Catechol-O-methyltransferase (COMT)-mediated metabolism of catechol estrogens: comparison of wild-type and variant COMT isoforms.

The oxidative metabolism of 17beta-estradiol (E2) and estrone (E1) to catechol estrogens (2-OHE2, 4-OHE2, 2-OHE1, and 4-OHE1) and estrogen quinones has been postulated to be a factor in mammary carcinogenesis. Catechol-O-methyltransferase (COMT) catalyzes the methylation of catechol estrogens to methoxy estrogens, which simultaneously lowers the potential for DNA damage and increases the concentration of 2-methoxyestradiol (2-MeOE2), an antiproliferative metabolite. We expressed two recombinant forms of COMT, the wild-type (108Val) and a common variant (108Met), to determine whether their catalytic efficiencies differ with respect to catechol estrogen inactivation. The His-tagged proteins were purified by nickel-nitrilo-triacetic acid chromatography and analyzed by electrophoresis and Western immunoblot. COMT activity was assessed by determining the methylation of 2-OHE2, 4-OHE2, 2-OHE1, and 4-OHE1, using gas chromatography/mass spectrometry for quantitation of the respective methoxy products. In the case of 2-OHE2 and 2-OHE1, methylation occurred at 2-OH and 3-OH groups, resulting in the formation of 2-MeOE2 and 2-OH-3-MeOE2, and 2-MeOE1 and 2-OH-3-MeOE1, respectively. In contrast, in the case of 4-OHE2 and 4-OHE1, methylation occurred only at the 4-OH group, yielding 4-MeOE2 and 4-MeOE1, respectively. Individual and competition experiments revealed the following order of product formation: 4-MeOE2 > 4-MeOE1 >> 2-MeOE2 > 2-MeOE1 > 2-OH-3-MeOE1 > 2-OH-3-MeOE2. The variant isoform differed from wild-type COMT by being thermolabile, leading to 2-3-fold lower levels of product formation. MCF-7 breast cancer cells with the variant COMT 108Met/Met genotype also displayed 2-3-fold lower catalytic activity than ZR-75 breast cancer cells with the wild-type COMT 108Val/Val genotype. Thus, inherited alterations in COMT catalytic activity are associated with significant differences in catechol estrogen and methoxy estrogen levels and, thereby, may contribute to interindividual differences in breast cancer risk associated with estrogen-mediated carcinogenicity.

Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity.

Activation of 17beta-estradiol (E2) through the formation of catechol estrogen metabolites, 2-OH-E2 and 4-OH-E2, and the C-16alpha hydroxylation product, 16alpha-OH-E2, has been postulated to be a factor in mammary carcinogenesis. Cytochrome P450 1B1 (CYP1B1) exceeds other P450 enzymes in both estrogen hydroxylation activity and expression level in breast tissue. To determine whether inherited variants of CYP1B1 differ from wild-type CYP1B1 in estrogen hydroxylase activity, we expressed recombinant wild-type and five polymorphic variants of CYP1B1: variant 1 (codon 48Arg-->Gly), variant 2 (codon 119Ala-->Ser), variant 3 (codon 432Val-->Leu), variant 4 (codon453Asn-->Ser), variant 5 (48Gly, 119Ser, 432Leu, 453Ser). The His-tagged proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography and analyzed by electrophoresis and spectrophotometry. We performed assays of E2 hydroxylation activity and quantitated production of 2-OH-E2, 4-OH-E2, and 16alpha-OH-E2 by gas chromatography/mass spectrometry. Wild-type CYP1B1 formed 4-OH-E2 as main product (Km, 40+/-8 microM; k(cat) 4.4+/-0.4, min(-1); k(cat)/Km, 110 mM(-1) min(-1)), followed by 2-OH-E2 (Km, 34+/-4 microM; k(cat), 1.9+/-0.1 min(-1); k(cat)/Km, 55 mM(-1)min(-1)) and 16alpha-OH-E2 (Km, 39+/-5.7 microM; k(cat), 0.30+/-0.02 min(-1); k(cat)/Km, 7.6 mM(-1)min(-1)). The CYP1B1 variants also formed 4-OH-E2 as the main product but displayed 2.4- to 3.4-fold higher catalytic efficiencies k(cat)/Km than the wild-type enzyme, ranging from 270 mM(-1)min(-1) for variant 4, to 370 mM(-1)min(-1) for variant 2. The variant enzymes also exceeded wild-type CYP1B1 with respect to 2- and 16alpha-hydroxylation activity. Thus, inherited alterations in CYP1B1 estrogen hydroxylation activity may be associated with significant changes in estrogen metabolism and, thereby, may possibly explain interindividual differences in breast cancer risk associated with estrogen-mediated carcinogenicity.

High plasma nortriptyline levels in the treatment of depression. I.

Following a 3-day single-dose kinetic study, 21 moderate to severely depressed inpatients were treated with 100 mg of nortriptyline nightly. Eighteen patients completed the 4-wk trial. The severity of depression was measured by weekly Hamilton Rating Scale and global rating. Blood for plasma nortriptyline estimation was taken at weekly intervals 12 h following the nighttime dose. There was a 6-fold variation in mean plasma nortriptyline levels, ranging from 120 microgram/L to 681 microgram/L. Patients with high plasma levels (greater than 200 microgram/L) showed significantly poorer clinical responses than those with levels in routine treatment, high plasma nortriptyline levels are significantly less effective than intermediate levels. Single-dose pharmacokinetic data obtained on the same patients showed a highly significant correlation with mean steady-state plasma levels obtained, which themselves correlated with clinical response. The value of predicting high plasma nortriptyline levels which are associated with poor response is discussed.

Nortriptyline metabolism in chronic renal failure: metabolite elimination.

Single oral dose kinetics of nortriptyline and of tis two major metabolites, conjugated and unconjugated 20-hydroxynortriptyline, were studied in eight healthy subjects and 15 patients with chronic renal failure, five of whom were being treated with hemodialysis. Nortriptyline kinetics were unaltered, but the elimination of the metabolites was reduced in both groups of patients. In chronic renal failure the excretion of nortriptyline metabolites appeared to be the rate-limiting step in nortriptyline elimination. Three depressed hemodialysis patients were treated with nortriptyline (75 mg at night) for 6 wk. The ratios of the steady-state plasma concentrations of unconjugated 10-hydroxynortriptyline to nortriptyline (0.74 to 2.30) were in the same range as those in a control group of depressed patients with adequate renal function (0.53 to 4.08) who were also receiving nortriptyline. Conjugated 10-hydroxynortriptyline in renal failure patients was slow to reach steady-state concentrations and these were 10 to 20 times as high as those of the control depressed patients. Conjugated 10-hydroxynortriptyline in dialysis fluid during treatment showed that a mean 43 +/- 7% (SD) of the dose was removed by a 10-hr dialysis. Dialysis clearance of conjugated 10-hydroxynortriptyline was 58 +/- 8 (SD) ml min-1, but nortriptyline and unconjugated 10-hydroxynortriptyline were not appreciably removed by dialysis. Hemodialysis is not likely to be of value in the management of acute nortriptyline poisoning.

Clinical pharmacokinetic considerations in the elderly. An update.

There are numerous studies of drug handling in the elderly, but it is difficult to assess the significance of changes seen in vitro, or after single-dose administration, because they are often compensated by other mechanisms at steady-state. However, a knowledge of these studies is important as the results alert the investigator to possible treatment problems. The high incidence of adverse drug reaction in the elderly population leaves no doubt that improvements in therapy are needed. Research has been directed at seeking patterns of abnormality in the elderly on which to base recommendations for alterations in dosage regimens. The major shortcoming of this approach has been the failure to distinguish between the effect of chronological age on drug pharmacokinetics, and drug kinetics in elderly people with multiple pathology. The latter concern appreciates the variety of factors involved and the importance of treating each patient as an individual: presentation of mean data is confusing and misleading. The objective of drug treatment in any age group, but particularly in the elderly, is to administer the smallest possible dose which gives adequate therapeutic benefit throughout the entire dosage interval with the minimum of side effects. For most drugs the safe starting dose in the elderly is one-third to half that recommended in the young. Vigilance for potential side effects with plasma concentration monitoring, if available, should help keep toxicity to a minimum. When other medications are added or changed, the possibility of interaction should be anticipated. Methods for individualisation of dosage regimens and the use of sustained-release formulations in the elderly are discussed. Dosage alteration in the elderly in terms of reduced dose frequency, rather than dose size, may help improve compliance. A knowledge of the pharmacokinetics of a drug helps determine which approach will be most beneficial.

Pharmacokinetics of single oral doses of nortriptyline in depressed elderly hospital patients and young healthy volunteers.

A single oral dose of 75mg nortriptyline was given to a group of 20 depressed elderly patients in hospital. Subsequent plasma nortriptyline concentrations were used to calculate the half-life and clearance of the drug. These measurements were compared with those made previously in 17 healthy young volunteer subjects. Plasma nortriptyline half-life was longer and clearance slower (p < 0.002) in the elderly group than in the volunteers. There was no correlation of age with either of these parameters within the 2 groups, and no differences in nortriptyline pharmacokinetics could be detected between the male and female volunteer subjects. The possible reasons for these findings and their clinical consequences are discussed.

Amitriptyline dosage prediction in elderly patients from plasma concentration at 24 hours after a single 100mg dose.

Fifteen depressed elderly patients (14 female, 1 male; mean age 85 years) received a single oral dose of amitriptyline. The concentration of amitriptyline plus nortriptyline in a blood sample taken 24 hours later was used to predict by means of a nomogram the amitriptyline dosage required for each patient. Each dose was selected to produce steady-state amitriptyline plus nortriptyline concentrations close to 140 micrograms/L. The daily dosage ranged from 20 to 100mg (mean 62mg). Patients received the individually calculated dose each night, and weekly blood samples were obtained for drug analysis. At 2 weeks, mean amitriptyline plus nortriptyline concentrations were 118 +/- 21 micrograms/L. Eight of the patients were studied for a further 2 weeks and the mean amitriptyline plus nortriptyline concentration was then 111 +/- 19 micrograms/L. The dose prediction test is easy to use and ensures each patient receives an adequate but safer dose of amitriptyline than might otherwise be prescribed routinely.

Plasma concentrations of mianserin after single dose and at steady-state in depressed elderly patients.

Plasma mianserin and desmethylmianserin concentrations were measured in 17 clinically depressed elderly patients after a single 30 mg dose of mianserin. The patients then received mianserin 30 mg daily for up to 6 weeks and the plasma concentrations were measured at weekly intervals. The relationship between concentrations of mianserin and desmethylmianserin at steady-state and at 16 and 24 hours after the single test dose was not good enough to be used for prediction of dosage requirements. Reasons for this finding are discussed.

Dosage adjustment from simple nortriptyline spot level predictor tests in depressed patients.

20 routine patients with endogenous depression were investigated in a kinetic and 4 week treatment study. Steady-state plasma nortriptyline concentrations above 200 microgram/L were associated with a highly significant poorer therapeutic outcome. The correlations between the 24, 48 and 72 hour concentrations and steady-state concentration were very good (r = 0.81, 0.97, 0.94; p less than 0.0001) and better than the correlation between half-life and steady-state (r = 0.65; p less than 0.01). The Spearman rank correlations (Rs) between amelioration of depression measured by the Hamilton Rating Scale (HRS) and the 24, 48 and 72 hour concentrations were highly significant (Rs = 0.74, 0.79, 0.79; p less than 0.001) but for half-life (Rs = 0.33) the correlation was not significant. The single 48 hour plasma nortriptyline concentration following a single oral dose is recommended as a reliable simplified monitoring test suitable for a busy clinic. The test is useful for dosage adjustment to maximise antidepressant action and minimise toxicity. A tentative dosage adjustment schedule for individualising antidepressant treatment with nortriptyline based on the 48 hour or the 24 hour plasma concentration is proposed.

Abolition of latent inhibition by a single 5 mg dose of d-amphetamine in man.

The performance of healthy volunteer subjects on an auditory latent inhibition (LI) paradigm was assessed following administration of a single oral dose of d-amphetamine or placebo. It was predicted that a low (5 mg), but not a high (10 mg), dose of d-amphetamine would disrupt LI. The prediction was supported with left ear presentation of the preexposed stimulus only. When the preexposed stimulus was presented to the right ear the predicted pattern of findings was not obtained. It is concluded that the dopaminergic system is involved in the mediation of LI in man and it is speculated that the interaction between amphetamine dose and ear of presentation of the preexposed stimulus may reflect normally occurring dopaminergic hemisphere asymmetry.

Sudden, unexpected cardiac or unexplained death in England: a national survey.

BACKGROUND: Post-mortem examinations of adults who were apparently healthy but died suddenly and unexpectedly sometimes reveal no morphological abnormalities to explain their deaths. The frequency of such unexplained deaths in relation to other causes of sudden cardiac death is not known. AIM: To estimate the frequency of sudden unexpected cardiac or unexplained death in England. DESIGN: Prospective survey using a stratified random sample of 83 of the 132 H.M. Coroner's jurisdictions in England. METHODS: Consecutive White Caucasians, aged 16-64 years, with no medical history of cardiac disease, seen alive within 12 h of death, on whom autopsy found either a cardiac or no identifiable cause of death, were included. The coroner's officer sent a copy of the post-mortem report and a completed case registration form to the investigators, with tissue samples. RESULTS: Sixty-seven (81%) coroners participated, each maintaining prospective surveillance for 4 months. Of 692 ascertained cases, case registration forms were received for 650 (94%), post-mortem reports for 682 (99%), blood samples for 569 (82%), myocardial slices for 517 (75%) and whole hearts for 47 (7%). In cases with myocardial tissue, death was ascribed to ischaemic heart disease in 465 (82.4%). In 43.1% the ischaemia was acute, in 19.1% there was myocardial scarring but no acute ischaemia, and 20.2% had coronary atheroma only. Death was due to left ventricular hypertrophy in 32 (5.7%), to other cardiac causes in 30 (5.3%) and in 23 (4.1%) there was no clear cause. Those with cardiac causes were 81% male, median ages 55.9 (male) and 56.6 (female) years. The 23 unexplained deaths were 57% female, median ages 40.5 (male) and 54.9 (female) years. The estimated annual frequency of sudden unexpected death due to cardiac or unidentified causes, in English adults of employment age, was 11/100,000 (3481 annual deaths). DISCUSSION: In 4.1% of sudden unexpected deaths under 65 years, no cause was found. Until it becomes accepted practice to identify these cases by a name, such as Sudden Adult Death Syndrome (SADS), it will not be possible to study their aetiology systematically.

Monitoring of tricyclic antidepressant therapy.

During the three-year period 1978-1980, 2141 plasma samples from 1055 patients receiving therapy with amitriptyline (77%) or nortriptyline (23%) were analysed using GLC with nitrogen selective detection. Compared to the recommended therapeutic ranges, wild inter-individual differences were observed in plasma drug concentration, even when corrections for dosage were made. Concentrations ranged from below the limit of sensitivity of the assay (5 microgram.1(-1)) to greater than 1 mg.1(-1). The reporting of toxic symptoms subjective side-effects) was found not to reliably predict high drug concentrations. Serious complications, however, were associated with high plasma drug concentrations. Neither nortriptyline nor amitriptyline displayed dose-dependent pharmacokinetics over the concentration ranges studied. Treatment with either drug produced age-related increases in drug concentration, which were more pronounced in female patients. With amitriptyline therapy, there was an age-related decrease in the plasma nortriptyline:amitriptyline ratio, suggesting that demethylation may be more influenced by increasing age than hydroxylation. Plasma drug monitoring of tricyclic antidepressant therapy is the only reliable means of ensuring that all patients receive a fair opportunity to benefit from these drugs.

Systemic absorption and abuse liability of snorted flunitrazepam.

Benzodiazepine hypnotics are widely abused as part of a polydrug misuse culture. This study set out to investigate some pharmacokinetic and pharmacodynamic characteristics of a novel method of abuse, snorting, of flunitrazepam. Twenty healthy volunteers took part: three took 0.5 mg, three took 1 mg, three took 1.5 mg, six took 2 mg and five took placebo. Blood was sampled and ratings of mood, bodily symptoms, strength and liking of drug effect were completed pre- and at 5, 15, 30, 60, 90, 120 and 240 minutes and at 24 hours post-drug. It was found that flunitrazepam could be detected in venous blood 5 minutes after intake. As the dose increased, the peak plasma concentration was higher but also occurred progressively later, the levels reached being comparable to oral or intramuscular administration at 110 minutes. Subjects reported sedation but complained of few side-effects. They liked the drug effects and subjective ratings of strength were correlated with liking and with plasma drug levels.

Value of serum diazepam and nordiazepam measurements in anxious patients.

The relationship between the dose and anxiolytic effects of diazepam and the serum concentrations of diazepam and nordiazepam were examined in groups of acutely and chronically anxious patients. The results showed a significant correlation between dose and serum nordiazepam concentrations after short-term (14-day) administration, but no significant association between clinical symptoms of anxiety and serum diazepam and nordiazepam. We conclude that the main value of serum benzodiazepine measurements in anxious patients in assessing compliance, particularly in patients suspected of taking more than the recommended dose. Serum nordiazepam is a more consistent index of dosage after chronic therapy than serum diazepam because it has a longer elimination half-time.

Application of an individually predicted dosage of amitriptyline to the treatment of depression.

The treatment of depressed patients with a fixed dose of amitriptyline is compared to treatment with an individualized dose calculated by means of a simple pharmacokinetic test. Clinical response and the development of side-effects are compared between the two groups of patients. Although the numbers in the groups were small, the clinical results lend little support to the concept of a therapeutic range of plasma drug concentrations for amitriptyline, and none of the plasma concentrations was high enough to produce serious toxic effects. No obvious clinical advantage was observed in the predicted dose treatment group as assessed by a reduction in depression ratings. The dose prediction test did however more than halve the variance in blood drug concentrations, and its usefulness in preventing high and potentially toxic concentrations is indisputable.

Measurement of ethylene glycol (ethane-1,2-diol) in biological specimens using derivatisation and gas-liquid chromatography with flame ionisation detection.

Ethylene glycol in plasma, urine or dialysis fluid is analysed as the phenylboronate derivative by mixing with acetonitrile/acidified 2,2-dimethoxypropane containing phenylboronic acid. After centrifugation, a portion of the supernatant is analysed directly by gas-liquid chromatography using a 3% OV-101 column at 150 degrees C and flame-ionisation detection. Propane-1,3-diol is used as a reactive internal standard. The limit of accurate measurement is at least 0.1 g/L and the linear range extends up to 5.0 g/L. No sources of interference have been identified.

Rapid measurement of basic drugs in blood applied to clinical and forensic toxicology.

A gas chromatographic method is presented to measure blood, serum or plasma concentrations of more than 40 basic drugs. The sensitivity is 0.05 mg/L or less, which represents medium-high therapeutic and overdose concentrations, and in many instances the major active metabolites are also quantified. The paper describes a single step extraction from basic solution into n-butyl acetate containing maprotiline internal standard. Disposable glass tubes are used, with direct chromatography of the upper organic layer. GLC analysis is conducted for 10 min isothermally on a packed column (3% SP2250) with nitrogen-phosphorus detection. The coefficient of variation (CV) of the assay is between 2% and 5%, and data on the reproducibility of retention times are presented.

Gas chromatographic measurement of cocaine in serum, plasma and whole blood.

A gas chromatographic method is presented to measure cocaine in serum, plasma or blood. To reduce the in-vitro chemical and enzymic hydrolysis of cocaine, samples should be collected into fluoride oxalate tubes, frozen immediately and stored at -20 degrees C until analysis. Extractions are carried out in disposable glass tubes immersed in an ice-bath. The method uses a single step extraction from a mildly basic solution into n-butyl acetate containing maprotiline internal standard. A portion of the upper organic layer is chromatographed for 5 min isothermally on a packed column (3% SP2250) with nitrogen-phosphorus detection. The coefficient of variation (CV) of the assay is below 6% at 0.1 mg/L and the limit of accurate measurement is 0.02 mg/L. A case of acute cocaine intoxication is described to illustrate the application of the method.

Systemic paraphenylenediamine (PPD) poisoning: a case report and review.

A case of suicidal poisoning with paraphenylenediamine (PPD) is reported. The patient presented with typical features of severe oropharyngeal oedema and rhabdomyolysis. He suffered sudden cardiac death within 4 hours of admission despite full supportive treatment. The diagnosis was only established after his death. Systemic poisoning with paraphenylenediamine (PPD) is rare in western countries, and therefore a high degree of awareness and circumstantial evidence is required to make an early diagnosis. The classical and other less commonly reported features of this poisoning are discussed. There is no specific antidote available but some guidelines for management of such a case are reviewed.

Physiologic effects of anesthesia induced and maintained by intravenous administration of a climazolam-ketamine combination in ponies premedicated with acepromazine and xylazine.

OBJECTIVE: To examine the physiologic and pharmacokinetic effects of a technique of total intravenous anesthesia in ponies. ANIMALS: 6 healthy ponies. PROCEDURE: Ponies were premedicated with acepromazine (0.03 mg/kg of body weight, IV) and xylazine (1.0 mg/kg, IV). Two minutes later, anesthesia was induced with ketamine (2.0 mg/kg, IV) followed by climazolam (0.2 mg/kg, IV). Anesthesia was maintained for 120 minutes by an infusion of climazolam (0.4 mg/kg/h) and ketamine (6.0 mg/kg/h). Oxygen (5 L/min) was supplemented. 20 minutes after the infusion was stopped sarmazenil (0.04 mg/kg, IV) was administered. Cardiovascular and respiratory function measurements were taken before and after premedication, and during anesthesia. Plasma cortsol, ACTH, and catecholamine concentrations were used to assess adrenal and pituitary gland function Ketamine and climazolam kinetics were calculated, on the basis of plasma drug concentrations. RESULTS: There were no significant changes from pre-xylazine values in heart rate, respiratory rate, arterial blood pressure, cardiac index, systemic vascular resistance, or arterial PO2, PCO2, and pH. Plasma cortisol concentration decreased during anesthesia, but plasma ACTH and catecholamine concentrations did not change. Recovery was fairly smooth, but some excitement and ataxia were noted in 2 ponies. CONCLUSION: Ketamine-climazolan infusion appeared suitable for maintenance of anesthesia in ponies, although recovery was not ideal in 2 of 6 ponies.