Christer von Bahr

Protein Binding of Diphenylhydantoin and Desmethylimipramine in Plasma from Patients with Poor Renal Function

Abstract The protein binding of diphenylhydantoin (DPH) and desmethylimipramine (DMI) was measured in plasma from azotemic and uremic patients by an Ultrafiltration technic. DPH binding was decreased in uremic plasma. The impairment of binding was strongly correlated (p less than 0.001) to the degree of azotemia and degree of physical disability of the patients but weakly correlated (p less than 0.05) to their concentrations of serum proteins. DMI binding was almost normal. Dialysis of normal and uremic plasma in tap water or hemodialysis solution did not alter the DPH binding. The binding of DPH by plasma proteins from azotemic patients appears to be decreased, probably owing to a change in the binding proteins. The possibility of alteration of drug binding by pathologic states should be considered when total plasma concentrations of protein-bound drugs are measured and the values used to establish or modify drug-dosage regimens.

[62] Glutathione transferases from human liver

Publisher Summary This chapter investigates glutathione transferases derived from human liver. The glutathione transferases are a group of related enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds bearing an electrophilic center. The proteins also act as intracellular binding proteins for a large number of lipophilic substances, including bilirubin. Human glutathione transferases have been purified from liver, erythrocytes, placenta, and lung. A simple and rapid procedure for the purification of basic (α-ɛ) and neutral (μ) glutathione transferases from human liver cytosol is described in the chapter. The enzyme activity during purification is determined spectrophotometrically at 340 nm by measuring the formation of the conjugate of glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The steps of the purification procedure include (1) preparation of cytosol fraction, (2) chromatography on Sephadex G-25, (3) chromatography on DEAE-cellulose, and (4) chromatography on Sephadex G-25.

Drug metabolism in human liver in vitro: Establishment of a human liver bank

Marked species differences in xenobiotics metabolism in the liver seriously limit extrapolations from animals to man. Because access to human liver is limited and periodic, we have set up a human “liver bank” available for metabolic studies. The liver tissue is obtained shortly after circulatory arrest from cadaveric (cerebral infarction) kidney transplant donors. Postmortem changes are minimal. Subcellular liver fractions are prepared immediately and part of this is used directly for assay. Intact pieces and subcellular fractions are stored in different media at −80°. Each liver is characterized by light and electron microscopy. Several enzymes, including cytochromes P‐450 and b5, NADPH‐cytochrome c reductase, demethylation of aminopyrine and amitriptyline, epoxidation of carbamazepine, oxidation of acetaminophen, and benzo[a]pyrene, were tested with freshly prepared fractions so that each liver got a “drug metabolic profile.” This “test battery” was repeated after storing to evaluate the effect of storage. Our preparation technique gave a well‐preserved microsomal fraction with minimal contamination. In freshly prepared microsomes the following activities (levels) were observed: cytochrome P‐450, 0.13 to 0.73 nmole/mg protein; NADPH‐cytochrome c reductase, 70 to 426 nmole/mg protein; demethylation of aminopyrine, 0.9 to 4.1, and of amitriptyline, 0.11 to 0.92 nmole/mg protein; carbamazepine‐10,11 epoxidation, 0.03 to 0.46 nmole/mg protein; oxidation of acetaminophen, 0.48 to 2.11, and of benzo[a]pyrene, 0.04 to 0.11 nmole/mg protein · min. These values are generally higher than in the literature. Our storage conditions were efficient: most of the activities were well preserved during storage for at least 6 mo. When pairs of enzyme activities (levels) were plotted against each other with fresh tissue there was good correlation between some but not all activities.

Plasma levels of thioridazine and metabolites are influenced by the debrisoquin hydroxylation phenotype.

The pharmacokinetics of thioridazine and its metabolites were studied in 19 healthy male subjects: 6 slow and 13 rapid hydroxylators of debrisoquin. The subjects received a single 25 mg oral dose of thioridazine, and blood samples were collected during 48 hours. Concentrations of thioridazine and metabolites in serum were measured by HPLC. Slow hydroxylators of debrisoquin obtained higher serum levels of thioridazine with a 2.4‐fold higher Cmax and a 4.5‐fold larger AUC(0‐∞) associated with a twofold longer half‐life compared with that of rapid hydroxylators. The side‐chain sulphoxide (mesoridazine) and sulphone (sulphoridazine), which are active metabolites, appeared more slowly in serum and had lower Cmax values, but comparable AUC. The thioridazine ring‐sulphoxide attained higher Cmax and 3.3‐fold higher AUC in slow hydroxylators than in rapid hydroxylators of debrisoquin. Thus the formation of mesoridazine from thioridazine and the 4‐hydroxylation of debrisoquin seem to be catalyzed by the same enzyme, whereas the formation of thioridazine ring‐sulphoxide is probably formed mainly by another enzyme.

Purification of a new glutathione S-transferase (transferase μ) from human liver having high activity with benzo(α)pyrene-4,5-oxide

A new glutathione S-transferase from human liver has been purified to homogeneity in good yield by use of ion-exchange chromatography on DEAE-cellulose, affinity chromatography on S-hexylglutathione coupled to epoxy-activated Sepharose 6B, and chromatography on hydroxyapatite. This new enzyme, transferase μ, is present in high concentration, but only in some individuals. It has an isoelectric point at about pH 6 to 6.5 and a different substrate specificity than the previously described alkaline transferases α-e from human liver. Especially noteworthy is the finding of high activity against benzo(α)pyrene-4,5-oxide. Glutathione S-transferase μ has about 20-fold higher activity with this substrate than have the alkaline transferases. The most pronounced difference was found with trans-4-phenyl-3-buten-2-one which was >100-fold better as substrate for transferase μ than for the previously described transferases.

Phenotypic consistency in hydroxylation of desmethylimipramine and debrisoquine in healthy subjects and in human liver microsomes

The 2‐hydroxylation of desmethylimipramine (DMI) and the 4‐hydroxylation of debrisoquine (D) were studied in healthy subjects and in human liver microsomes. A single oral dose of DMI (25 mg) was given to 18 healthy subjects previously phenotyped with D (13 rapid and five slow hydroxylators). Urine was collected for 24 hr and DMI and total 2‐hydroxydesmethylimipramine (2‐OH‐DMI) levels were determined by HPLC. The urinary ratio DMI/2‐OH‐DMI correlated strongly (r = 0.92) with the urinary ratio of D to 4‐hydroxydebrisoquine (D/4‐OH‐D). The two hydroxydations were also studied in human liver microsomes from 10 different subjects. Formation rates of the hydroxylated metabolites correlated strongly (r = 0.869). Moreover, D competitively inhibited the 2‐hydroxylation of DMI. These findings suggest that both are hydroxylated by the same cytochrome P‐450 isozyme.

Effect of pentobarbital on the disposition of alprenolol.

Alprenolol was administered orally and intravenously to 5 healthy subjects before and after IO to 14 daily doses of 0.1 gm pentobarbital. The area under the plasma concentration time curve after an oral 200‐mg dose decreased from 706 ± 277 to 154 ± 48 ng/ml . hr (mean and SD) with the barbiturate treatment, but there was no significant change in elimination rate. The change in area corresponded to an increase in extraction by the liver from 0.72 ± 0.13 to 0.93 ± 0.01. The disposition ofa 5.0‐mg intravenous dose of alprenolol did not change significantly after pentobarbital treatment. There was no indication of a marked change in hepatic blood flow estimated from the clearance of alprenolol after intravenous administration. It is concluded that pentobarbital administration induces the metabolism ()f alprenolol in man and that the pharmacokinetic theories derived for hepatic extraction of drugs subject to a high metabolic clearance can be successfully applied.

Orally given melatonin may serve as a probe drug for cytochrome P450 1A2 activity in vivo: A pilot study

Melatonin is a hormone that is metabolized by cytochrome P450 (CYP) 1A2 to its main primary metabolite 6‐hydroxymelatonin. We therefore evaluated the utility of oral melatonin as a marker of hepatic CYP1A2 activity.

Simultaneous determination of d- and l-propranolol in human plasma by high-performance liquid chromatography.

A method for the determination of d- and l-propranolol in human plasma is described. The method involves extraction of propranolol from plasma, and the formation of diastereomeric derivatives with the chiral reagent N-trifluoroacetyl-1-prolylchloride. Separation and quantitation of the diastereomeric propranolol derivatives are carried out by a reversed-phase high-performance liquid-chromatographic system with fluorimetric detection. The reproducibility in the determination of d- and l-propranolol in human plasma was 4.5% (relative standard deviation) at drug levels of 10 ng/ml. In two subjects who received a single 40-mg tablet of racemic propranolol the plasma levels of the d-isomer were lower than of the l-propranolol. The half-lives of d- and l-propranolol were similar.

Time course of enzyme induction in humans: Effect of pentobarbital on nortriptyline metabolism

To study the effect of induction we gave six male volunteers 10 mg nortriptyline three times a day for 4 weeks and 0.2 gm pentobarbital on days 8 to 21. Plasma and urinary levels of nortriptyline and metabolites were measured. The rate and extent of induction of the enzyme(s) were estimated by a model with use of nortriptyline concentrations. There was a marked decrease of nortriptyline levels after 2 days of pentobarbital treatment. Total clearance of nortriptyline increased more than twofold (range, 1.6‐fold to 4.1‐fold). Apparent metabolic clearance by 10‐hydroxylation increased markedly. The decrease in nortriptyline levels was more rapid than the increase after pentobarbital cessation, fitting with the theory of the model. The induction of nortriptyline metabolism is probably mainly the result of an increase in a non‐CYP 2D6 P450 isozyme, possibly CYP 3A4 or a CYP 2C form. More knowledge of induction characteristics of drugs should lead to better predictions of decreased effects and appearance of adverse effects. The kinetic model used for analysis of our data could then be useful.