Biochemical modulation of 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine incorporation into DNA in VX2 tumor-bearing rabbits.

The thymidine analogues 5-bromo-2'-deoxyuridine (Brd-Urd) and 5-iodo-2'-deoxyuridine (IdUrd) compete with thymidine for incorporation into the DNA of replicating cells. This incorporation results in radiosensitizing effects which are directly related to the degree of analogue substitution. In vitro and in vivo evidence suggests that preadministration or coadministration of the thymidylate synthetase inhibitors fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) can modulate analogue incorporation into DNA. We have evaluated in the rabbit VX2 tumor model the effects of thymidylate synthetase inhibitor (fluorouracil or FdUrd) coadministration (as 24-hour, intravenous infusions) on the incorporation of BrdUrd or IdUrd into the DNA of relevant normal tissues (bone marrow, gut mucosa) and intrahepatic VX2 tumor. Tissues were harvested and processed for gas chromatography-mass spectrometry analysis of the thymine, 5-bromouracil, and 5-iodouracil contents in hydrolyzed DNA. Coadministration of FdUrd resulted in statistically significant (P less than .01) enhancement of IdUrd incorporation into the DNA of intrahepatic VX2 tumor and normal (bone marrow and duodenal mucosa) rabbit tissues. Coadministered fluorouracil, on the other hand, significantly enhanced IdUrd incorporation only into DNA of intrahepatic VX2 tumor. Statistically significant enhancement of BrdUrd incorporation was achieved only with FdUrd coadministration and then only into the DNA of intrahepatic VX2 tumor. The percent of thymine replaced by analogue (I) is related to the steady-state arterial plasma drug concentration (C) by the Michaelis-Menten equation: I = I(MAX.) C/(C50 + C). The primary effect of FdUrd coadministration on BrdUrd incorporation into VX2 tumor DNA was a reduction of the C50 parameter (plasma BrdUrd concentration eliciting I = I(MAX)/2) from 8.17 microM to 1.78 microM. On the other hand, the I(MAX) parameter (I as C approaches infinity) was only slightly affected (29.7% to 25.2%). Thus, the degree to which the modulator enhanced analogue incorporation varied inversely with the analogue's steady-state plasma concentration. These results, which describe potential tissue specificity of modulator efficacy and characterize the effects of thymidylate synthetase inhibitor modulation on thymidine analogue incorporation pharmacodynamics, should provide guidance as to dose scheduling of BrdUrd and IdUrd in clinical trials for improved tumor specificity of uptake.

Comparative bioavailability study of two tablet formulations of cimetidine.

A two-way crossover bioavailability study of two commercial cimetidine formulations was performed on 24 healthy male volunteers. Drug was administered after an overnight fast and plasma samples were withdrawn periodically for 12 h. Urine was collected throughout the study period. Results indicated that the two formulations were bioequivalent since no statistically significant difference in means was detected for any of the parameters studied. Extensive interpatient variation in cimetidine blood concentration was observed during both treatments.

Nonlinear pharmacokinetics of CI-912 in adult epileptic patients.

We studied the pharmacokinetics of CI-912 (1,2-benziosoxazole-3-methanesulfonamide) in 10 adults with refractory partial seizures during an open-label pilot study. Plasma and whole blood concentrations were measured by a high-performance liquid chromatograph method after a single dose and up to and at steady state with one or two dosage regimens. Steady-state clearances averaged only 42% (range 20-60%) of single-dose clearances. Vm and Km values of the Michaelis-Menten equation were calculated for 9 of the 10 patients by a new method: Vm averaged 1,272 (range 500-1,973) mg/day and Km averaged 25.1 (range 9.23-52.5) micrograms/ml. The best initial dosage for a new patient is 300 mg CI-912 every 12 h.

Determination of myocardial and serum digoxin concentrations in children by specific and nonspecific assay methods.

After obtaining samples at open heart surgery, serum and right atrial digoxin concentrations were measured in 25 children by a nonspecific, direct radioimmunoassay method (NS) and by a specific method in which digoxin was separated from its metabolites by HPLC before radioimmunoassay was applied to the digoxin fraction (S). Digoxin was detectable by S assay (sensitivity 0.1 ng/g) in 16 heart specimens and 22 serum samples. The mean and range of the S/NS ratio was 0.74 (0.23 to 2.63) for serum and 0.81 (0.068 to 1.38) for atrial tissue. By NS assay the mean and range of the atrial/serum ratio was 78.1 (2.4 to 340, n = 21) and by S assay the corresponding values were 100 (10.7 to 318, n = 15). A multiple linear regression indicated that 72.5% of the variance of the heart digoxin concentrations measured by S assay were accounted for by the variables height, body weight, daily digoxin dose before operation, plasma digoxin concentration by S assay, and BUN.

The assessment of the intrasubject variability in digoxin absorption in man from two oral dosage forms.

The reproducibility of drug absorption within a given subject as well as the evaluation of bioavailability of two digoxin dosage forms were studied. The data showed (a) a higher initial plasma digoxin concentration after the soft elastic gelatin (SEG) capsule; (b) a more irregular absorption after the tablet; (c) on the average, the coefficients of variation of individual plasma concentrations were lower after the capsule; and (d) for the capsule, the intrasubject variations of the peak plasma concentrations, time of peak, area under plasma concentrations-versus-time curve (AUC), and amount digoxin excreted in urine (Ae) were on the average 60 per cent of the variations in the tablet parameters. The ratios of AUC and Ae for capsule/tablet were essentially unity, indicating that the amount digoxin absorbed from the 0.4-mg digoxin SEG capsule is identical to that from a 0.5-mg standard reference tablet.

Evidence of nonlinearity in digoxin pharmacokinetics.

Six normal male volunteers received 0.5 mg label doses of digoxin as (a) a bolus intravenous injection over 2 min, (b) a constant rate intravenous infusion over 1 hr, (c) a constant rate intravenous infusion over 3 h, and (d) a solution in 5% dextrose given orally. Plasma concentrations of digoxin were measured by radioimmunoassay for a 4 day period and urinary excretion for a 6 day period after the single doses. The mean (coefficient of variation) total areas under the plasma concentration-time curves per 0.5 mg of digoxin were (a) 35.55 (14.8%), (b) 30.20 (27.7%), (c) 25.80 (35.5%), and (d) 15.47 (49.9%); the means differed significantly (0.01 greater than p greater than 0.005). The mean (coefficient of variation) total amounts excreted in the urine as a fraction of the dose were (a) 0.689 (6.31%), (b) 0.517 (20.4%), (c) 0.588 (16.8%), and (d) 0.374 (23.4%); the means differed significantly (p less than 0.001). Both the total clearance and the nonrenal clearance of digoxin differed significantly with the method of intravenous administration. The slower the rate of input of digoxin to the body, the greater were both the total clearance and the nonrenal clearance of the drug, which strongly suggests nonlinear pharmacokinetics.

Plasma protein-binding parameters of prednisolone in immune disease patients receiving long-term prednisone therapy.

Prednisone and prednisolone bind in plasma to albumin and transcortin. In am attempt to determine whether prednisone side effects and/or type of disease correlated with prednisolone plasma protein binding, multiple plasma samples from 17 patients (three asthma, eight SLE, three RA, two PSS, one PAN) receiving long-term prednisone therapy were monitored during an interval between two prednisone doses. Prednisolone plasma protein binding was nonlinear and exhibited large intrapatient and interpatient variability. For the group, mean association constants of the prednisolone-albumin complex and the prednisolone-transcortin complex were 2.3 X 10(3) M-1 and 2.9 X 10(7) M-1, with coefficients of variation of 82% and 127%, respectively. SLE patients tended to have lower mean prednisolone association constants for albumin and transcortin than did other patients. The presence of corticosteroid side effects did not correlate with prednisolone plasma protein-binding parameters. The wide range of prednisolone free fraction noted in plasma from patients who achieved comparable total prednisolone plasma concentrations implies that administration of a uniform prednisone dose will not lead to a predictable clinical response.

Specific and sensitive assays for digoxin in plasma, urine and heart tissue.

A combined internal standard and marker was added directly to urine, then after mixing, an aliquot was injected directly onto a C18 mu-Bondapak HPLC column. The 4.5 ml digoxin fraction was collected over 1.5 min starting 1 min after the appearance of the marker peak (UV detection), and the digoxin determined in the fraction by radioimmunoassay using 3H-digoxin. Digoxin was extracted from alkalinized plasma into dichloromethane. After evaporating the extract and addition of internal standard the remainder of the assay was as described above for urine. Multiple samples of urine and plasma collected in a single dose bioavailability trial in normal human volunteers were assayed by one of these specific methods as well as by the usual direct nonspecific RIA method. There were no significant differences in results by the specific and nonspecific methods. Heart tissue was homogenized with internal standard and phosphate buffer than the mixture was centrifuged. The supernatant was extracted with dichloromethane, the solvent evaporated from the extract, the residue redissolved in mobile phase and the remainder of the assay carried out as for urine and plasma.

Suppression of plasma hydrocortisone levels. A poor estimate of rectal absorption of glucocorticoids.

The relative degree of absorption of methylprednisolone acetate when administered by the rectal and oral routes was studied in 12 normal male subjects. The plasma samples were assayed for methylprednisolone and endogenous hydrocortisone concentrations. After the administration of equal doses of methylprednisolone acetate by the rectal and oral routes, the plasma hydrocortisone levels were not significantly different from each other; but the plasma concentrations of methylprednisolone were significantly lower after rectal administration than after oral administration. We conclude that the practice of utilizing the degree of suppression of endogenous hydrocortisone levels as an indicator of the extent of absorption of rectally administered glucocorticoids is inappropriate.

Rectal and oral absorption of methylprednisolone acetate.

Rectal absorption of methylprednisolone acetate and oral absorption of methylprednisolone and methylprednisolone acetate were investigated in a single-dose 3-way crossover study of 12 normal male volunteers. The median value of bioavailability (relative to oral dose) of methylprednisolone acetate based on unchanged methylprednisolone plasma levels was 14.2% after rectal administration, suggesting that the drug exerts its therapeutic effect topically rather than systemically. In contrast, the median of total radioactivity in urine (as a percentage of rectal dose) was 34.3% (range, 4.52% to 58.8%), suggesting partial bacterial metabolism in the rectum prior to absorption. Mean bioavailability (relative to oral administration of methylprednisolone acetate) of methylprednisolone after oral administration was 89.9%, indicating somewhat better systemic availability of the ester than the alcohol. The average apparent elimination rate constant for methylprednisolone after oral administration of both ester and alcohol was 0.290 hr-1, corresponding to a half-life of 2.39 hr.

Comparison of the in vitro and in vivo release of digoxin from four different soft gelatin capsule formulations.

A blinded, four-treatment crossover study in 16 normal adult male volunteers compared plasma concentrations and urinary excretion of digoxin, measured by radioimmunoassay, after oral administration of soft gelatin capsule formulations of digoxin. Four 0.4-mg formulations with different in vitro "burst times" and dissolution rates were administered, with 2-week intervals between treatments. The two capsules with lowest in vitro burst times (2.9 and 16 min) gave comparable in vivo results. The other two capsules, with in vitro burst times of 62 and 229 min, produced significant delays in digoxin absorption. In vitro-in vivo correlations were obtained by comparing the logarithm of the in vitro burst time with time to peak plasma level and the time to the first measurable plasma level (> or = 0. 05 ng/ml). Also, the mean time to peak plasma level correlated with the logarithm of the time required to release either 50% or 85% of the digoxin in vitro. No significant changes were found in the amount of digoxin absorbed from each capsule as determined by urinary excretion or AUC0-infinity.

Influence of kaolin--pectin suspension on digoxin bioavailability.

The effect of a kaolin--pectin suspension on the bioavailability of orally administered digoxin was evaluated when both drugs were given concomitantly and when their time of administration was separated by 2 hr. Coadministration of the antidiarrheal with the cardiac glycoside delayed absorption of the latter and, at the same time, decreased by 62% the amount of drug absorbed. Intersubject variation in digoxin bioavailability also was increased more than twofold. When the kaolin--pectin suspension was given 2 hr before the cardiac glycoside, the digoxin absorption rate was not affected, although its relative extent of absorption was reduced by about 20%. In contrast, when the antidiarrheal was given 2 hr after digoxin, neither the rate nor the extent of absorption of the cardiac glycoside was perturbed. No change in the intersubject variability in digoxin bioavailability was noted whether the antidiarrheal was given 2 hr before or 2 hr after the cardiac glycoside.

Preliminary in vitro and in vivo investigations on methylprednisolone and its acetate.

Sodium fluoride (6--8 mg/ml) inhibits hydrolysis of methylprednisolone acetate to methylprednisolone. An HPLC method for simultaneous determination of hydrocortisone, methylprednisolone and methylprednisolone acetate in plasma is presented. Analysis of plasma samples (containing NaF) for methylprednisolone acetate shows no significant change in concentration over extended periods of storage at -20 degrees C. In vitro hydrolysis of methylprednisolone acetate at 37 degrees C in human whole blood is rapid (average t1/2 = 19 min). In one cat, the bioavailabilities of methylprednisolone acetate rectally was 13% and of methylprednisolone (alcohol) rectally was 26%, relative to intravenous administration of methylprednisolone. In the same cat, the bioavailabilities of methylprednisolone acetate orally was 93% and of methylprednisolone was 82%, relative to intravenous administration of methylprednisolone. All samples collected after oral administration of methylprednisolone acetate to a human subject were found to contain only methylprednisolone (alcohol) indicating hydrolysis of the drug during absorption through the gastrointestinal membrane and/or in the liver. If the ester had the same half-life in blood in vivo as measured in vitro, it would have been measurable in plasma.

Pharmacokinetics of digoxin in the cat and comparisons with man and the dog.

Digoxin doses of 14.3, 5.72 or 1.43 mcg/kg were administered to 8 cats and the plasma digoxin concentrations (concns) were measured as a function of time for 96 hr. Pharmacokinetic linearity was evidenced by: (1) mean plasma clearance (Clp) of 4.05 ml/(kg x min) with a coefficient of variation of 34.6% and a range of 2.69-6.71; (2) linear relationship between total area under the plasma concn-time curve and the administered dose. Further evidence for linearity was the lack of dose-dependence of apparent elimination half-life (beta). The volume of distribution and beta for cats were respectively (mean +/- s.d.) 20.4 +/- 5.35 L/kg and 0.0120 +/- 0.0030 hr-1. Results from cats were compared with literature data for dogs and man. The order of Clp in units of ml/(kg x min) were: infant greater than dog greater than cat greater than adult man.

Linear and nonlinear assessment of tolmetin pharmacokinetics.

Plasma concentration-time data in man after oral dosing of the nonsteroidal antiinflammatory agent, tolmetin sodium, were fitted to both linear and nonlinear pharmacokinetic equations. The apparent elimination half-life of tolmetin is shown to be 4.5 to 6.0 hours. An analytical method is presented which allows measurement of plasma concentrations for at least 32 hr (compared with a previous limit of about 8 hr) after a single oral dose of the equivalent of 400 mg of tolmetin acid.

Sensitive radioimmunoassay for digoxin in plasma and urine.

A reproducible and sensitive radioimmunoassay for digoxin in either serum, plasma or urine is described. Using 0.5 ml of serum or plasma, the assay sensitivity is 0.05 ng of digoxin/ml. The antiserum and tracer solutions employed are available in a kit sold in the United States. All other reagents were prepared in the laboratory. The assay allows measurement of digoxin in plasma or serum for 96 hours after single 0.5 mg doses of digoxin; this is necessary in human bioavailability studies to accurately estimate the total area under the digoxin concentration, time curve from zero to infinite time. In contrast, with the kit assay, employing 0.2 ml of plasma or serum, it has been reported that the 12 hr serum digoxin levels, after single 0.5 mg doses, are, in most subjects, below the sensitivity limit (about 0.5 ng/ml) of the assay.