathological situations. The aim of this study was to investigate effects of diabetes mellitus on function and expression of multidrug resistance-associated protein 2 (Mrp2) in rat liver, kidney and intestine. Diabetic rats were induced by an intraperitoneal administration of streptozotocin (65 mg/kg) and randomly divided into diabetic (DM) rats and insulin-treated diabetic rats. Sulfobromophthalein (BSP), a substrate of Mrp2, was used to evaluate Mrp2 function in vivo. Data from excretion experiments demonstrated that compared with normal rats, diabetes markedly enhanced BSP excretion via bile, urine and intestinal perfusate, which contributed to the elevated plasma clearance of BSP after intravenous administration of 45 µmol/kg BSP. Western blot results showed higher levels of hepatic, renal and intestinal Mrp2 protein in DM rats, although no difference was observed in renal Mrp2. Insulin treatment partly reversed these alterations. Induction of Mrp2 by diabetes was in parallel with the increase in bile flow, levels of biliary and plasma total bile acid (TBA), and plasma conjugated bilirubin in DM rats. Diabetes may enhance Mrp2 function and expression in liver, kidney and intestine, which might be due to insulin deficiency, increased TBA and conjugated bilirubin.

Pharmacokinetics, tissue distribution and excretion of a new photodynamic drug deuxemether.

Deuxemether was a new photodynamic drug effective for many kinds of solid tumor therapy, which was mainly composed of 3-(or 8-)-(1-methoxyethyl)-8-(or 3-)-(1-hydroxyethyl)-deutero-porphyrin IX (MHD) and 3,8-di(1-methoxyethyl)-deuteroporphyrin IX (DMD). The aims of this study were to elucidate its pharmacokinetic characteristics, tissue distribution, plasma protein binding and excretion properties and underlying mechanisms of deuxemether in rats based on the simultaneous determination of MHD and DMD. The pharmacokinetic profiles of both MHD and DMD in rats after intravenous doses were linear and best fitted to a two compartment model, characterized with a rapid distribution phase (MHD: t(1/2)alpha, 0.09-0.14 h; DMD: t 1/2 alpha, 0.07-0.11h) and a relatively slow elimination phase (MHD: t 1/2 beta, 2.03-3.20 h; DMD: t 1/2 beta, 2.51-3.20 h). The tissue distributions of MHD and DMD in rats were rather limited as evidenced from their low distribution volume (0.75-1.70 L/kg) and the results of tissue distribution study. Protein binding of MHD and DMD were moderate (65.36-89.99% for MHD; 45.43-76.23% for DMD), independent of drug concentrations and similar between human and rat plasma over a concentration range of 0.50-50.0 microg/mL. Both MHD and DMD were predominantly (>74.1%) eliminated from rats as the parent drugs through the hepatobiliary systems and finally excreted into the feces. The multidrug resistance-associated proteins 2 (MRP2) inhibitors, bromosulfophthalein and probenecid, substantially inhibited the hepatobiliary elimination of MHD and DMD while the P-gp inhibitor digoxin had little effect, suggesting that MRP2 may contribute to the rapid and extensive hepatobiliary excretion of deuxemether. There were no significant differences between MHD and DMD for all pharmacokinetic characteristics studied. In conclusion, this study provided firstly the full pharmacokinetic characteristics and mechanisms of deuxemether, which would be helpful for its clinical regiment design.

Down-regulation of hepatic transporters for BSP in rats with indomethacin-induced intestinal injury.

Previous reports have demonstrated that an intestinal injury causes hypofunctions of the liver associated with down-regulations of cytochrome P450, but an influence on hepatic transporters remains unclear. Here, we tested hepatic transporter functions in a rat model of bowel injury using indomethacin (IDM). After administration of IDM (8.5 mg/kg, i.p., 3 d), the rats suffered the intestinal impairment indicated by a reduction of alkaline phosphatase activity in mucosa. In vivo pharmacokinetic experiments of bromosulfophthalein (BSP) showed that there was a reduction in its plasma elimination rate and cumulative biliary excretion in IDM-treated rats and systemic and biliary clearances reduced to nearly 50% of the control group. Protein expressions in plasma membrane and mRNA levels of organic anion transporting polypeptide 1b2 (Oatp1b2) and multidrug resistance-associated protein 2 (Mrp2), which play hepatic BSP uptake and biliary excretion, respectively, in the liver were significantly reduced following the IDM treatment. In portal plasma, the levels of proinflammatory cytokines were unchanged, while the level of nitric oxide metabolites (NO2- + NO3-) increased to 6.5-fold that of the control. The time-course on IDM treatment indicated that, firstly, intestinal injury was induced, the NO level increased, and the hepatic Oatp1b2 and Mrp2 expression began to fall followed by an increase in plasma ALT. In conclusion, IDM-induced injury to the small intestine causes the hypofunction of hepatic Oatp1b2 and Mrp2 independently on the hepatic impairment, and NO arising from bowel injury may be one of key factors for it through the remote effect.

[Pharmacokinetic study of cancer chemotherapy].

We reported that the rate of conversion of lactone to carboxylate forms of irinotecan (CPT-11) and its metabolites plays a major role in the biliary excretion of these compounds. Sulfobromophthalein partially inhibited the secretion of SN-38-glucronide into the gastrointestinal lumen, whereas little change was seen in that of active metabolite SN-38. Co-administration of sulphobromophthalein with CPT-11 might lower the late-onset gastrointestinal toxicity observed during treatment with CPT-11 without lowering anticancer activity. In the ileum, the level of transport in the direction form the serosal layer to mucosal layer was significantly greater than that in the direction form the mucosal layer to serosal layer, whereas a significant difference was not observed in the jejunum. This secretory transport required metabolic energy was diminished by sulfobromophthalein. A specific transport system plays a major role in the secretion of SN-38 and that this secretory transport system predominantly exists in the ileum. Uptake of SN-38 was significantly reduced at 4 degrees C. Baicalin inhibited the uptake of SN-38. A specific transport system mediates the uptake of SN-38 across the apical membrane in Caco-2 cells. Inhibition of this transporter would be a useful means for reducing late-onset diarrhea.

Effects of organic anions on biliary glutathione excretion in rats.

The effects of various organic anions (2 mumol/100 g body weight) on biliary glutathione excretion were studied in rats. Bromosulfophthalein markedly inhibited glutathione excretion, whereas dibromosulfophthalein was a less potent inhibitor. Experiments with different bromosulfophthalein doses revealed that the extent of inhibition of glutathione excretion was related to the total bromosulfophthalein excretion. Indocyanine green also inhibited biliary glutathione excretion. Dinitrophenyl glutathione and pravastatin did not inhibit biliary glutathione excretion. These findings indicate the existence of several pathways of biliary excretion of organic anions and suggest that one of them is a pathway for biliary glutathione excretion.

Mitigation of nitrofurantoin-induced toxicity in the perfused rat liver.

1. Nitrofurantoin is an antimicrobial agent which produces hepatotoxicity caused by the redox cycling of the nitro group and its radical anion. This futile cycling triggers a complex series of events known collectively as oxidative stress. 2. Our goal was to determine treatment strategies which could mitigate nitrofurantoin-induced toxicity in the isolated perfused rat liver. We co-infused various agents which blocked early or late events in the progression to toxicity. Tissue levels of glutathione and protein thiols were measured as indicators of the progression to toxicity and lactate dehydrogenase leakage into the perfusate was used as a marker of irreversible cell death. 3. Five treatments significantly (P < 0.05) decreased LDH leakage (reported as thousands of units accumulated in perfusate at 300 min, mean+/-standard error, n = 3-4) when compared to nitrofurantoin alone (274 +/- 37). These treatments were adenosine-2'-monophosphate (120 +/- 53), penicillamine (90 +/- 29), N-(2-mercaptopropionyl)-glycine (120 +/- 49) and bromosulfophthalein with (80 +/- 29) or without 5,5'-difluro-1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraace tic acid (101 +/- 46). Two other treatments, N-acetylcysteine (183 +/- 7) and dithiothreitol (166 +/- 59) delayed the onset of toxicity. Finally, calpeptin (319 +/- 34) which blocks activation of nonlysosomal proteases was ineffective. 4. We concluded that early intervention on the pathway to toxicity was most effective. The strategies detailed here may prove beneficial in treating hepatotoxicity seen following nitrofurantoin therapy.

Does paracellular permeability play a role in cholephilic dye-induced cholestasis?

Changes in hepatic paracellular permeability were investigated during the development of cholephilic dye-induced cholestasis in rats. For this purpose, four dyes with different cholestatic potency (phenol red, sulfobromophthalein, bromcresol green and rose bengal) were infused at a high, potentially damaging dose (280 nmol/min per 100 g body wt., i.v.), and changes in paracellular permeability were continuously monitored by measuring the access into bile of the permeability probe -14C-sucrose. The cholestatic potency of the different dyes was: rose bengal > bromcresol green > sulfobromophthalein > phenol red. All dyes increased [14C]sucrose bile-to-plasma ratio, producing a displacement towards curves of higher permeability. The capability of the dyes to increase biliary permeability followed the same order as their respective cholestatic potencies. The possible implications of the present results for cholephilic dye-induced cholestasis are discussed.

Partial characterization of regulation of biliary lecithin hydrophobicity: association with organic anion-induced solute cholestasis in rats.

We examined the effects of the depletion of bile salts and of the intravenous infusion of sodium taurocholate (STC) with or without bromosulphophthalein (BSP) in rats on the biliary secretion of lipids to clarify the regulatory mechanism(s). Each rat was equipped with a bile-duct cannula to collect bile. After the endogenous bile salt pool was depleted, STC was infused at a constant rate (160 nmol/min per 100 g body wt.) with or without BSP (50, 100, or 150 nmol/min per 100 g body wt.). BSP reduced the biliary secretion of cholesterol and phospholipids dose-dependently without affecting the secretion of bile salts (uncoupling phenomenon). Compared with the physiological and STC-infused condition, the biliary cholesterol/phospholipid ratio and saturated/unsaturated fatty acid ratio increased under the bile salts depletion and uncoupling phenomenon. Data indicate that the hydrophobicity of biliary lecithin increases with a decrease in the bile salt micelle capacity to induce biliary lipid secretion, resulting in a higher packing density of biliary vesicle. The cholesterol-holding capacity of the biliary vesicle is therefore enhanced during the depletion of bile salts and the uncoupling phenomenon.

Comparison of bromosulphthalein and indocyanine green clearance in sheep.

The kinetics of indocyanine green removal from the blood after a single intravenous injection were measured in sheep and compared with those of a single dose of bromosulphthalein given to the same animals 48 hours later. A single dose of 1.5 mg kg-1 of indocyanine green (ICG) was cleared in a double exponential manner. The hepatic uptake of ICG was not significantly different from that of bromosulphthalein (BSP) but its rate of excretion in bile was significantly lower (P < 0.05) and its rate of plasma reflux significantly higher (P < 0.05). The plasma distribution volumes of the two compounds were similar but the mean (SD) plasma clearance of ICG (435 [228.6] ml min-1) was significantly lower than that of BSP (626 [291.9] ml min-1) (P < 0.05). The retention of ICG 30 minutes after the injection was significantly higher than that of BSP (P < 0.01).

Absorption of organic anions as model drugs following application to rat liver surface in-vivo.

Absorption of organic anions (phenol red, bromphenol blue and bromosulphonphthalein) has been studied after their application to rat liver surface in-vivo, employing a cylindrical glass cell (i.d. 9 mm, area 0.64 cm2). Each drug appeared gradually in the blood with the peak level at about 1 h, after which its concentration declined slowly. Absorbed model drug was efficiently excreted into the bile. These observations appear to indicate the possibility of drug absorption from liver surface membrane. Absorption of model drugs was estimated to be more than 59% in 6 h. The biliary recovery and metabolism of phenol red did not change as compared with that after intravenous administration.

Concurrent bromosulphophthalein and antipyrine administration to assess liver blood flow and hepatic enzymes in rats.

This study investigated the feasibility of using concurrent iv administration of antipyrine (15 mg/kg body weight) and bromosulphophthalein (BSP; 25 mg/kg) in the rat. Antipyrine is used as an index of hepatic drug metabolism and BSP is used to assess hepatic blood flow. Plasma concentrations of BSP were described using biexponential phases, while antipyrine plasma concentrations were monoexponential. No significant difference was observed between antipyrine pharmacokinetic parameters in concurrent BSP rats when compared with controls. There was also no significant difference between BSP pharmacokinetic parameters in concurrent antipyrine rats when compared with controls, except in the alpha value (P less than 0.05). This indicates that BSP distribution may be affected by concurrent antipyrine administration. Therefore, simultaneous administration of both substrates is not acceptable to study hepatic blood flow. Another iv combination dose (25 mg BSP/kg body weight, followed by 15 mg antipyrine/kg 0.5 hr later) and a dose of 15 mg antipyrine/kg body weight only was administered to rats pretreated with phenobarbital (90 mg/kg body weight) for 6 days. Pharmacokinetic parameters of BSP, beta, k21, k23 and plasma clearance, in the pretreated rats were significantly different from non-pretreated rats. No significant difference was observed in the pharmacokinetic parameters of antipyrine between the combination dose and antipyrine dose in the phenobarbital-pretreated rats. The half-lives of antipyrine in both pretreated groups decreased approximately by 70%, while the clearance increased four times compared with controls. The volume of distribution in these animals did not change as a result of phenobarbital pretreatment. This suggests that a 25 mg BSP/kg body weight dose followed by 15 mg antipyrine/kg 0.5 hr later may be a feasible approach to study liver blood flow, as well as hepatic efficiency in rats.

Effect of Haemonchus contortus infection on the clearance of antipyrine, sulfobromophthalein, chloramphenicol, and sulfathiazole in lambs.

A study was made to determine the effect of Haemonchus contortus parasitic infection in lambs on the clearance of several IV administered drugs. Clearance of sulfobromophthalein or sulfathiazole from the plasma of lambs was unaffected by infection with H contortus. Clearance of antipyrine was enhanced by the infection, and thiabendazole treatment did not alter this effect. Clearance of chloramphenicol (CAP), administered as the succinate ester (CAPS), was not changed by the infection, but it was increased after treatment with thiabendazole. Changes in the mean body residence time and initial plasma concentration of CAPS and CAP after treatment with thiabendazole indicate that hydrolysis of CAPS to CAP was reduced. High concentrations of CAPS apparently enhanced its own elimination directly rather than via the expected sequence involving hydrolysis, glucuronidation, and excretion of CAP-glucuronide. Enhanced clearance of antipyrine following infection of lambs with H contortus can be explained in at least 2 ways. First, it is possible that the lambs did not have mature amounts of hepatic drug metabolizing enzyme activity as reported by other investigators, which may be explained by breed differences or animal husbandry practices. Second, infection of lambs by H contortus may have triggered an inductive response in hepatic cytochrome P-450-mediated activities, which might result via a generalized enhancement in hepatic protein synthesis associated with the physiologic response to replace plasma proteins and other blood components lost through gastrointestinal hemorrhage caused by the active feeding of adult worms. Other phase-II reactions such as acetylation, glucuronidation, and glutathione-S-transferase apparently were not affected.

Determination of sulfobromophthalein excretion in rabbits.

The excretion of sulfobromophthalein (BSP) is determined by hepatic uptake (storage capacity) and secretion (transport maximum). We calculated the BSP excretion data in six rabbits by measuring the rate of rise of BSP at various infusion rates. The relative storage capacity was determined to be 0.34 +/- 0.13 mg.mg-1.dl-1.kg-1. The transport maximum was calculated to be 0.91 +/- 0.12 mg.min-1.kg-1. These results compare closely with previously reported results using more direct measurements that require anesthesia and surgical cannulation of the biliary tree. Repeated studies were easily performed. Our method provides a reliable and simple technique to study the effects of various conditions or substances on BSP excretion in conscious, unanesthetized rabbits.

Alteration in sulfobromphthalein hepatic storage capacity (S) in non-pregnant women previously affected with intrahepatic cholestasis of pregnancy (ICP).

The pathogenesis of intrahepatic cholestasis of pregnancy (ICP) is still unknown, although it is currently accepted that the disease represents an abnormal reaction of genetically predisposed maternal liver, to estrogen hormones. To gain a better insight into the hepatic handling of cholephilic anions outside of pregnancy itself, we determined, using the perfusion technique of Wheeler et al. (1960), the hepatic maximum excretory rate (Tm) and the storage capacity (S) of sulfobromphthalein (BSP) in 6 women with a past history of ICP and in 6 controls, matched for age and parity, but with no history of ICP or other liver disease. The BSP Tm in the group with previous ICP did not differ significantly from that measured in the control group (9.22 +/- 2.37 vs 7.92 +/- 1.20). By contrast, BSP S values in the 'previous ICP' group was critically higher than that of the controls (120.43 +/- 55.89 vs 39.15 +/- 16.17: t-test p less than 0.005, U-test p less than 0.001). A possible explanation for this result could be the existence of a metabolic defect responsible for an increased intrahepatic concentration of the cholephilic substances inside the liver cells.

Hepatic transport and biliary excretion of sulfobromophthalein in aflatoxin B1-treated rats.

The effects of a single dose of aflatoxin B1 (AFB1) on sulfobromophthalein (BSP) plasma disappearance, hepatic transport maximum (Tm), and relative storage capacity (S), were examined in rats 48 hr after AFB1 injection. BSP plasma concentration decay was delayed, and the BSP biliary excretion was diminished in treated animals. S and Tm values were unaltered. However, the Tm was reached in treated rats at a higher infusion rate. A Lineweaver-Burk plot of BSP biliary excretion rate vs BSP serum concentration curve showed a higher apparent Km in the AFB1-treated rats.