The cytochromes P450 (CYPs) responsible for the oxidative metabolism of tacrolimus and its in vitro major metabolite M-I, the 13-O-mono-demethylated metabolite, were characterized in human liver microsomes. Human liver microsomes and ten human CYPs expressed in Hep G2 cells were used in the experiments.
1. When ¹⁴C-labeled tacrolimus (¹⁴C-tacrolimus) was incubated with human liver microsomes, M-I formation was mainly observed in the early stage of incubation, while many peaks of the more polar metabolites, including M-VII, the 13, 15-O-di-demethylated metabolite, were detected in the late stage of incubation. When the microsomes were incubated with ¹⁴C-M-I, formation of M-VII and unidentified metabolites were observed. The rates of formation of M-I from ¹⁴C-tacrolimus in 10 different human liver microsomes correlated well with the activities of testosterone 6β-hydroxylation and tolbutamide methyl-hydroxylation, but not with the marker enzyme activities of other CYPs. The metabolism of both tacrolimus and M-I by human liver microsomes were inhibited by ketoconazole and anti-CYP3A4 antiserum.
2. After incubation of ¹⁴C-tacrolimus with Hep G2 cell lysates containing expressed human CYPs, M-I formation was observed in the reaction system with CYP3A3, 3A4 and 3A5, but not with CYP1A2, 2A6, 2B6, 2C8, 2C9, 2D6 or 2E1. After incubation of the lysates with ¹⁴C-M-I, M-VII formation was again observed only in the system with the CYP3A subfamily. Substrate disappearance of both tacrolimus and M-I was the most efficiently catalyzed by CYP3A4.
3. These results suggest that tacrolimus is metabolized to polar metabolites through M-I by human liver microsomes, and that the metabolism of both tacrolimus and M-I are mainly catalyzed by CYP3A4.

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A computer program, MOMENT (EXCEL), for moment analysis was developed on Microsoft Excel®. MOMENT (EXCEL) written in Visual Basic for Applications® can handle time course not only of blood concentration but also of urinaly or bilialy excretion according to linear or linear-logarthmic trapezoidal rule. MOMENT(EXCEL) calculates and tabulates moment parameters, such as AUC, MRT and clearance accompained with the graphical output. A method of AIC (Akaike's Information Criteion) based determination of terminal points for automatic extrapolation to infinite time were also equipped in this program, which was investigated the validation by analysis of the artificial time courses simulated with 15% random error in the obserbation. The moment values estimated using the AIC based method by MOMENT (EXCEL) were in good agreement with the theoretical ones. MOMENT (EXCEL), with using Excel, can reduce errors occurred during data handling and shorten time for routine analysis.
MOMENT(EXCEL) can be down loaded from the following internet web site; http://bunseki02.pharm.kyoto-u.ac.jp/.

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The changes in pharmacokinetics of a cephalosporin derivative (Cefluprenam; CFLP) with age were studied. CFLP was administered intravenously to infant (at 3 weeks of age) or adult beagle dogs (at 8 months of age) once a day for 4 weeks at a dose of 300 mg/kg.
In adult dogs, the plasma elimination half-life (t_1/2) on the first day and on the 27th day of treatment was 0.91±0.030 hr and 0.90±0.031 hr, and total body clearance of CFLP (CLtot) was 0.27±0.009 l/hr/kg and 0.28±0.007 l/hr/kg, respectively. The pharmacokinetics of CFLP in adult dogs showed no change after repeated administration. Creatinine clearance (normalized per body weight) in adult dogs was 0.21 ± 0.010 l/hr/kg. These findings indicate that CFLP is excreted mainly by glomerular filtration in the kidneys.
In infant dogs, on the other hand, CFLP disappeared from the plasma on the 27th day of treatment faster than on the first day. The t_1/2 of CFLP on the first day (at 3 weeks of age) and on the 27th day (at 7 weeks of age) were 0.90 ± 0.15 hr and 0.61 ± 0.02 hr, respectively, and CLtot were 0.45 ± 0.03 l/hr/kg and 0.64 ±0.01 l/hr/kg, respectively. There was no notable difference in the distribution volume VI or Vss between the first day and the 27th day of treatment. Creatinine clearance which was 0.28 ± 0.05 l/hr/kg for the CFLP 300 mg/kg group of infant dogs on the first day of administration increased to 0.49 ± 0.13 l/hr/kg on the 27th day of treatment. The increase in Mot on the 27th day of treatment was resulted from an increase in creatinine clearance, that is, glomerular filtration. Since creatinine clearance was similar in the CFLPtreated group and the untreated control group, the increase in creatinine clearance was not due to repeated administration of CFLP, but to the growth of the infant dogs.
The plasma levels of CFLP in infant dogs were lower than those in adult dogs, and the distribution volume Vss was higher than those in adult dogs. Because CFLP is distributed in extracellular fluid, these results seem to be caused by the water content ratio of the body which is higher in infant dogs than in adult ones.
These results suggest that the changes in pharmacokinetics of CFLP are related to physiological changes in renal function and the water content of their body with age. Glomerular filtration was low in infant dogs at 3 weeks of age, but increased in a steep curve at 7 weeks of age. It appears that glomerular filtration of the drug gradually declines after the age of 7 weeks.

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The absorption, distribution, and excretion of radioactivity were investigated following a single oral administration of ¹⁴C-pramipexole to rats at a dose of 0.5 mg/kg. The protein binding of the drug was also investigated in vitro and in vivo.
1. The total radioactivity in plasma reached the maximum at about 1.5 hr after oral administration of ¹⁴C-pramipexole to male and female rats. The half-lives of radioactivity in plasma were about 48 and 41 hr in male and female rats, respectively, as determined from the data at 24 to 72 hr after administration. The C_max was decreased in the presence of food, but total absorption of radioactivity remained unaffected. ¹⁴CPramipexole was well absorbed throughout the duodenum, jejunum and ileum, but not in the stomach. 2. The radioactivity in most tissues after oral administration of ¹⁴C-pramipexole to male rats reached Cmax at 2 hr, being higher in liver, kidney, hypophysis and salivary gland. The radioactivity in the whole brain was higher than that in plasma at 2 and 6 hr after administration. At 48 hr after oral administration, the radioactivity in most tissues except blood had decreased to below 1/10 of the respective maximum concentration. The ratio of the concentration in blood cells to that in plasma was in the range of 1.63-7.15 at 0.5 to 48 hr after oral administration.
3. The binding of ¹⁴C-pramipexole at concentrations of 10-1000 ng/ml to rat plasma protein in vitro was about 15-18% and that at concentrations of 0.5-50 ng/ml to human serum protein in vitro was about 17-26%.
The percentage of radioactivity bound to rat plasma protein within 2 hr after oral administration of ¹⁴Cpramipexole to male rats was below 20%. After 2 hr, the ratio of plasma protein binding increased with time, and then the percentage of radioactivity bound to rat plasma protein was about 67% at 48 hr.
4. The amount of radioactivity excreted in urine and feces within 168 hr after oral administration of ¹⁴C-pramipexole to male rats were 59.0% and 44.0% of the dose, respectively. Within 48 hr after intraduodenal injection of the bile obtained from other rats which had been administered ¹⁴C-pramipexole (0.1 mg/kg) intravenously, about 4% of the injected radioactivity was excreted in the bile.
5. The radioactivity in milk after oral administration to lactating rats was 2-5 times higher than that in plasma at all times from 0.5 to 48 hr, but the half-life of radioactivity in milk was shorter than that in plasma.

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The absorption and distribution of radioactivity were investigated following a 14-day period of daily oral administration of ¹⁴C-pramipexole (0.5 mg/kg/day) to male rats.
1. When measured 24 hr after each of 14 repeated daily administrations of ¹⁴C-pramipexole to male rats, the level of radioactivity in plasma rose as the number of doses increased, and reached a steady state after 12 or 13 doses. The elimination of radioactivity from the plasma after the last dose was similar to that after a single administration.
2. At 1 hr after the 14th administration, the level of radioactivity reached the maximum in most tissues. High levels were observed in the liver and kidney, being about 22 and 14 times higher than that in the plasma, respectively. At 168 hr after the last dose, the levels of radioactivity in the liver and kidney were very much higher than that in plasma. The elimination of radioactivity from most tissues after the last dose was parallel to that from plasma, except the spleen and kidney, where an accumulation was observed.
3. After the last administration, the ratio of the concentration in blood cells to that in plasma gradually increased with the lapse of time. The elimination of radioactivity from blood cells was very much slower than that from plasma. The relative increase of radioactivity in blood cells at the late stage after drug administration may be attributed to metabolites of pramipexole.

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Peptide and protein drugs are becoming a very important class of therapeutic agents. However, the oral bioavailability of peptide and protein drugs is generally poor because they are extensively degraded by proteases in the gastrointestinal tract and impermeable through the intestinal mucosa. For systemic delivery of peptide and protein drugs, parenteral administration is currently required to achieve their therapeutic activities. However, these administration are poorly accepted by patients and may cause allergic reactions and serious side effects. Therefore, various approaches have been examined to overcome the delivery problems of these peptides when they administered orally. These approaches include (1) to use additives such as absorption enhancers and protease inhibitors, (2) to develop an administration method for peptides that can serve as an alternative to oral and injection administration, (3) to modify the molecular structure of peptide and protein drugs to produce prodrugs and analogues, and (4) to use the dosage forms to these peptide drugs. Of all these approaches, we demonstrated that transmucosal absorption of various peptides including insulin, calcitonin, tetragastrin and thyrotropin releasing hormone (TRH) could be improved by using these approaches. Therefore, these approaches may give us basic information to improve the transmucosal absorption of peptide and protein drugs.

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