Tokuzo Minouchi

Predictive performance of serum digoxin concentration in patients with congestive heart failure by a hyperbolic model based on creatinine clearance

Objectives:  To formulate a simple equation for determining the daily dose requirements of digoxin by inclusion of creatinine clearance (Ccr) values as an explanatory variable.

Influence of Glycerol-induced Acute Renal Failure on the Pharmacokinetics of Cyclosporin in Rats

Although it is widely believed that renal dysfunction has no effect on the pharmacokinetics of cyclosporin, many clinical reports suggest that renal dysfunction after renal transplantation is closely related to the pharmacokinetics of cyclosporin. To clarify the relationship between renal dysfunction and the pharmacokinetics of cyclosporin, we examined the influence of acute renal failure (ARF) on its pharmacokinetics in glycerol‐induced ARF rats.

Probable Metabolic Interaction of Doxifluridine with Phenytoin

OBJECTIVE: To report the marked elevation of the serum phenytoin concentration during treatment with antineoplastic agents. CASE SUMMARY: A 51-year-old Japanese woman, who was diagnosed with multiple brain metastatic tumors, was placed on oral phenytoin at a maintenance dose of 200 mg/d (3.8 mg/kg/d) to prevent seizures. The serum concentration of phenytoin was well controlled within the therapeutic range; no seizures occurred. Four months later, combination therapy with doxifluridine (5′-DFUR) 800 mg/d, cyclophosphamide 100 mg/d, and medroxyprogesterone acetate 800 mg/d was initiated because of further metastasis. Approximately 1 month after the start of concurrent treatment with the antineoplastic agents, the serum phenytoin concentration was elevated to fourfold of the original concentration. Staggering was observed at that time, but toxic symptoms gradually subsided with the decline in the serum phenytoin concentration after its withdrawal. DISCUSSION: A probable explanation for the marked elevation of serum phenytoin concentration is a reduction of the capacity of CYP2C-dependent phenytoin metabolism, and the antineoplastic agents could be involved in this event. The interaction of fluorouracil and phenytoin is known in clinical practice, and it is reported that the expression of hepatic CYP2C enzymes is depressed by exposure of rats to fluorouracil. 5′-DFUR, a prodrug of fluorouracil, was considered the likeliest candidate responsible for the interaction. This interaction was of clinical significance because of the great extent of changes in the serum phenytoin concentration. CONCLUSIONS: Clinicians should be aware of the elevation of serum phenytoin concentrations when phenytoin is given in combination with fluorouracil derivatives, including 5′-DFUR.

Decrease in oral bioavailability of ciclosporin by intravenous pulse of methylprednisolone succinate in rats.

We examined the effects of high‐dose methylprednisolone on the bioavailability of orally administered ciclosporin in rats. To emulate the clinical protocol of methylprednisolone pulse therapy, methylprednisolone sodium succinate (MPS), a prodrug of methylprednisolone, was intravenously administered as repeated doses (66.3 mg kg−1) for 3 days. The area under the blood ciclosporin concentration versus time curve after oral administration was significantly reduced by 60% by pulse treatment with MPS. Based on our previous finding that the total body clearance of ciclosporin was reduced by about 20% by the same methylprednisolone pulse protocol, the extent of reduction in the oral bioavailability of ciclosporin was estimated to be approximately 50%, indicating a drug interaction between high‐dose methylprednisolone and orally administered ciclosporin, which affected the absorption process. In rats treated with MPS, an in‐situ efflux experiment using rhodamine‐123 demonstrated that the reverse transport function of P‐glycoprotein (P‐gp) in the small intestine was significantly enhanced, although there was no significant increase in the intestinal microsomal activity of triazolam α‐ and 4‐hydroxylation, metabolic probes for CYP3A. In addition, a significant decrease was observed in the amount of secreted bile acids serving as an enhancer of gastrointestinal absorption of ciclosporin in MPS treatment. To directly estimate the absorptive capacity, an in‐situ absorption test was conducted using a closed‐loop of small intestine in control and MPS‐treated rats. Intestinal absorption of ciclosporin was significantly decreased, not only in the absence of bile flow but also by treatment with MPS, which well reflected the change in the in‐vivo pharmacokinetic behaviour of ciclosporin after methylprednisolone pulsing. These results demonstrate that bioavailability of ciclosporin is markedly reduced by MPS pulse treatment, and the mechanism of this interaction was confirmed to involve enhancement of small‐intestinal P‐gp function and decrease in bile secretion.

Factors that affect absorption behavior of cyclosporin a in gentamicin-induced acute renal failure in rats.

Factors that affect the absorption of cyclosporin A (CsA) were examined in gentamicin-induced acute renal failure (ARF) rats. In ARF rats, the area under the blood CsA concentration-time curve after oral administration was significantly decreased in comparison with that of control rats; 5.81 ± 0.55 vs 11.30 ± 1.59 mg h mL−1 (mean ± s.e.m.), respectively, and the relative bioavailabilities in ARF and control rats after oral administration were 15.2% and 43.4%, respectively. The flow rate of bile and the amount of bile acids in ARF rats were markedly decreased to about 61% of control, and 41% of control, respectively. The amount of CsA uptaken into the evened sac of jejunum, transferred to serosal side, and metabolized in tissues was significantly decreased in ARF rats without verapamil, while with 0.3 mM verapamil, the amount in ARF rats recovered to the levels of control rats. The absorption clearance of CsA in ARF rats was significantly decreased, however it was significantly improved by adding bile or bile acid. Adenosine triphosphate released from enterocytes in ARF rats was significantly decreased in the presence of 2.0 1−M CsA, 0.3 mM verapamil, or both, in comparison with control rats. From these findings, we concluded that a reduction of CsA bioavailability during ARF is caused by depression in bile excretion and renal function-dependent depression of uptake from intestinal tract via maybe P-gLycoprotein in enterocytes. They are main two factors that reduce the absorbed fraction of CsA in ARF rats.

Depression of phenytoin metabolic capacity by 5-fluorouracil and doxifluridine in rats.

It has been found in clinical practice that the serum level of phenytoin, of which metabolism is mediated by hepatic CYP2C enzymes, was markedly elevated by co‐administration of 5‐fluorouracil (5‐FU) and doxifluridine (5′‐deoxy‐5‐fluorouridine; 5′‐DFUR), a prodrug of 5‐FU, but the detailed mechanisms are unclear. A study using rats was undertaken to examine the effects of 5‐FU and 5′‐DFUR on phenytoin metabolism in hepatic microsomes and phenytoin pharmacokinetics in‐vivo. Neither 5‐FU nor 5′‐DFUR exhibited direct inhibitory effects on hepatic microsomal phenytoin p‐hydroxylation, a major metabolic route catalysed by CYP2C in rats, as in humans. 5‐FU and 5′‐DFUR were injected intraperitoneally into male rats as single doses (1.68 mmol kg−1) and repeated doses (0.24 mmol kg−1 for 7 days). Control rats received vehicle alone. A significant reduction in the activity of phenytoin p‐hydroxylation was observed 4 days after the last administration irrespective of the agents and their treatment regimens, although the activity was unchanged on Day 1. Pharmacokinetic analysis of phenytoin revealed that the elimination rate constant and the total clearance was decreased by 70–75 % in both the 5′‐DFUR‐treated and 5‐FU‐treated rats, indicating that the decrease in the metabolic capacity of phenytoin was responsible for the change in phenytoin disposition in‐vivo. On the other hand, 5‐FU significantly depressed the total P450 content, NADPH cytochrome c reductase activity and activities of progesterone hydroxylations. However, the depressive effects of 5′‐DFUR were not very potent relative to those of 5‐FU, which can be explained by the fact that 5‐FU is derived from 5′‐DFUR to only a small extent. According to a recent report, phenytoin p‐hydroxylation and progesterone 2α‐/21‐hydroxylations share common CYP2C enzymes as their catalysts. Because there was a difference in the modulation profiles between phenytoin p‐hydroxylation and progesterone 2α‐/21‐hydroxylations after exposure to 5′‐DFUR, 5′‐DFUR might modulate phenytoin metabolism without loss of catalytic ability for other substrates, unlike 5‐FU. The present study suggested that the down‐regulation of hepatic CYP2C enzymes occurs by 5‐FU exposure even at a low level, and provided a fundamental explanation for the drug interaction encountered in clinical practice.

Fluorimetric determination of mexiletine in serum by high-performance liquid chromatography using pre-column derivatization with fluorescamine.

A simple, specific and sensitive micro-scale method for the assay of the antiarrhythmic agent mexiletine in human serum is described. The method uses high-performance liquid chromatography, with pre-column fluorimetric derivatization by fluorescamine. Following extraction with diethyl ether, mexiletine and 4-methylmexiletine (an internal standard) were derivatized with fluorescamine under weakly alkaline condition (pH 9.0) and chromatographed on a reversed-phase column with aqueous methanol-2-propanol as the mobile phase. The two fluorescent derivatives of mexiletine and the internal standard were separated as clear single peaks, and no interfering peaks were observed on the chromatograms. The detection limit for mexiletine was 0.005 micrograms/ml from only 100 microliters of serum, and the calibration curves in the range 0.01-5 micrograms/ml were linear, with an overall coefficient of variation of less than 5%. The analytical recovery of a known amount of mexiletine added to serum was almost 100%. This method proved to be effective in the rapid monitoring of the serum concentrations in patients who received this potent antiarrhythmic drug.

Influence of intravenous methylprednisolone pulse treatment on the disposition of ciclosporin and hepatic CYP3A activity in rats.

We examined the effects of high‐dose methylprednisolone (MP) on the disposition of ciclosporin (CsA) and hepatic microsomal CYP3A activity using rats. Methylprednisolone sodium succinate (MPS), a prodrug of MP, was intravenously administered as repeated doses (66.3 mg kg−1) for 3 days or as a single dose. In MP‐treated rats, a significant increase was observed in the total body clearance (CLtot) and elimination rate constant (Ke) of intravenously administered CsA. The enzyme activities of triazolam hydroxylations and erythromycin N‐demethylation in hepatic microsomes were also enhanced by about 50% by MP treatment, suggesting that the alteration in the CsA pharmacokinetics was due to significant induction of the hepatic CYP3A responsible for the metabolic conversion of CsA. In contrast, no significant changes in the values of CLtot and Ke were found following a single treatment with MP. On the other hand, MP inhibited the CYP3A‐mediated triazolam hydroxylations in a concentration‐dependent manner. The difference between the in‐vivo and in‐vitro inhibitory behaviours of MP was attributed to the rapid elimination of MP after biotransformation from MPS because the plasma MP concentration decreased with a half‐life of 15 min immediately after reaching a level close to the inhibition constant for the triazolam 4‐hydroxylation reaction (32.4 μm). Although there is a general consideration that MP cannot act as an enzyme inducer at maintenance doses, the present results strongly suggest that high‐dose MP is likely to interact pharmacokinetically with CsA by inducing hepatic CYP3A. These results may provide basic explanations for the clinical experience that blood CsA levels are reduced during MP pulse therapy.

Preferential inhibition of CYP1A enzymes in hepatic microsomes by mexiletine

SummaryWe examined the inhibitory behavior of theophylline oxidations and a variety of cytochrome P450 (P450)-dependent metabolism in the presence of mexiletine (MEX), using hepatic microsomes from both control mice and mice exposed to β-naphthoflavone (β-NF). Theophylline metabolism, which is mainly catalyzed by CYP1A2, was susceptible to competitive inhibition by MEX. The calculated inhibition constants (Ki) for theophylline 3-demethylation and its 8-hydroxylation were 4.3 μM and 8.3 μM, respectively, which are comparable to the recommended therapeutic serum range for MEX. The inhibitory potency of MEX on cytochrome P450-dependent enzyme activities diverged among the several metabolic reactions, which were probes for CYP1A, 2A, 2C, 2D, 2E, and 3A subfamilies. The Ki value (6.7 μM) for methoxyresorufin O-demethylation mediated by CYP1A2 agreed with those from theophylline oxidations. These metabolic reactions exhibited the smallest Ki values, 1–3 orders of magnitude lower than activities of other constitutive cytochrome P450 species. Similar degrees of inhibition were observed in CYP1A1, a β-NF-inducible isoform with a relatively high conformity to CYP1A2. These results indicate that MEX acts as a selective and potent inhibitor of the CYP1A enzymes responsible for oxidative biotransformation of chemicals such as theophylline. This evidence provides a fundamental explanation for the pharmacokinetic interactions experienced in clinical practice.

Evaluation of fluorescence polarization immunoassay for determination of cyclosporin in plasma.

The fluorescence polarization immunoassay (FPIA) method for determination of cyclosporin in plasma was evaluated and compared with the high-performance liquid chromatography (HPLC) and the radioimmunoassay (RIA) methods. The coefficients of variation for the within-run and between-run precision were <5 and <8%, respectively, for samples ranging in concentration from 50 to 600 ng/ml. Recoveries were determined by adding cyclosporin at concentrations from 25 to 1,000 ng/ml to patient plasma; they were, on average, 98.5%. The calibration curve was stable throughout a 10-week study period. There was no clinically significant interference due to hemolysis, icterus, lipemia, or other commonly used drugs. There was considerable variation of the ratio of the FPIA result to the HPLC result, whereas there was a good correlation between the FPIA and the RIA results (r = 0.975, n = 25, y = 1.2x – 36.4), when evaluated using specimens from renal transplant patients receiving cyclosporin orally. It was concluded that the FPIA is an appropriate, rapid method for patient cyclosporin analysis in plasma and serves as a practical alternative to the RIA.