Evaluation of absorption kinetics of orally administered theophylline in rats based on gastrointestinal transit monitoring by gamma scintigraphy.

The gastrointestinal (GI) transit and absorption of orally administered theophylline, a highly absorbable drug without presystemic elimination, were investigated under fasted and fed conditions using three rats in a crossover study. To evaluate the GI transit rate for each segment in vivo, a noninvasive technique, gamma scintigraphy, was employed using a nonabsorbable compound, (99m)Tc-labeled diethylenetriamine pentaacetic acid (DTPA). Using a gamma scintigraphic technique it is possible to simultaneously evaluate the GI transit and absorption of orally administered drug in the same individual. Theophylline was simultaneously administered along with [(99m)Tc]DTPA to animals in the fasted and fed states. Each GI transit pattern, simulated using the GI transit-kinetic model with a lag time factor, was well fitted to the experimental data. Gastric emptying rate varied in each study, even under the same experimental condition. The GI transit pattern for each segment was highly variable, especially in animals in the fed state. This inconsistency in transit pattern was mainly due to the variability in gastric emptying, which was much slower in animals in the fed compared with the fasted state. However, in spite of a large variability of GI transit kinetics, the plasma concentration-time curves of theophylline were well predicted by the GI transit-absorption model using the individual GI transit parameters obtained in the study. The absorption rate of theophylline was considerably reduced in animals in the fed state, because of the reduction of gastric emptying rate. Analysis using GI transit-absorption model and gamma scintigraphic technique made it possible to estimate the variable absorption kinetics regulated by GI transit with huge variability.

Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression.

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics. III: mechanisms for sinusoidal efflux of 4-methylumbelliferone sulfate.

1. To elucidate the mechanisms involved in the sinusoidal efflux of sulfate and glucuronide metabolites of 4-methylumbelliferone (4MU), isolated rat liver perfusion studies were performed under several conditions. 2. The effect of sodium azide on the hepatic handling of both conjugates was examined. The net sinusoidal efflux clearance (CL(eff)) based on the unbound concentration in the liver did not change for 4MU glucuronide (4MUG) or significantly increase for 4MU sulfate (4MUS), suggesting that the sinusoidal efflux of both conjugates is not mediated by the transport systems dependent on adenosine triphosphate. 3. Under Cl(-)-depleted conditions, the CL(eff) of 4MUG significantly decreased, but the saturation of its sinusoidal efflux rather than the transport system dependent on Cl(-) might be involved because the hepatic concentration of 4MUG was extensively higher than that of the control study due to the extremely attenuated biliary excretion. The CL(eff) of 4MUS also significantly decreased, but its hepatic concentration was not different from that in the control study, suggesting that the transport system using Cl(-) is involved in the sinusoidal efflux of 4MUS. 4. The effect of glutathione was examined. CL(eff) of 4MUG was not affected by the additional glutathione, but CL(eff) of 4MUS decreased significantly, suggesting that some transport system sensitive to glutathione is involved in the sinusoidal efflux of 4MUS, but not of 4MUG. 5. Transporters such as Oatp1, Oatp2 and/or Npt1 might be involved in the sinusoidal efflux of 4MUS, but 4MUG is secreted from the sinusoidal membrane via the systems that are totally different from those for 4MUS.

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of drugs. I: studies by in vivo constant infusion.

1. The hepatic and renal handling of glucuronides and sulphates of three phenolic compounds, 4-methylumbelliferone (4-MU), p-nitrophenol (pNP) and acetaminophen (APAP), were evaluated pharmacokinetically by in vivo constant infusion experiments in rat. It was shown that the urinary excretion rate at steady-state was larger than the biliary excretion rate for both glucuronides and sulfates, and sulfates, in particular, were extensively excreted into the urine. 2. For each glucuronide, however, biliary excretion clearances (CL(b)) calculated based on the total concentration and unbound concentration in the liver were much larger than the corresponding renal excretion clearances (CL(r)). Even in the case of sulfates, there was not any large difference between CL(r) and CL(b) based on the total and unbound concentration in tissues, which could not explain their extensive urinary excretion. From these results, these excretion clearances were recognized not to reflect necessarily the actual excretion rate obtained. 3. On the other hand, the tissue-to-plasma concentration ratio (K(p)) of both glucuronides and sulfates for every phenolic compound was much higher in the kidney than that in the liver. The results suggested that one of the most important determinants for the preferential excretion of these conjugates into the bile or urine is the extent of disposition of each compound to the liver or kidney. 4. In addition, K(p) of both glucuronides and sulfates in the liver, where these conjugates are mainly formed, was small. The K(p) of sulfates was quite low, suggesting that sulfates generated in the liver were subject to extensive sinusoidal efflux.

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics II: Studies with isolated perfused rat liver.

1. To elucidate the determining factors for elimination pathways of sulfate and glucuronide metabolites of xenobiotics, a single-pass perfusion of 4-methylumbelliferone (4MU) or p-nitrophenol (pNP) was performed with an isolated rat liver preparation. 2. Without bovine serum albumin in the perfusion system, clearance calculated based on the unbound concentration in the liver clearly showed that the net efflux clearances (CLeff) of sulfates from the sinusoidal membrane were much higher than those of glucuronides and that the biliary excretion clearances (CLb) of glucuronides were approximately two times larger than those of sulfates. 3. The ratios of CLeff to CLb were much higher for sulfates than those for glucuronides. The bile-oriented elimination of glucuronides or sinusoidal efflux-oriented elimination of sulfates was observed even using the perfusate including 3% bovine serum albumin, but the sinusoidal efflux of sulfates was extensively enhanced by bovine serum albumin in the perfusate. The mechanisms behind this stimulatory effect remain to be elucidated. 4. For both compounds, CLb of glucuronide was comparable with CLb of sulfate, meaning that CLb is not responsible for the biliary excretion of glucuronides at extensively higher rate than sulfates. 5. Higher concentration of glucuronides in the liver, partly caused by much lower CLeff of glucuronides than that of sulfates, is likely responsible for the bile-oriented excretion of glucuronides. The extensive sinusoidal efflux of sulfates, leading to the urine-oriented excretion, is attributed to the substantially higher CLeff than CLb. 6. In conclusion, the sinusoidal efflux is an important factor for determining elimination pathways of both sulfates and glucuronides, although further studies are needed to clarify the mechanisms of the sinusoidal efflux.

Analysis and prediction of absorption profile including hepatic first-pass metabolism of N-methyltyramine, a potent stimulant of gastrin release present in beer, after oral ingestion in rats by gastrointestinal-transit-absorption model.

The prediction method for the plasma concentration-time profile of N-methyltyramine (NMT), a potent stimulant of gastrin release present in beer after oral ingestion in rats was examined using the previously developed Gastrointestinal (GI)-Transit-Absorption Model, with the addition of a process of hepatic first-pass metabolism. Phenol red was used as a nonabsorbable marker for estimation of the GI transit rate constant for eight segments in the GI tract. The first order absorption rate constant for each segment was estimated by means of a conventional in situ closed loop method. The results of in situ absorption experiments showed that NMT is well absorbed in the small intestine, especially in the duodenum and jejunum. Using the GI-Transit-Absorption Model, it was demonstrated that more than 90% of orally ingested NMT is absorbed in the small intestine, and that the substantial absorption site for NMT in vivo is the lower jejunum and the ileum. However, the observed bioavailability was only 39.0%. The in vitro metabolism study clarified that NMT is metabolized in the liver, but not in the small-intestinal mucosa. With the hepatic intrinsic clearance value (2.0 liters/h) calculated from the rate of metabolism in vitro, the hepatic availability was estimated to be 0.510 on the basis of a well stirred model, which was validated by two other methods to calculate the hepatic availability of NMT. The plasma concentration-time curve and bioavailability of NMT after oral ingestion were well predicted by the GI-Transit-Absorption Model with the hepatic first-pass metabolism process.

Pharmacokinetic evaluation of high pulmonary disposition of clarithromycin after systemic administration.

1. The distribution characteristics of clarithromycin to the lung were investigated in vivo and in isolated lung perfusion experiments. The in-vivo integration plot analysis showed that the pulmonary uptake and extracellular distribution in the lung were significantly higher for clarithromycin than for erythromycin. 2. In the rat lung single-pass perfusion study, the pulmonary extraction ratio (E(ss)) of clarithromycin at steady-state was significantly higher than that of erythromycin, and the E(ss) of clarithromycin tended to decrease as the inflow concentration increased, suggesting the involvement of carrier-mediated transport in the pulmonary disposition of clarithromycin. 3. The outflow patterns of clarithromycin or erythromycin at various inflow concentrations were simultaneously analysed based on a pharmacokinetic model, which consists of the non-specific binding site, the specific binding site and the subsequent uptake process. The parameters obtained suggested that clarithromycin would have the higher affinity and higher capacity for the specific binding site, and the higher equilibrium constant for the non-specific binding site than erythromycin. 4. The simulation study using those parameters demonstrated that clarithromycin could be bound to the specific binding site and subsequently taken up more extensively than erythromycin. 5. A multiple-indicator dilution study also indicated that clarithromycin was more readily associated and extracted with the lung than with erythromycin. In the inhibition study, it was suggested that the pulmonary uptake of clarithromycin could be ascribed not only to the non-specific binding depending on its lipophilic nature, but also in part to some specialized mechanisms such as organic cation transporters.