Effects of oxidative stress on the pharmacokinetics and hepatic metabolism of atazanavir in rats.

We studied the effects of oxidative stress (OS) on the pharmacokinetics of atazanavir (ATV), particularly the distribution of ATV in the plasma and its metabolism in hepatic microsomes, using a rat model of ferric-nitrilotriacetate-induced OS (OS rats). The areas under the plasma concentration-time curves for intravenous bolus, oral, and intraportal administration of ATV in the OS rats were significantly greater than those in the control rats, whereas blood clearance of ATV after intravenous bolus injection in the OS rats (0.94 ± 0.04 L/h/kg) was approximately half of that in the control rats (2.08 ± 0.20 L/h/kg). Moreover, the intrinsic clearance (CLint), which is determined by in vitro metabolic studies using hepatic microsomal fractions of rats, was approximately 43% lower in the OS rats (0.489 ± 0.017 mL/min/mg protein) than in the control rats (0.851 ± 0.004 mL/min/mg protein). ATV concentrations in both the plasma-bound fraction and erythrocytes of the OS rats were significantly greater than those in the control rats. These results suggest that the hepatic metabolism of ATV may be reduced in patients under OS.

Effects of renal function on the pharmacokinetics and pharmacodynamics of prophylactic cefazolin in cardiothoracic surgery.

The purpose of this investigation was to study the effects of renal function on the pharmacokinetics and pharmacodynamics (PK-PD) of free cefazolin administered prophylactically in cardiothoracic surgery. Patients received an initial 2-g dose of cefazolin, followed by 1-g doses 6, 12, 18 and 24 h after the first dose. In patients who underwent cardiopulmonary bypass, 1 g was added to the priming solution. In 35 patients with a normal estimated creatinine clearance (CLcr) ≥50 ml/min, a free cefazolin concentration <4 µg/ml was observed in 11.4, 5.7 and 54.3% of patients before the second dose, at the end and 24 h after operation, respectively. In contrast, only 7.4% of 27 patients with CLcr <49 ml/min had a free cefazolin concentration <4 µg/ml 24 h after the operation. There was a high negative correlation between CLcr and time above the target minimal inhibitory concentration (MIC) when the CLcr was <50 ml/min (r(2) = 0.807), and no correlation when the CLcr was ≥50 ml/min. Renal function has a significant impact on the PK-PD of prophylactic cefazolin in cardiothoracic surgery. The postoperative drug dosing intervals should be <6 h in order to achieve a 100% time above the MIC in patients with CLcr ≥ 50 ml/min.

Effect of plasma uric acid on pharmacokinetics of cyclosporine A in living-related renal transplant recipients and pharmacokinetic study in rats with experimental hyperuricaemia.

OBJECTIVES: Renal transplant recipients are thought to have an increased risk of hyperuricaemia (HU); therefore, the effects of plasma uric acid (UA) on the pharmacokinetics (PK) of cyclosporine A (CyA), an immunosuppressant, in renal transplant recipients and experimental animals were investigated. METHODS: An open-label, non-randomized, retrospective study was performed in renal transplant recipients. Data from 76 subjects who received a renal transplantation with CyA medication were included. We compared the PK of CyA of recipients showing a high UA level with the other recipients. In addition, PK studies were performed using hyperuricaemic-model rats (HU rats) prepared by subcutaneous injection of the uricase inhibitor, potassium oxonate and intraperitoneal injection of UA. RESULTS: The area under the blood concentration vs. time curve (AUC) up to 9 h, the blood level at 2 h after dose and peak level in high UA recipients (UA > 7.0 mg/dL) was significantly lower (about 10-16%) than that in the other recipients, although there were no differences in dose, and the trough blood level. On the contrary, there were no differences in PK parameters after intravenous administration of CyA between HU and control rats; however, AUC, peak level and bioavailability in HU rats (2.01 ± 0.56 µg h/mL, 0.47 ± 0.26 µg/mL and 0.186 ± 0.05, respectively) after oral administration were significantly lower than in the control animals (6.13 ± 0.97 µg h/mL, 0.82 ± 0.17 µg/mL and 0.458 ± 0.07 µg/mL, respectively). In addition, the absorptions of CyA and midazolam, an ideal probe for CYP3A, from the intestinal loop in HU rats were significantly less (about 50% and 37%, respectively) than in the controls. CONCLUSIONS: The absorption of CyA was affected by plasma UA in transplant recipients and experimental rats. The contribution of intestinal metabolism by CYP3A to decreasing CyA absorption in HU rats was significant. These results suggest that transplant recipients with high UA may have poor absorption of CyA.

Pharmacokinetic and pharmacodynamic studies following percutaneous absorption of erythropoietin micropiles to rats.

To ascertain the pharmacological activity of erythropoietin (EPO) administered by self-dissolving micropiles (SDMP), four kinds of EPO SDMPs were prepared and were administered to rats in 4 consecutive days at 200, 500, 1000 and 2300 IU/kg. After the start of the experiment, blood samples were obtained once a day for 10 days and percent circulating reticulocytes were counted using Miller technique. At the lower doses, 200 and 500 IU/kg, pharmacological activity of EPO was not obtained. By increasing EPO dose to 1000 IU/kg, circulating reticulocytes significantly increased at days 4, 5, 6 and 7 after the start of the experiment and the average value for the change in reticulocyte levels during day 1 and day 5 was 170.9%. With the highest dose, 2300 IU/kg, higher circulating reticulocytes levels started to increase at the 4th day after the start of the experiment and maintained from day 5 to day 10. The average of the changes in reticulocyte from day 5 to day 10 was 251%. Dose-dependent circulating reticulocytes increase was observed at the higher dose range, 1000 and 2300 IU/kg. To study the linearity on the serum EPO level vs. time curves, pharmacokinetic experiment was performed with rats. After the administration of EPO SDMPs to rats, 200, 500, 1000 and 2300 IU/kg, serum EPO levels gradually increased and reached to the maximum level, C(max), at 18 h after administration. The C(max)s were 100.4+/-11.7 mIU/ml (200 IU/kg), 346.6+/-11.8 mIU/ml (500 IU/kg), 391.6+/-17.6 mIU/ml (1000 IU/kg), and 1094.9+/-114.8 mIU/ml (2300 IU/kg), respectively. AUCs were 1407+/-231, 3843+/-402, 5363+/-482 and 15,566+/-1894 mIU h/ml. Linear relation was obtained between serum EPO level and EPO dose administered as SDMP. With histological study, any adverse effect was not found out on the skin where SDMPs were administered for consecutive 4 days. These results suggest the usefulness of SDMP as a new percutaneous delivery system of EPO.

Pharmacokinetic and pharmacodynamic studies following oral administration of erythropoietin mucoadhesive tablets to beagle dogs.

Oral administration of mucoadhesive tablets containing erythropoietin (EPO) and an absorption enhancer Labrasol was studied in rats and dogs. Mucoadhesive tablets were prepared using Sylysia 550 holding the absorption enhancer and Carbopol 974P as a mucoadhesive agent. Mucoadhesive tablets were covered with a water-insoluble backing layer made of cellulose acetate and a pH-sensitive covering layer made of Eudragit L/Eudragit S. Tablet was administered into the rat jejunum at EPO dose of 100 IU/kg and serum samples were collected for 6h. Serum EPO level was analysed with a standard ELISA procedure. After administration, rats showed a maximum serum EPO level of C(max) 70.6 +/- 8.9 mIU/ml. Oral administration of a single tablet containing 100 IU/kg EPO to beagle dogs showed a C(max) of 24.6 +/- 4.1. When EPO dose was increased to 500 IU/kg and the number of tablets was also increased to 5, the C(max) was 54.8 +/- 9.0 mIU/ml. However, when EPO, 100 IU/kg dose was divided into five tablets, the C(max) was 15.5 +/- 1.8 mIU/ml. In the absence of absorption enhancer, the C(max) was 35.8 +/- 3.8 with 500 IU/kg dose distributed among five tablets. Pharmacodynamic studies were carried out following oral administration of mucoadhesive tablets for 6 consecutive days at an EPO dose of 500 IU/kg. Whole blood samples were collected and percent circulating reticulocytes were counted using Miller technique. The increase in percent circulating reticulocytes was found to be 1.7% on day 8 following oral administration. As a control study, EPO was administered by i.v. route at a dose of 300 IU/kg for 3 consecutive days and the percent circulating reticulocytes were counted. Mucoadhesive tablets showed promising results as an oral drug delivery system for protein therapeutics.

Age-related alteration of carbamazepine-serum protein binding in man.

To determine whether biological maturation influences the kinetics of carbamazepine-serum protein binding, the carbamazepine free fraction (%) was investigated in the serum of 66 patients, ranging from 4 to 83 years, with epilepsy or trigeminal neuralgia, treated with carbamazepine alone or carbamazepine in combination with phenytoin, phenobarbital, and/or valproic acid, over a relatively long period. Biochemical parameters such as levels of albumin and non-glycated albumin showed a significant relationship with carbamazepine free fraction (r = -0.521, P < 0.001 for albumin; r = -0.700, P < 0.001 for non-glycated albumin). Non-glycated albumin was more strongly correlated with carbamazepine free fraction. The biochemical parameters showed a significant relationship with age (r =-0.243, P < 0.1 for albumin; r =0.666, P < 0.001 for glycated albumin; r = -0.459, P < 0.001 for non-glycated albumin; r = 0.640, P < 0.001 for carbamazepine free fraction). Glycated albumin (%), non-glycated albumin and carbamazepine free fraction (%) were strongly correlated with age, whereas albumin showed only a weak correlation with age. To evaluate the effects of ageing on carbamazepine-serum protein binding, the patients were divided into three groups according to age: children, 4-15 years; adults, 16-64 years; elderly, 65-83 years. Albumin and non-glycated albumin were much lower, and glycated albumin (%) and carbamazepine free fraction (%) much higher in the elderly group than in the other two groups. The results of this study showed that the major ligand of carbamazepine in the serum was non-glycated albumin, which decreased with age. These observations suggested that in elderly patients, the elevation of free carbamazepine concentrations in the serum caused by reduced non-glycated albumin levels, induces increases in the sensitivity of the pharmacological effects of carbamazepine and the risk of drug interactions.

DAFS measurements using the image-plate Weissenberg method.

An instrumental technique for DAFS measurements which can provide site-specific information is proposed. The approach uses (i) focusing optics with parabolic mirrors and a double-crystal monochromator, (ii) the Laue and Bragg settings and (iii) data collection by the image-plate Weissenberg method. Six image exposures are recorded per plate at five intrinsic energies and one reference energy. The single-crystal measurements were performed at the Co K-absorption edge, and the 200, 220 and 311 reflections of CoO and 511 and 911 reflections of Co(3)O(4) were used for analysis. The regression analysis of chi(k), Fourier transforms of k(3)chi(k) and back-Fourier filtering have been performed.

Pharmacokinetic studies of mycophenolate mofetil in rat kidney allograft recipients.

BACKGROUND: A pharmacokinetic estimation of the immunosuppressive activity of mycophenolate mofetil (MPM) was performed in rats with transplanted kidney allografts. METHODS: Kidney allografts from Brown Norway rats were transplanted into Lewis rats using a microsurgical technique. MPM, at doses of 5, 10, 15 and 25 mg/kg/day, was administered orally every day after transplantation. Pharmacokinetic studies were performed on days 7 and 14, and thereafter every 2 weeks. RESULTS: After the administration of MPM, the plasma concentration of mycophenolic acid (MPA) increased rapidly, peaking at 15 to 30 minutes, and then decreased biexponentially. The peak concentration of MPA and the area under the plasma MPA concentration versus time curves (AUC) significantly correlated with the dosage. MPM administration at 5 and 10 mg/kg/day significantly prolonged the graft survival time from 7.1 days to 18.5 days and 85.0 days, respectively. The AUC values on day 7 after transplantation were 32.7 micrograms.h/mL and 38.6 micrograms.h/mL in rats receiving 5 and 10 mg/kg/day, respectively. However, in rats receiving 15 mg/kg/day of MPM or more, the AUC value at 7 days was 78.8 micrograms.h/mL, and almost all of these rats died from gastrointestinal toxicity. CONCLUSION: MPM monotherapy significantly prolonged rat kidney allograft survival, however, high dosages of MPM caused gastrointestinal toxicity. AUC measurements of the MPA concentration are suitable for the pharmacokinetic monitoring of MPM.

Effect of glycosylation on carbamazepine-serum protein binding in humans.

Effect of glycosylation on carbamazepine-serum protein binding was investigated in vitro using the serum from 24 diabetics and 10 healthy subjects, and in vivo using the serum from 49 patients receiving carbamazepine. In both binding studies, nonglycosylated albumin levels were strongly correlated with the carbamazepine free fraction (%). To evaluate the effect of glycosylation in vivo, the patients were divided into two groups according to glycosylated albumin levels (%): a healthy group (10-15) and a high group (15 and over). The high group had decreased nonglycosylated albumin levels and an increased carbamazepine free fraction. Our results suggest that one should not use total concentrations for the monitoring of serum carbamazepine concentrations, but free concentrations, especially in poorly controlled diabetics.

Effects of glycosylation of hypoglycaemic drug binding to serum albumin.

The binding properties of hypoglycaemic drugs to glycosylated human serum albumin (G-HSA) were investigated using a fluorescence quenching method. Displacement patterns between tolbutamide and Sudlow's-site-specific drugs to G-HSA were also investigated. The order of the binding affinities of these drugs to HSA was glibenclamide > acetohexamide > tolbutamide > or = glicrazide > metfolmin. The order of the binding affinities were the same for G-HSA as for HSA. The ability of G-HSA to bind hypoglycaemic drugs, however, was much lower than that of HSA. Scatchard plots for the binding of tolbutamide to both albumins were biphasic. The glycosylation affected saturable binding sites (I and II), whereas it did not influence non-saturable binding sites. The displacement patterns of tolbutamide binding between both albumins were not affected in the presence of site-I- or III-specific drugs, whereas the relative binding of tolbutamide to site-II-specific drugs between the two albumins was remarkably changed. The glycosylation of HSA not only increases the unbound drug concentration but also changes the displacement pattern at site II. Our results suggest that the extensive glycosylation of plasma proteins in diabetic patients complicates drug-drug interactions beyond those seen in normal people.

Age-dependent alteration of the serum-unbound fraction of nicardipine, a calcium-channel blocker, in man.

To determine whether the age-dependent increase in the pharmacological effect of calcium-channel blockers is a result of age-dependent alteration of the unbound fraction the drug in serum, the unbound fraction of the nicardipine was investigated in the serum of 38 adults. The unbound concentration of nicardipine in serum to which nicardipine (205.4 ng mL-1) had been added was determined by ultracentrifugation to range from 0.49 to 4.01% (mean +/- s.d., 1.55 +/- 0.78%). Non-glycosylated albumin was most strongly correlated with age (r = 0.901). Total bilirubin was weakly correlated with age whereas levels of alpha-1-acid glycoprotein, triglycerides and glycosylated albumin were not correlated with age. A significant (P < 0.01) linear correlation was obtained between the unbound fraction of nicardipine and parameters such as age, albumin, albumin/globulin ratio, albumin/glycosylated albumin ratio, non-glycosylated albumin and total bilirubin. To assess the relative effect of each variable on the unbound fraction of nicardipine, stepwise multiple linear regression was performed using age and biochemical parameters. The three variables (non-glycosylated albumin, total bilirubin and age) were entered into the regression equation. The results of this study showed that the major ligand of nicardipine in serum was non-glycosylated albumin, which decreased with age. It was, moreover, shown that the serum-unbound concentration of nicardipine increased with age. This finding would be one factor accounting for the increase in the pharmacological effect of nicardipine with age. In addition, our predicted model for the unbound fraction of nicardipine might be useful in determining the appropriate nicardipine dose for the elderly.

Determination of L-buthionin (SR)-sulfoximine, gamma-glutamylcysteine synthetase inhibitor in rat plasma with HPLC after prelabeling with dansyl chloride.

L-(SR)-Buthionin sulfoximine (L-(SR)-BSO) is a potent and specific inhibitor of gamma-glutamylcysteine synthetase, which catalyzes the first reaction of glutathione biosynthesis. A selective, sensitive, and simple high-performance liquid chromatographic method was developed for the determination of L-(SR)-BSO in rat plasma. After the compound was labeled with dansyl chloride (Dns-Cl) under optimal conditions, it was separated in a Zolbax-ODS column with a mobile phase that consisted of 0.01M phosphate buffer, methanol, and acetonitrile (8:1:3, v/v). The compound was detected with a fluorescence detector at an excitation wavelength of 335 nm and an emission wavelength of 525 nm using a xenon lamp. The coefficients of variation (DV) from the interassay in the low and high concentrations (10 and 500 micrograms/mL of L-(SR)-BSO in rat plasma) were 2.5 and 4.8%, respectively. The CVs from the intra-assay in the low and high concentrations were 3.2 and 5.6%, respectively. The minimum concentration of L-(SR)-BSO that could be determined was 10 micrograms/mL when 100-microL serum samples were used. The detection limit was 50 ng per injection volume. This method enables pharmacokinetic and pharmacodynamic studies in rats.

Pharmacokinetics of mycophenolate mofetil, a new immunosuppressant, in rats.

Mycophenolate mofetil (MPM), a new immunosuppressant, is the morpholinoethyl ester of mycophenolic acid (MPA). The distribution in blood and pharmacokinetics of MPA after administration of MPM were examined. The plasma to erythrocyte concentration ratio was low (0.10-0.15). MPA existed in rat plasma as the highly bound form (bound fraction was 9.79 +/- 0.57%). MPA disappeared from the systemic circulation with biexponential decay. After i.v. administration of MPM at the doses of 8.3, 16.7, and 33.3 mg/kg, the total clearance of MPA was 0.241 +/- 0.056, 0.321 +/- 0.126, and 0.317 +/- 0.092 L/h/kg, respectively. The terminal elimination half-live were 5.17 +/- 1.44, 8.89 +/- 2.76, and 7.94 +/- 2.94 h, respectively. After i.d. administration of MPM at the doses of 8.3, 16.7, 33.3, and 50.0 mg/kg, the terminal elimination half-live were 6.41 +/- 4.16, 4.49 +/- 2.20, 7.58 +/- 3.72, and 8.18 +/- 1.32 h, respectively. The mean peak times were within 30 min. The systemic availability of MPA after i.d. administration of MPM (at 8.3, 16.7, and 33.3 mg/kg) was calculated using the corresponding mean AUCiv, and the values were 84.3 +/- 35.0%, 69.9 +/- 25.7%, and 63.6 +/- 8.8%, respectively.

Stability and pharmacokinetic studies of a new immunosuppressant, mycophenolate mofetil (RS-61443), in rats.

Mycophenolate mofetil (MPM), a new immunosuppressant, is a morpholinoethyl ester of mycophenolic acid (MPA). The enzymatic and non-enzymatic hydrolysis was studied in an artificial digestive fluid, rat plasma, and tissue homogenates. MPM was chemically stable in the artificial digestive fluid. In rat tissue homogenates and plasma, MPM was rapidly hydrolysed to MPA. The conversion rate of MPM to MPA in various rat tissue homogenates was in the order of liver > kidney > plasma > small-intestine epithelial cells. After the intravenous injection of MPM at 16.7 mg kg-1, the terminal elimination half-life, t1/2 beta, was 4.74 +/- 0.33 (mean +/- SD)h, and the area under the plasma concentration versus time curve, AUC, was 48.78 +/- 6.01 micrograms h mL-1. After intraduodenal (ID) administration of MPM at 16.7 mg kg-1, t1/2 beta was 3.92 +/- 1.05 h, and the AUC was 38.08 +/- 8.30 micrograms h mL-1. The systemic availability of MPA after ID MPM dosing was 1.52 times higher than that after ID administration of MPA. This result supports the usefulness of MPM as an oral prodrug of MPA as a new oral immunosuppressant.

Effects of lansoprazole on pharmacokinetics and metabolism of theophylline.

The effect of the new substituted benzimidizole proton pump inhibitor, lansoprazole, on pharmacokinetics and metabolism of theophylline has been studied in healthy adults given oral lansoprazole 30 mg once daily for 11 days. On Days 4 and 11 of 300 mg aminophylline was simultaneously administered orally and blood samples for theophylline analysis were taken over 24 h. Urine samples were collected for up to 24 h and were assayed for theophylline and its major metabolites 1,3-dimethyluric acid (1,3-DMU), 1-methyluric acid (1-MU) and 3-methylxanthine (3-MX). The pharmacokinetic parameters of theophylline were determined, and the urinary recovery of unchanged theophylline and its major metabolites were calculated. After administration of lansoprazole for 4 days, no significant alteration in the terminal elimination half-life (t1/2beta) or the mean resistance time (MRT) was detected. However, there was a significant decrease of about 13% in the area under the plasma concentration-time curve (AUC) and a significant increase of about 19% in the apparent clearance (CLapp). Lansoprazole treatment for 11 days caused a significant decrease of approximately 12% in t1/2beta and about 10% in the MRT of theophylline, although neither AUC nor CLapp showed a significant alteration. The excretion of 3-MX in the urine was significantly increased by about 20% after lansoprazole treatment for 4 and 11 days, although there was no significant alteration in the excretion of unchanged theophylline, 1,3-DMU or 1-MU. The results indicate that repeated administration of lansoprazole to humans induces the hepatic microsomal P-450-dependent drug oxidation system that mediates N-1-demethylation of theophylline, consequently increasing its metabolism.

Determination of a new immunosuppressant, mycophenolate mofetil, and its active metabolite, mycophenolic acid, in rat and human body fluids by high-performance liquid chromatography.

Mycophenolate mofetil, a new immunosuppressant, is a morpholinoethyl ester of mycophenolic acid. A new selective, sensitive and simple high-performance liquid chromatographic method was developed for the determination of mycophenolic acid and mycophenolate mofetil in biological samples. The preparation of samples was based on liquid-liquid extraction. The compounds were separated on a CN column using acetonitrile-0.01 M phosphate buffer (1:4, v/v) as the mobile phase. UV detection was used at wavelengths 215 and 304 nm. The detection limit was 5 ng per injection volume. This method enabled pharmacokinetic and pharmacodynamic studies in humans and rats.

Inhibitory effect of diltiazem on diazepam metabolism in the mouse hepatic microsomes.

In order to elucidate the drug interaction between diltiazem and diazepam, the effect of diltiazem on the N-demethylation of diazepam in the mouse hepatic microsomes was investigated. Kinetic study showed that diltiazem noncompetitively inhibited the N-demethylation of diazepam with inhibition constant (Ki) value of 247.8 microM, indicating that diltiazem exhibits an inhibitory effect on the hepatic oxidative metabolism of diazepam. It was therefore suggested that diltiazem may impair the metabolism of diazepam in vivo.