Pyridostigmine kinetics in healthy subjects and patients with myasthenia gravis.

Comparative pyridostigmine kinetics in plasma were measured in 10 healthy subjects given 4 mg iv and 60 mg oral pyridostigmine bromide. As determined from the AUC ratio, oral availability was 11.5% to 18.9% (means = 14.3%). Mean t 1/2 of the plasma level decline after oral dosing was 200 minutes, twice as long as the terminal elimination t1/2 after intravenous infusion (97 minutes). Thus absorption may proceed at a slower rate than elimination. Comparison of intraindividual data revealed strict dependence of the AUC on the infused dose (2, 4, and 8 mg) in one subject and variability in AUC up to a factor of two when two subjects took oral pyridostigmine three times. Patients with myasthenia who were receiving continuous therapy with oral pyridostigmine had AUC values per unit dose corresponding to those in healthy subjects. Storage stability of pyridostigmine in plasma required acidification of samples and storage at -75 degrees C. When native plasma was kept at -20 degrees C, there was appreciable loss of pyridostigmine within 1 to 2 months, the extent of which depended on the initial concentration.

Enantioselective amitriptyline metabolism in patients phenotyped for two cytochrome P450 isozymes.

In 26 hospitalized patients with depression, a combined pharmacogenetic test with dextromethorphan, a substrate of cytochrome P450IID6, and mephenytoin, the S-form of which is hydroxylated by a P450IIC isozyme, was carried out before amitriptyline therapy. Metabolites were determined in 24-hour urine samples collected on treatment day 8, and the contributions of individual compounds, including the four isomers of 10-hydroxyamitriptyline and 10-hydroxynortriptyline to total excretion were calculated. Formation of (-)-E-10-hydroxyamitriptyline and (-)-E-10-hydroxynortriptyline apparently depends on the activity of cytochrome P450IID6 because negative correlations existed between the log metabolic ratio of dextromethorphan and the relative quantities of these enantiomers. In contrast, correlations were positive for nortriptyline, (+)-E-10-hydroxynortriptyline, (-)-Z-10-hydroxynortriptyline, and (+)-Z-10-hydroxynortriptyline. The mephenytoin hydroxylase seems to participate in side-chain demethylation to the secondary and primary amines, because the log metabolic ratio of mephenytoin correlated negatively with the relative quantity of E-10-hydroxydidesmethylamitriptyline and positively with that of amitriptyline and its N-glucuronide.

High-affinity stereoselective reduction of the enantiomers of ketotifen and of ketonic nortriptyline metabolites by aldo-keto reductases from human liver.

Aldo-keto reductases (AKR) form an enzyme superfamily catalyzing the reduction of carbonyl compounds and in some cases the reverse oxidation of alcohols as well. In particular, a role in drug metabolism has been considered for the AKR1C family, but published data failed to reveal low Km drug substrates. Moreover, structure activity relationships using chemically related substrates have not been established. In the present investigation, a modified procedure was developed for the isolation of AKR1C1, 1C2, and 1C4 (dihydrodiol dehydrogenases 1, 2, and 4) from human liver cytosol along with carbonyl reductase (EC 1.1.1.184), a member of the short-chain alcohol dehydrogenase superfamily. The kinetics of NADPH-dependent reduction by the closely related enzymes AKR1C1 and 1C2 were studied with the structurally similar substrates (R)- and (S)-ketotifen and E- and Z-10-oxonortriptyline by HPLC measurement of the products. Km values varied between 2.6 and 53 microM and Vmax values between 5 and 313 mU/mg protein; substrate inhibition with Ki around 30 microM occurred in the reduction of E- and Z-10-oxonortriptyline by AKR1C1. The reactions were strictly stereospecific with production of one enantiomeric alcohol from each ketotifen enantiomer and of the (+)-enantiomers of E- and Z-10-hydroxynortriptyline. Enzymatic NADP+ -dependent oxidation of the alcohols mirrored the reduction with regard to stereochemical specificity. All four ketones were no or poor substrates of carbonyl reductase, whereas haloperidol was reduced by this enzyme with low affinity, but high efficiency.

Increased binding of desmethylimipramine in plasma of phenobarbital-treated rats.

In plasma of untreated male Wistar rats the mean free fraction of desmethylimipramine (DMI) amounted to 10.5%. A five-day oral treatment with phenobarbital (PB) reduced it to 6.2%. A similar effect was produced in female Wistar rats and in male, but not in female Sprague-Dawley rats. Pretreatment with DDT did not alter DMI binding. The PB effect not be attributed to the presence of PB or its metabolites in plasma nor to lower levels of endogenous compounds attached to plasma proteins. Studies on single DMI-binding proteins in plasma of male Wistar rats disclosed unchanged concentrations and binding properties of albumin and total lipoproteins following PB administration, while alpha 1-acid glycoprotein isolated from plasma of PB-treated animals bound DMI stronger than that from controls and contained a higher percentage of N-acetylneuraminic acid. The enhanced binding to a chemically altered alpha 1-acid glycoprotein species is at least one factor responsible for decreased tissue-plasma concentration ratios of DMI in PB-treated rats and constitutes an unusual type of drug interaction.

Urinary metabolites of amitriptylinoxide and amitriptyline in single-dose experiments and during continuous therapy.

In a cross-over design, six healthy volunteers received 50 mg amitriptylinoxide (AT-NO) IV and orally and 50 mg amitriptyline (AT) IV. Urine was collected completely for 8 h and occasionally up to 48 h. In addition, five patients each under treatment with AT-NO or AT for tension headache collected 24-h urine samples. The following compounds were analysed by HPLC: AT-NO, E- and Z-10-hydroxy-AT-NO (E- and Z-10-OH-AT-NO), free and conjugated AT, E- and Z-10-OH-AT and their mono- and didemethylated analogues, and 2-OH-nortriptyline (2-OH-NT). Unchanged AT-NO in urine accounted for an average of 34% and 22% of the single IV and oral doses, respectively, and for 28% in continuous therapy, with a further 8-9% being excreted as E- and Z-10-OH-AT-NO. The remaining part was converted to the same metabolites as was AT. In the steady state the measured compounds accounted for 74% and 77% of the daily AT-NO and AT doses, respectively. The renal plasma clearance of AT-NO varied between 75 and 265 ml/min in the six volunteers. Tubular secretion must play an important part in the renal excretion of AT-NO.

Single-dose kinetics of the neuroleptic drug perazine in psychotic patients.

Eight male and two female unmedicated psychotic patients received 100 mg perazine orally and seven blood samples were taken within 25 h. Plasma levels of perazine and its demethylated metabolite were analyzed by HPLC with electrochemical detection. They exhibited large interindividual variations, with maximal concentrations as well as AUC values of perazine differing more than 10-fold. From the decay of plasma levels during the last 12-18 h half-lives were estimated to be between 7.5 and 10 h; they did not correlate with AUC. There was a significant positive correlation between AUC and age. Desmethylperazine was consistently present at lower concentrations than the parent drug during the first 12 h.

Do urinary MHPG and plasma drug levels correlate with response to amitriptyline therapy?

Twenty-nine inpatients with primary affective disorder were treated with 150 mg amitriptyline (AT) daily for 28 days. Pretreatment urinary excretion of 3-methoxy-4-hydroxyphenylglycol (MHPG) was measured in two or three 24-h urine samples. Plasma levels of AT and nortriptyline (NT) were determined after 14, 21, and 28 days of treatment. MHPG excretion was significantly correlated with clinical response to treatment. Responders defined by two different methods showed higher pretreatment MHPG excretion than nonresponders. Correspondingly, high MHPG excretors (median split) showed significantly more improvement than low excretors. These relationships were even more apparent when possibly incomplete urine samples (creatinine excretion below 1000 mg/24h) were excluded. The high and low MHPG subgroups did not significantly differ from each other in their plasma levels of AT, NT, or AT plus NT. A significant rank correlation between clinical response and plasma levels of AT and/or NT did not exist, but there was a trend towards lower levels in responders.

Antidepressive effect and pharmacokinetics of amitriptyline with consideration of unbound drug and 10-hydroxynortriptyline plasma levels.

In 27 inpatients with primary affective disorder the urinary excretion of 3-methoxy-4-hydroxyphenylglycol (MHPG) was measured prior to a 4-week treatment with 150 mg amitriptyline (AT)/day. Ratings according to the Hamilton depression scale were performed before therapy and repeated after 2 and 4 weeks. Plasma levels of AT, nortriptyline (NT), and E-10-hydroxynortriptyline (OHNT) were assayed weekly, and binding of AT to plasma proteins was determined in one sample. Better therapeutic results were obtained at intermediate, as compared to low and high concentrations of AT or AT plus NT. Independent evaluation of AT and metabolite levels revealed that patients with AT of 50--125 ng/ml responded particularly well when NT did not exceed 95 ng/ml or when NT plus OHNT was below 150 ng/ml. Outside this "therapeutic window' the outcome was markedly poorer. Interindividual variation of AT binding was much smaller than variation of total concentrations. Evaluation of free, instead of total levels did not help to clarify the relationship between clinical and pharmacokinetic variables. Plasma levels within the optimal ranges were found in more patients with high than with low MHPG excretion. The free fraction of OHNT in plasma of healthy subjects was about 35%.

The contribution of alpha 1-acid glycoprotein, lipoproteins, and albumin to the plasma binding of perazine, amitriptyline, and nortriptyline in healthy man.

Parameters for the binding of perazine (PER), amitriptyline (AT) and nortriptyline (NT) to plasma and to single plasma proteins were determined by equilibrium dialysis. The highest affinity (K at least 10(5) M-1) and lowest capacity (first site 1 mol/mol) towards all three drugs was exhibited by alpha 1-acid glycoprotein (alpha 1-AGP). From the parameters, alpha 1-AGP was estimated to contribute 43% to total binding of PER and 49 and 31%, respectively, to AT and NT binding in samples with normal protein concentrations. Fractions bound to total lipoproteins would amount to 32% (PER), 40 (AT) and 52% (NT), respectively, while the contribution of albumin would range from 11% (AT) to 25% (PER). The extent of the binding to plasma was compared with that to single proteins and their mixtures. Binding to combinations of alpha 1-AGP, lipoproteins and albumin exceeded that to plasma with PER but not with AT and NT. This leads to the assumption that additional plasma constituents interfere with PER binding.

Serum albumin stimulates serotonin uptake into human blood platelets.

Active uptake of serotonin (5-hydroxytryptamine, 5-HT) into blood platelets from healthy donors exhibited a lower Vmax value in buffer media than in plasma. Also in plasma ultrafiltrate Vmax was reduced, but it returned to the level measured in plasma upon addition of human serum albumin (40 milligrams) containing fatty acids. Fatty-acid-free albumin was even more stimulatory and when added to platelets in a phosphate-buffered medium, it increased Vmax beyond the value observed in plasma. Km values calculated on the basis of unbound 5-HT were not affected by the media except for a slight decrease in ultrafiltrate as compared to plasma. The fraction of 5-HT (0.5 mumol/l) bound to 40 milligrams albumin was 17% with the preparation containing fatty acids and 22% with fatty-acid-free albumin, while total plasma proteins dissolved in phosphate buffer bound 24%. The uptake of 1 mumol/l 5-HT was enhanced by both albumin preparations already at 1 milligram and near-maximal effects occurred at 10 milligrams.

Elevated norharman plasma levels in alcoholic patients and controls resulting from tobacco smoking.

Plasma norharman and harman levels were measured by solvent extraction and HPLC with fluorescence detection in alcohol-dependent patients undergoing in-patient abstinence treatment and in control subjects. In both groups, randomly collected samples from smokers contained higher mean norharman levels than those from non-smokers. In three volunteers norharman concentrations rose sharply after smoking of one or two cigarettes and declined to near-basal levels within one hour after one cigarette. When 12 patients kept a smoking-free interval of at least 6 h, they had similarly low plasma norharman concentrations (20 +/- 8 pg/ml) as 18 non-smoking control subjects (17 +/- 8 pg/ml) or as 13 smoking controls who had abstained from smoking (20 +/- 6 pg/ml). Ten of the patients smoked one cigarette and within 5-10 min attained norharman levels of 177 +/- 147 pg/ml plasma. The high prevalence of smokers among chronic alcoholics probably explains the previous finding of elevated norharman plasma levels in these patients.

Plasma levels, psychophysiological variables, and clinical response to amitriptyline.

Hamilton depression scale ratings and physiological measurements were made for 37 patients with primary depression before treatment with amitriptyline (150 mg/day) and again after 2 and 4 weeks of treatment; plasma drug levels were determined weekly. Improvement was maximal at mean amitriptyline + nortriptyline concentrations of 125-200 ng/ml (14 patients), while at lower levels the outcome was significantly poorer (12 patients). Highly variable results were seen in 11 patients with levels between 200 and 301 ng/ml, with lesser improvement occurring in those patients who exhibited poor habituation of the skin resistance response before treatment. Other psychophysiological variables showed significant changes during treatment, but no correlation with clinical results or drug levels.

Carbonyl reduction of naltrexone and dolasetron by oxidoreductases isolated from human liver cytosol.

The opioid receptor antagonist naltrexone and the antiemetic 5-HT(3) receptor antagonist dolasetron are ketonic drugs that are efficiently reduced to their corresponding alcohols in-vivo. These experiments aimed at characterizing the role in these reactions of individual oxidoreductases present in human liver cytosol. Aldo-keto reductases (AKRs) and carbonyl reductase (CR, EC 1.1.1.184) purified from human liver cytosol were incubated with varying substrate concentrations and 6beta-naltrexol or reduced dolasetron were analysed by HPLC. AKR1C1, AKR1C2, and AKR1C4 were able to reduce both substrates. On the basis of k(cat)/K(m) values, AKR1C4 was nearly 1000-fold more efficient in reducing naltrexone than was AKR1C1, while AKR1C2 was of intermediate efficiency. Substrate inhibition was observed on incubating AKR1C2 or AKR1C4 with naltrexone. In contrast, dolasetron was also a substrate of CR. AKR1C1 and AKR1C4 were the most efficient enzymes in producing reduced dolasetron. We concluded that the efficient reduction of naltrexone by AKR1C4 probably causes the high 6beta-naltrexol/naltrexone ratio in man. The rapid disappearance from human plasma of dolasetron given intravenously and its virtual absence after oral dosage are explained by its liability to reduction by several enzymes, including CR which shows widespread expression in human tissues.

Comparison of procedures for measuring the quaternary N-glucuronides of amitriptyline and diphenhydramine in human urine with and without hydrolysis.

The activities of beta-glucuronidases from Helix pomatia, Escherichia coli and rat towards the N-glucuronides of amitriptyline and diphenhydramine were considerably lower than those towards standard substrates. The two N-glucuronides were analysed in urine samples by the following procedures: HPLC of the intact conjugate after solid-phase extraction on a cation exchanger cartridge or after direct injection of urine; HPLC of the aglycone after hydrolysis with beta-glucuronidase from H. pomatia or E. coli or after alkaline hydrolysis. Solid-phase extraction led to the highest recovery and precision, and sensitivity can be improved by extracting a larger volume of urine. On application to samples from patients under treatment with amitriptyline, the results of all procedures except alkaline hydrolysis were in good agreement. When diphenhydramine N-glucuronide was analysed in urine samples of volunteers, solid-phase extraction, hydrolysis by E. coli glucuronidase and alkaline hydrolysis resulted in similar values.

Variability of diphenhydramine N-glucuronidation in healthy subjects.

The H1-antagonist diphenhydramine can undergo direct glucuronidation at its tertiary amino group with formation of a quaternary ammonium glucuronide. The intraindividual variability in the amount of N-glucuronide excretion in urine was investigated in two female volunteers who repeatedly took single doses of 25 mg diphenhydramine hydrochloride without and with concomitant administration of ascorbic acid or ammonium chloride for urine acidification. Another two female and four male subjects underwent single tests without and with additional ascorbic acid. Diphenhydramine N-glucuronide quantities in urine differed significantly among subjects and ranged between 2.7% and 14.8% of the dose within 8 h. Neither ascorbic acid nor ammonium chloride significantly influenced the quantity of N-glucuronide in urine, but ammonium chloride, that in contrast to ascorbic acid proved effective in lowering urinary pH, increased the excretion of the parent drug.

[Amalgam--"etiology" of psychiatric disorders?]. / Amalgam - "Ursache" psychischer Störungen?

In a liability lawsuit an expertise had to answer the question whether a mania in the course of an affective psychosis could have been caused by chronic mercury intoxication resulting from dental amalgam fillings. On the basis of current psychiatric and toxicological knowledge, such an association can be disproved. Mercury intake from amalgam fillings does not lead to toxic concentrations in organs or body fluids. Therefore physicians and dentists should avoid alarming patients and thus causing iatrogenic harm.