Steady-state plasma levels of nortriptyline and its 10-hydroxy metabolite: relationship to theCYP2D6 genotype

The relationship between theCYP2D6 genotype and the steady state plasma levels of nortriptyline (NT), its main active metabolite 10-hydroxynortriptyline (10-OH-NT) and the NT/10-OH-NT ratio were studied in 21 Caucasian depressed patients treated with 100–150 mg NT daily. The patients had participated in a previously published study investigating the role of NT and 10-OH-NT for the therapeutic effect of NT, and the plasma level data were from that study. In the present follow-up study, the patients were genotyped with respect to the polymorphicCYP2D6 by allelespecific PCR amplification andEcoRIRFLP. One poor metabolizer (PM) was identified and she had the highest plasma concentration of NT. Among the 20 extensive metabolizers (EM), the genotype (homozygous versus heterozygous EM) alone was not found to explain the variance in dose-corrected NT concentrations, but contributed significantly when gender was also taken into account. Together, these factors accounted for 59% of the variability in NT levels. Female patients had higher plasma levels of NT than male patients. 10-OH-NT levels were influenced by genotype, and NT/10-OH-NT ratio by genotype and gender. The present follow-up study confirms a relationship between theCYP2D6 genotype and the plasma levels of NT and its active metabolite. Identification of PM by genotyping should be of value for the prediction of the plasma levels and, consequently, the lower than average dose of NT required for optimal therapy. Also among EM, the genotype contributes to the variability in NT and 10-OH-NT levels but alone is of limited practical value for the prediction of optimal dosage.     

CSF/plasma ratio of 10-hydroxynortriptyline is influenced by sex and body height

The CSF/plasma ratios of nortriptyline (NT) and its major metabolite 10-hydroxy-NT (10-OH-NT) were investigated retrospectively in 25 depressed patients. For 10-OH-NT (but not NT), a significant influence of sex and body height was found, most conspicuously in males, in whom the ratio related to body height curvilinearly (N=8;R=0.93;P<0.01). In males, the NT/10-OH-NT ratio in plasma correlated with body height (N=8;r=0.80;P<0.05). Hypothetically, CSF circulation is partly influenced by body height, which accounts for a steeper gradient of 10-OH-NT across the blood-brain barrier in taller persons. From the lumbar site, the more polar 10-OH-NT is assumed to be eliminated by bulk flow via the villi, while the less polar NT exits by diffusion in the choroid plexus. Prospective studies are urgently needed to further evaluate the distribution of antidepressants in the CSF.     

Interindividual differences in plasma protein binding of nortriptyline in man — A twin study

Summary The binding ratio,r _A, of plasma proteins for nortriptyline (NT), a tricyclic antidepressant drug, was investigated by a plasma-plasma equilibrium dialysis technique at 4°C in 7 identical and 10 fraternal sets of twins. The mean steady-state plasma concentrations of NT during oral administration of NT (0.2 mg per kg body weight, t.i.d. for 8 days) were known for all subjects from a previous study. — A two-fold variation inr _A was found (from 0.86 to 1.55), and there were significant intrapair differences inr _A, both in identical (P<0.01) and fraternal pairs (P<0.0005). The variance ofr _A between individuals within pairs was significantly greater in dizygotic than in monozygotic twinships (P<0.025). — It is concluded that plasma protein binding of NT is influenced both by environmental and genetic factors. The interindividual differences in NT-binding to plasma proteins could not be correlated with variations in its steady-state plasma concentrations.     

Isomers of 10-hydroxynortriptyline in geriatric depression

In geriatric, depressed inpatients treated with nortriptyline (NT), total unconjugated plasma concentrations of the Z isomer of the 10-hydroxylated metabolite (10-OH-NT) were determined simultaneously with concentrations of the E isomer by high-performance liquid chromatography. Z-10-OH-NT concentrations averaged 14% of E-10-OH-NT concentrations.     

CSF and plasma levels of nortriptyline and its 10-hydroxy metabolite.

After 3 weeks' nortriptyline (NT) treatment the mean plasma concentration of its 10-hydroxy metabolite (10-OH-NT) (599 +/- 207 nmol l-1) was higher than that of the parent drug (433 +/- 199 nmol l-1) in 25 depressed patients. Also in the cerebrospinal fluid (CSF) the mean level of 10-OH-NT (67 +/- 20 nmol l-1) was higher than that of NT (39 +/- 23 nmol l-1). There was a strong correlation (P less than 0.001) between the CSF and plasma concentration of both NT (r = 0.92) and 10-OH-NT (r = 0.77). The interindividual variation in the CSF/plasma ratio of both compounds was small, compared to the variation in plasma levels. These results show that 10-OH-NT passes the blood-brain barrier as it is present in concentrations higher than those of NT in the CSF. 10-OH-NT has previously been shown to be a potent blocker of noradrenaline uptake and to have much less affinity for muscarinic receptors than NT itself. This active metabolite might therefore be a potential antidepressant with less disturbing anticholinergic side-effects.     

Synthesis,characterization, and monoamine transporter activity of the new psychoactive substance 3′,4′‐methylenedioxy‐4‐methylaminorex (MDMAR)

The recent occurrence of deaths associated with the psychostimulant cis‐4,4′‐dimethylaminorex (4,4′‐DMAR) in Europe indicated the presence of a newly emerged psychoactive substance on the market. Subsequently, the existence of 3,4‐methylenedioxy‐4‐methylaminorex (MDMAR) has come to the authors' attention and this study describes the synthesis of cis‐ and trans‐MDMAR followed by extensive characterization by chromatographic, spectroscopic, mass spectrometric platforms and crystal structure analysis. MDMAR obtained from an online vendor was subsequently identified as predominantly the cis‐isomer (90%). Exposure of the cis‐isomer to the mobile phase conditions (acetonitrile/water 1:1 with 0.1% formic acid) employed for high performance liquid chromatography analysis showed an artificially induced conversion to the trans‐isomer, which was not observed when characterized by gas chromatography. Monoamine release activities of both MDMAR isomers were compared with the non‐selective monoamine releasing agent (+)‐3,4‐methylenedioxymethamphetamine (MDMA) as a standard reference compound. For additional comparison, both cis‐ and trans‐4,4′‐DMAR, were assessed under identical conditions. cis‐MDMAR, trans‐MDMAR, cis‐4,4′‐DMAR and trans‐4,4′‐DMAR were more potent than MDMA in their ability to function as efficacious substrate‐type releasers at the dopamine (DAT) and norepinephrine (NET) transporters in rat brain tissue. While cis‐4,4′‐DMAR, cis‐MDMAR and trans‐MDMAR were fully efficacious releasing agents at the serotonin transporter (SERT), trans‐4,4′‐DMAR acted as a fully efficacious uptake blocker. Currently, little information is available about the presence of MDMAR on the market but the high potency of ring‐substituted methylaminorex analogues at all three monoamine transporters investigated here might be relevant when assessing the potential for serious side‐effects after high dose exposure. Copyright © 2014 John Wiley & Sons, Ltd.     

Stereoselective inhibition of nortriptyline hydroxylation in man by quinidine

1. Four volunteers phenotyped as extensive metabolizers of sparteine took 25?mg nortriptyline hydrochloride and collected urine for 72–80?h. Total free and conjugated 10-hydroxynortriptyline (10-OH-NT) accounted for 54–58% of the dose and it was reduced to 25–40% when 50?mg quinidine sulphate was ingested on the first and second day.

2. Of the four isomers of 10-OH-NT, (-)-E-10-OH-NT was selectively decreased in quantity by quinidine coadministration, while the (+)-isomer and (-)- and (+)-Z-10-OH-NT were found in unchanged or slightly increased quantities. The contribution of (-)-E-10-OH-NT to total E-10-OH-NT and the E-/Z-ratio in total 10-OH-NT were significantly reduced.

3. The quantity of the phenol, 2-hydroxynortriptyline in urine was decreased by quinidine; the relative amounts of metabolites with a primary amino group were not affected.

4. Liver microsomes from a donor in which cytochrome P450IID6 was shown to be present by in vitro phenotyping metabolized NT to E-10-OH-NT containing 86% of the (-)-isomer. Quinidine reduced the hydroxylation rate in (-)-E-10-position much more than that in (+)-E-10-position.

5. Since quinidine selectively impairs the function of cytochrome P450IID6, it is concluded that this isoform catalyses NT hydroxylation predominantly in (-)-E-10-and in 2-position     

Metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide by cytochrome P450 enzymes in human liver microsomes

The in vitro metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9‐oxidized metabolite were determined using gas chromatography‐mass spectrometry (GC‐MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide. CYP1A2 also efficiently oxidized (‐)‐cis‐rose oxide at the 9‐position but not (‐)‐trans‐rose oxide. α‐Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti‐CYP2B6 antibody inhibited (‐)‐cis‐rose oxide 9‐hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (‐)‐trans‐rose oxide was suppressed by thioTEPA and anti‐CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (‐)‐9‐hydroxy‐cis‐ and (‐)‐9‐hydroxy‐trans‐rose oxide formations correlated with (S)‐mephenytoin N‐demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide in human liver microsomes. Copyright © 2015 John Wiley & Sons, Ltd.     

NMR INVESTIGATIONS OF PROLINE AND ITS DERIVATIVES.

The proton magnetic resonance (PMR) spectrum of acetyl-proline amide in D₂O solution has been analysed by computer simulation. The spectra of the cis and the trans isomers have been separated and their PMR parameters (chemical shift and coupling constants) are given. Vicinal coupling constants of the pyrrolidine ring are interpreted by means of a Karplus zone relation. The chemical shift effect of the anisotropy of both peptide planes is considered. It follows that both isomers are puckered with Cγin an endo position, but the cis isomer is more rigid than the trans isomer, which moreover undergoes a small interconversion of the Cγand Cδatoms between two extreme spatial positions. The dihedral angle SP has different values in both isomers. Thus, the dihedral angle between the two peptide planes is smaller in the trans isomer than in the cis isomer.     

Doxepin and its metabolites in plasma and cerebrospinal fluid in depressed patients

Little information exists on the concentrations of antidepressants and their metabolites in CSF. We measured plasma and CSF levels of trans-doxepin (trans-DOX) and DOX metabolites in 12 depressed patients treated with DOX (250 mg/day) for 6 days. Spinal taps and blood samples were taken on day 7, 10 h after drug administration. Trans-DOX, cis-desmethyldoxepin (cis-DM-DOX), trans-desmethyldoxepin (trans-DM-DOX) and di-desmethyldoxepin (DDM-DOX) were analyzed in CSF and plasma samples by HPLC with column-switching. Although DOX was given as a mixture of 85% trans-DOX and 15% of the pharmacologically more active cis-DOX, we found similar amounts of cis-DM-DOX and trans-DM-DOX in plasma (59.8 ± 45.1 versus 72.0 ± 60.0 ng/ml; NS), suggesting that isomerization of DOX had taken place. Trans-DOX and DOX metabolites could be detected in CSF of most patients. Relatively low CSF concentrations of the active metabolite cis-DM-DOX were measured. Clinical efficacy, as assessed by HAMD scores, was not significantly related to plasma or CSF concentrations of trans-DOX or its metabolites. Trans-DOX and DOX metabolites were distributed differently between plasma and CSF. It is concluded that isomerization of DOX is not only relevant for neuronal uptake inhibition, but also for the transport of the metabolites. Received: 13 May 1996/Final version: 16 December 1996     

Stereoselective inhibition of nortriptyline hydroxylation in man by quinidine.

1. Four volunteers phenotyped as extensive metabolizers of sparteine took 25 mg nortriptyline hydrochloride and collected urine for 72-80 h. Total free and conjugated 10-hydroxynortriptyline (10-OH-NT) accounted for 54-58% of the dose and it was reduced to 25-40% when 50 mg quinidine sulphate was ingested on the first and second day. 2. Of the four isomers of 10-OH-NT, (-)-E-10-OH-NT was selectively decreased in quantity by quinidine coadministration, while the (+)-isomer and (-)- and (+)-Z-10-OH-NT were found in unchanged or slightly increased quantities. The contribution of (-)-E-10-OH-NT to total E-10-OH-NT and the E-/Z-ratio in total 10-OH-NT were significantly reduced. 3. The quantity of the phenol, 2-hydroxynortriptyline in urine was decreased by quinidine; the relative amounts of metabolites with a primary amino group were not affected. 4. Liver microsomes from a donor in which cytochrome P450IID6 was shown to be present by in vitro phenotyping metabolized NT to E-10-OH-NT containing 86% of the (-)-isomer. Quinidine reduced the hydroxylation rate in (-)-E-10-position much more than that in (+)-E-10-position. 5. Since quinidine selectively impairs the function of cytochrome P450IID6, it is concluded that this isoform catalyses NT hydroxylation predominantly in (-)-E-10- and in 2-position.     

Free radical scavenging activity of conjugated linoleic acid as single or mixed isomers

Context: Conjugated linoleic acids (CLAs) are a mixture of positional and geometric isomers of linoleic acid (LA) and believed to have many positive biological activities.

Objective: The present study was undertaken to assess the antioxidant activity of cis-9, trans-11 and trans-10, cis-12 as single or mixed CLA isomers at two ratios, 1:6 and 1:13 (trans-10, cis-12/cis-9, trans-11).

Materials and methods: A microplate reader was used to determine the free radical scavenging properties of CLAs against DPPH radical in ethanol.

Results: The kinetic reactions of CLA-DPPH^? showed that all tested CLAs have exerted radical scavenging activities in a dose-dependent manner and observed to immediately react and quench DPPH radicals at all tested levels and no lag phase was noticed in CLA-DPPH^? reactions. The median inhibitory concentration (IC₅₀) value for cis-9, trans-11 CLA was observed to be more effective than other tested CLA. Total antioxidant capacity (TAC) of all tested CLAs were less effective radical scavengers as compared to vitamin E and butylated hydroxytoluene, although all tested CLAs were quenched a high amount (P?<?0.05) of DPPH free radicals.

Discussion and conclusion: All tested CLAs have the ability to directly react and quench DPPH free radicals in ethanol. Furthermore, trans-10, cis-12 CLA has greater maximal efficacy than other tested CLAs as free radical scavenger, while cis-9, trans-11 CLA is the most potent isomer to directly react and quench free radicals at low concentrations in the system, suggesting that the free radical scavenging activity of CLA isomers may contribute to their diverse biological activities.     

The Toxicokinetics of (1R,Cis)- and (1R,Trans)-Tetramethrin in the Isolated Perfused Rat Liver

1. The toxicokinetics of cis- and trans-tetramethrin isomers were investigated using the isolated perfused rat liver preparation.

2. The concentration of cis- and trans-tetramethrin decreased rapidly in the plasma perfusate and was initially replaced by N-(hydroxymethyl)3,4,5,6-tetrahydrophthalimide (MTI) and then by 3,4,5,6-tetrahydrophthalimide (TPI). Plasma perfusate concentrations of the intact cis-isomer were higher than those of the trans-isomer. Concentrations of MTI and TPI were higher in livers treated with the trans-isomer.

3. Tetramethrin and its metabolites were rapidly excreted in the bile. Bile from livers perfused with trans-isomer contained higher concentrations of parent isomer and metabolites MTI and TPI, than did bile from livers treated with the cis-isomer     

Treatment of depression with E-10-hydroxynortriptyline — a pilot study on biochemical effects and pharmacokinetics

The major metabolite of nortriptyline, i.e. E-10-hydroxynortriptyline (E-10-OH-NT), was given as a racemate in increasing doses from 75 to 225 mg/day to five patients with major depressive episode. Plasma concentrations of both the (–)- and (+)-enantiomers were linearly related to the doses. The mean ratio between them was 3.6±0.53, indicating stereospecific kinetics during maintenance treatment. Lumbar punctures were performed in four of the patients before and after 3 weeks of E-10-OH-NT treatment. There was a 18% mean decrease (P<0.01) in the noradrenaline metabolite HMPG in cerebrospinal fluid (CSF), supporting previous in vitro data showing that E-10-OH-NT inhibits noradrenaline uptake in vivo. During treatment, the median depression score measured by the Montgomery-Åsberg Depression Rating Scale declined from 32 to 14 (P<0.05). As the study was open, the clinical outcome is not conclusive but does not contradict the hypothesis that E-10-OH-NT has antidepressant properties. If present at all, side effects were mild and did not interfere with the treatment.     

An automated method for the determination of nortriptyline and its isomeric 10-hydroxylated metabolites in plasma by high pressure liquid chromatography

A high pressure liquid chromatography assay for determination of the tricyclic antidepressant nortriptyline (NT) and its two major metabolites, Z- and E-10-Hydroxy-NT, in plasma is described. Sample preparation included addition of internal standard and a single step extraction procedure. Run time was approximately 14 min. with a LC-18. 5 mu 250 x 4.6 mm column, a mobile phase consisting of aqueous ammonium: methanol: acetonitrile (0.8:6.2:93, v/v), and flow of 1.3 ml/min. NT, Z- and E-10-OH-NT, amitriptyline, Z- and E-10-OH-AT and the internal standard desipramine, were adequately separated within this time span. In our laboratory, the assay has been employed mainly for pharmacokinetic and toxicologic studies in experimental animals at relatively high concentrations.     

Structure/Effect Studies of Fatty Acid Isomers as Skin Penetration Enhancers and Skin Irritants

Comparisons were made of branched vs unbranched saturated fatty acids and cis vs trans unsaturated fatty acids as skin penetration enhancers and primary skin irritants. Skin penetration studies used naloxone base as the diffusant, propylene glycol as the vehicle, and human skin. Maximum naloxone flux was with C_9–12-branched and unbranched fatty acids. For C_5–14 fatty acids, branched and unbranched isomers had similar effects. One branched C₁₈ fatty acid isomer (C₁₆-branched isostearic acid) was more effective in enhancing skin penetration than a differently branched (C₂-branched isostearic acid) or unbranched C₁₈ isomer (stearic acid). There was no significant difference between cis and trans unsaturated C_16–18 fatty acid isomers in their effects on naloxone flux, and all unsaturated fatty acids were more effective enhancers than the corresponding saturated isomers. Several of these fatty acid/propylene glycol vehicles were evaluated in a rabbit primary skin irritation test. Irritation indices were poorly correlated with the effectiveness of the vehicles in enhancing naloxone flux. It was possible to enhance naloxone skin penetration greatly with a vehicle with only minimal skin irritation potential.     

Human dose-excretion studies with the pyrethroid insecticide,cypermethrin

1. An analytical method for monitoring human exposure to cypermethrin has been developed, based on the detection of the free and conjugated forms of the urinary metabolite, the cyclopropanecarboxylie acid.

2. Four male subjects were given a single oral dose, ranging from 0.25?mg to 1.5mg, of a 1:1 cis/trans mixture of cypermethrin, and urine was monitored for the free and conjugated cyclopropanecarboxylic acid. Urinary excretion of the individual metabolites (cis and trans isomers) was similar for the different dosages. Subjects excreted, on average, 78% of the trans isomer dose, and 49% of the cis isomer dose respectively in 24h.

3. Thus, as in other mammals, ester cleavage and elimination of the cis and trans cyclopropanecarboxylic acid moieties in the free and conjugated form is a major route of metabolism of cypermethrin in man.     

Synthesis and in Vitro Receptor Binding Studies of Fluorotamoxifen Analogues

We describe the synthesis of new fluorotamoxifen analogues with the fluorine atom positioned on the end of the aliphatic chain of tamoxifen. The binding of fluorotamoxifens to cytosol estrogen receptors of rat uteri was determined with [³H]estradiol (5 nM). The fluorotamoxifens had similar or superior binding affinities compared with tamoxifen. The IC₅₀ value was as follows: tamoxifen, 5 × 10^–7 M; fluorotamoxifen (VII), 5 × 10^–7 M; N,N-diethylfluorotamoxifen (IV)—cis, 1 × 10^–6 M, and trans, 2 × 10^–7 M; and (cis) fluoromethyl-N,N-diethyltamoxifen (VI) 1 × 10^–7 M. Therefore, the fluorinated tamoxifens have potential use in imaging estrogen receptors by PET.     

Early stage autoinduction of carbamazepine metabolism in humans

Six healthy young adult male volunteers were given two 600 mg (2540 moles) oral doses of carbamazepine (CBZ) 5 days apart. Serial concentrations of CBZ and its 10,11-epoxy (CBZ-epoxide) and 10,11-dihydro-10,11-trans-dihydroxy (CBZ-diol) metabolites in plasma, and daily excretions of these substances and the 2-hydroxy (2-OH-CBZ), 3-hydroxy (3-OH-CBZ) and 9-hydroxymethyl-10-carbamoylacridan (acridan) metabolites in urine were followed for 5 days after each dose.Pharmacokinetic analysis showed that autoinduction of CBZ metabolism was present within 6–10 days of the initial drug dose. The mean oral clearance of CBZ increased from 1.48 to 1.74 l·h^– (difference 0.26 l·h^–, 95% confidence interval 0.11 to 0.41 l·h^–) and the mean percentage urinary recovery of the amount of CBZ eliminated increased from 41.8% to 44.6% (difference 2.8%, 95% confidence interval 0.5 to 5%) between the two studies 5 days apart.The data for daily clearance to metabolite and the time-courses of the plasma CBZ-epoxide to CBZ and CBZ-diol to CBZ concentration ratios suggested that autoinduction had begun by the second day after CBZ intake, and involved not only the epoxide-diol pathway but, to a lesser extent, the oxidations to phenolic derivatives.     

In silico identification of the active conformation of open-chain enaminones with anticonvulsant activity

A study about the relationship between molecular properties of open-chain enaminones and their anticonvulsant activity is presented in this paper. Geometry optimizations of the enaminones were performed at HF and DFT/B3LYP levels of theory using 6-31 + G(d) basis set. The HOMO and LUMO energies were obtained at the same level of theory. The solvent effect was studied through IPCM. A natural bond orbital (NBO) analysis was performed to analyze the possible association between the stability and the intramolecular hydrogen bond interaction energies. The stability order of the isomers in gas phase was the following: cis-1 > trans-4 > cis-2 > trans-3. The IPCM method showed that the trans-3 isomers are more stable that the cis-2 when the solvent effect was taken into account. Two important intramolecular hydrogen bonds were found by NBO analysis. According to our findings, these interactions could affect the activity of the two most stable isomers (cis-1 and trans-4). By contrast, trans-3 isomers did not present this type of interaction. Therefore, the latter isomers have a large flexibility and can adopt a conformation similar to the conformation of active ringed enaminones. In addition, HOMO and LUMO energies suggested that the trans-3 isomers could be the most reactive species.