Peter Seideman

Trace analysis of methotrexate and 7-hydroxymethotrexate in human plasma and urine by a novel high-performance liquid chromatographic method.

A new high-performance liquid chromatographic method for the quantitative determination of methotrexate (MTX) and its metabolite 7-hydroxymethotrexate (7-OHMTX) in blood plasma and urine was developed. The method utilized a solid-phase extraction procedure (Certify II cartridges) for the simultaneous isolation of MTX and 7-OHMTX. Chromatographic separation was achieved using a C18 reversed-phase column with isocratic elution. The eluent was irradiated with UV light of 254 nm, which converted MTX and 7-OHMTX by photolytic oxidation to fluorescent products. The limits of detection of MTX and 7-OHMTX in plasma were approximately 0.2 and 1 nmol/L, respectively. The intraday variability in the quantitation of MTX and 7-OHMTX was less than 8% down to 1 nmol/L and 4.6 nmol/L, respectively. Both MTX and 7-OHMTX could be detected in plasma from a patient being treated for rheumatoid arthritis 1 week after the last dose (10 mg orally).

Simultaneous quantitation of methotrexate and its two main metabolites in biological fluids by a novel solid-phase extraction procedure using high-performance liquid chromatography

We have developed an assay for the simultaneous determination of methotrexate (MTX) and its main metabolites, 7-hydroxymethotrexate (7-OHMTX) and 2,4-diamino-N10-methylpteroic acid (DAMPA) in plasma, urine and saliva meeting the requirement of rapidity for routine use in high-dose MTX therapy and the requirement of sensitivity for its potential use in therapeutic drug monitoring in low-dose MTX therapy. Sample preparation is based on solid-phase extraction using C8 Isolute cartridges. Chromatographic separation was achieved with a reversed-phase column (C18), and quantitation by subsequent exposure to UV light of 254 nm, which converted MTX and its two metabolites by photolytic oxidation to fluorescent products. The recoveries of MTX, 7-OHMTX and DAMPA from plasma at 100 nmol/l were 85.8, 91.1 and 102.3%, respectively. The limits of detection for MTX, 7-OHMTX and DAMPA in plasma and saliva were 0.1 nmol/l. In urine the limit of detection was 10 nmol/l for all compounds. The limits of quantitation in plasma and saliva were 0.5 nmol/l for all compounds.

Prazosin kinetics in hypertension

Prazosin kinetics were studied after single doses (intravenous and oral, 0.5 mg) and after increasing multiple doses (0.5 to 5 mg three times daily) in eight patients with hypertension. After intravenous administration the kinetics could be described by a linear two‐compartment open model. Terminal half‐life (t½β) was about 3 hr and apparent volume of distribution (Vdβ) about 0.6 l/kg. After oral doses bioavailability ranged between 55% and 82%. Since total plasma clearance was low (0.14 l/kg x hr) incomplete bioavailability was the result of incomplete absorption rather than of first‐pass liver metabolism. The estimated extraction ratio was about 14%. Renal clearance was negligible; only 1% to 2% of the dose was recovered unchanged in urine. Binding to plasma proteins to both albumin and abatid glycoprotein was substantial (97%), with albumin being most important. Increasing multiple doses showed that prazosin followed first‐order kinetics with a linear correlation between dose and steady‐state plasma concentration (P < 0.001). There were substantial variations in plasma concentrations between patients and there were also day‐to‐day variations in concentration within the same patient.

Naproxen and paracetamol compared with naproxen only in coxarthrosis: Increased effect of the combination in 18 patients

In a double-blind study of 18 patients with coxarthrosis the effect of 3 naproxen doses (0.5, 1.0, and 1.5 g daily) and 2 naproxen doses combined with paracetamol (0.5 g + 4 g daily and 1.0 g + 4 g daily) was investigated. Plasma levels of naproxen and paracetamol were measured (HPLC), and clinical assessment of pain, joint movement, activity of daily life and side-effects were performed at the end of the 5 treatment periods. A relationship was found between the 3 naproxen doses, naproxen plasma levels, pain at rest, and pain during movement. The combined treatment was more effective than treatment with the same naproxen dose alone. The effect of naproxen (0.5 g daily) combined with paracetamol (4 g daily) did not differ from that obtained during treatment with higher naproxen doses only. Furthermore, the effect of the highest naproxen dose was not better than the effect of the lower naproxen dose (1.0 g daily) combined with paracetamol. The main finding was that treatment with naproxen and paracetamol is more effective than treatment with higher naproxen doses alone.

Methotrexate in juvenile rheumatoid arthritis

Children with juvenile rheumatoid arthritis (JRA) have been reported to require higher doses (per kg body weight) of methotrexate (MTX) than adults with rheumatoid arthritis to control their disease. The purpose of the present study was to characterise the plasma pharmacokinetics of MTX and its major metabolite, 7-hydroxymethotrexate (7-OHMTX) in children, and to compare the results with those previously obtained in adults. Thirteen patients (age 5–16 y) with JRA (median disease duration 5.5 y) were studied after once weekly oral administration of MTX (median 0.21 mg·kg−1).The analytical method was sufficiently sensitive to permit determination of plasma and urinary concentrations of MTX and 7-OHMTX during the entire dose interval in most of the patients.The dose normalized area under the plasma concentration versus time-curve (AUC) of MTX increased with the age of the children and was lower than previously found in adults. The dose normalized AUC of 7-OHMTX was not dependent on age. No correlation was found between the AUCs of MTX and 7-OHMTX.The results suggest that the age-dependence of the pharmacokinetics of MTX might explain the observation that at least some children require higher doses of MTX than adults to obtain a sufficient therapeutic effect.

α‐Receptor function changes after the first dose of prazosin

The effect of oral prazosin on blood pressure and antagonism of phenylephrine‐induced blood pressure increase was investigated in six healthy subjects during a dosing interval after the first dose and 3 days after the first dose of the drug. Prazosin lowered standing blood pressure more after the first dose than after the same dose 3 days later, despite similar plasma levels. Blood pressure decrease correlated with plasma prazosin levels during the elimination phase at the first dose, but not after 3 days of therapy. The phenylephrine log dose‐response curves shifted to the right after prazosin, which indicates α‐receptor antagonism of the drug. On day 4, the phenylephrine curve before prazosin dose shifted to the right of the pretreatment curve on day 1, despite very low prazosin plasma levels. On day 4 after prazosin dosing the phenylephrine dose‐response curves were shifted to the left of that on day 1. Our data indicate tolerance to prazosin effect on blood pressure and to phenylephrine agonism after 3 days dosing. Our data suggest that this might be due to de sensitization of α1‐adrenoceptors with differential effects of agonists and antagonists.

Influence of pentobarbital on metoprolol plasma levels

Induction of microsomal enzymes with barbiturates in rats has little effect on the metabolism of metoprolol, compared with propranolol and alprenolol, which undergo extensive hepatic extraction in animals and man. Our study was designed to examine whether the metabolism of metoprolol is inducable by barbiturate in man. In 8 healthy subjects the area under the plasma concentration/time curve after 0.1 gm metoprolol was reduced by a mean of 32% after treatment with 0.1 gm pentobarbital at bedtime for 10 days. There was considerable interindividual variability in the reduction after pentobarbital treatment (2% to 46%).

ADDITIVE INTRAOCULAR PRESSURE REDUCING EFFECT OF TOPICAL TIMOLOL DURING SYSTEMIC β-BLOCKADE

The effect on intraocular pressure (IOP) of topical timolol was studied in 30 patients with arterial hypertension treated with oral alprenolol, metoprolol or timolol. No patient had any known eye disorder. Topical administration of timolol induced a significant reduction of IOP regardless if the patients had systemic β‐blockade or not. Treatment with topical timolol alone was equally effective in reducing IOP as combined topical and oral therapy. Patient compliance was checked with plasma concentrations of the different β‐blockers.

Methotrexate in Rheumatoid Arthritis—a Limited Sampling Strategy for Estimation of the Area Under the Plasma Concentration Versus Time Curve

A limited sampling strategy for determination of the area under the plasma concentration versus time curve (AUC) of methotrexate (MTX) in patients with rheumatoid arthritis (RA), treated with weekly oral doses, has been validated. Stepwise linear regression analysis was used for optimal inclusion of data points in mathematical models to estimate AUC. A new plot for evaluation of the accuracy and precision of the estimated AUC values was introduced in the present study. By plotting the ratio of determined/estimated AUC values versus estimated AUC values, the influence of number of sampling points on the precision and accuracy of estimated AUC values was easily validated. Our results show that AUC values of MTX in RA patients can be estimated from a single plasma sample at 3 h or preferably, due to increased precision, by additional samplings at 5 and 1 h. A further increase of the number of sampling points increased the precision of the AUC estimates only to a minor extent. The accuracy of the estimated AUC values was independent of the number of sampling points. A limited sampling procedure can now be used for further studies on the relationship between MTX levels and its effects.

Prazosin dynamics in hypertension: relationship to plasma concentration.

The antihypertensive effects of prazosin in relation to its kinetics were studied after single doses (intravenous and oral, 0.5 mg) and during increasing multiple doses (0.5 to 5 mg three times a day). There was a fall in systolic and diastolic blood pressures of 10% to 14%, which was greater in the standing than in the sitting position. Prazosin plasma concentrations correlated with dose (P < 0.001). After intravenous prazosin the fall in systolic and diastolic blood pressure and prazosin plasma concentration correlated (P < 0.01) during the β‐elimination phase in all patients. In only five of eight patients, however, did mean plasma concentration and antihypertensive effect during continuous treatment with different doses correlate. The maximal fall in systolic blood pressure correlated (P < 0.01) with that after the first oral steady‐state dose (0.5 mg three times daily), which indicates limited possibility of early identification of prazosin responders. There were no signs of overshoot of blood pressure when prazosin was withdrawn for a week. On rechallenge with a single oral dose of 2.5 mg prazosin there were no signs of enhanced hypotensive effect.