Spectroscopic, electrochemical and molecular docking studies of dothiepin and doxepin with bovine serum albumin and DNA base.

The interaction of dothiepin (DOT) and doxepin (DOX) with bovine serum albumin (BSA) and a DNA base (adenine) was studied using UV-visible, fluorescence, attenuated total reflection-infra-red (ATR-IR), cyclic voltammetry and molecular docking methods. Strong fluorescence quenching was observed upon interaction of DOT and DOX with BSA/adenine and the mechanism suggested static quenching. Hydrophobic and hydrogen bonding interactions were the predominant intermolecular forces needed to stabilize the copolymer. Upon addition of the drugs: (i) the tautomeric equilibrium structure of the adenine was changed; and (ii) the oxidation and the reduction peaks of the adenine/BSA interaction shifted towards high and low potentials, respectively. In ATR-IR, the band shift of amides I and II indicated a change in secondary structure of BSA upon binding to DOT and DOX drugs. The reduction in voltammetric current in the presence of BSA/adenine was attributed to slow diffusion of BSA/adenine binding with DOX/DOT. The docking method indicated that the drug moiety interacted with the BSA molecule. Copyright © 2016 John Wiley & Sons, Ltd.

Effect of cytochrome P450 2C19 and 2C9 amino acid residues 72 and 241 on metabolism of tricyclic antidepressant drugs.

Although cytochromes P450 2C9 (CYP2C9) and 2C19 (CYP2C19) have 91% amino acid identity, they have different substrate specificities. Previous studies have suggested that several amino acid residues may be involved in substrate specificity. In this study, we focused on the roles of two amino acids, residues 72 and 241. The amino acids in these positions have opposite charges in CYP2C9 and 2C19; the former has lysines in both positions (Lys72 and Lys241), and the latter has glutamic acids (Glu72 and Glu241). Reciprocal mutants for both CYP2C19 and 2C9 were produced, and their metabolic activities and spectroscopic properties were examined using three tricyclic antidepressant (TCA) drugs: amitriptyline, imipramine, and dothiepin. Although CYP2C19 wild-type (WT) had a high metabolic activity for all three drugs, the E72K mutation decreased enzymatic activity by 29-37%, while binding affinities were diminished 2.5- to 20-fold. On the other hand, low activity and low affinity of CYP2C9 WT were recovered notably by K72E mutation. The metabolic activities and binding affinities were minimally affected by CYP2C19 E241K and CYP2C9 K241E mutations. We could also show linear correlations between metabolic activities and binding affinities, and hence we conclude that amino acid residue 72 plays a key role in TCA drug metabolism by limiting the binding affinities of CYP2C19 and CYP2C9.

Comparison of cytochrome p450 mediated metabolism of three central nervous system acting drugs.

The investigation of cytochrome P450 (CYP) mediated metabolism reactions by determination of enzyme kinetic parameters, Michaelis constant (K(m)), maximum reaction velocity (V(max)), and intrinsic clearance (CL(int)) is important aspects in discovery and development of drugs. The kinetic parameters can be used to predict the clearance prior to human administration and for better understanding the mechanism of clearance in vivo. In this study, the metabolic activities of three major hepatic CYP isoforms (2C19, 2D6, and 3A4) were investigated on structurally different central nervous system (CNS) acting drugs, amitriptyline, fluphenazine, and dothiepin. By using our novel in vitro evaluation system, we could compare the kinetic parameters for the metabolism of fluphenazine and dothiepin for the first time. Comparing CL(int) values thus obtained, we concluded that 2C19 could be predominant for metabolic activity on tricyclic antidepressants as expected, but not on phenothiazine-related antipsychotic drugs. Since the metabolism of CNS drugs is susceptible to single nucleotide polymorphisms of human gene, our results suggest that phenothiazine could be an alternative to clinical application of CNS drugs.

Binding of imipramine, dosulepin, and opipramol to liposomes for overdose treatment.

Polymer shielded liposomes were investigated as detoxifying agents for the weak bases imipramine and dosulepin and the diprotic drug opipramol. In vitro binding measurements in the presence of human serum samples revealed that the liposomes reduced the free drug concentration of the weak bases (corrected for protein binding) by 88-93%. The reduction for opipramol was around 76%. The results demonstrate that polymer shielded liposomes composed of anionic lipids are widely useful for drug overdose treatment. Polyethylene glycol chain lengths of 2000 and 5000 for the polymer coatings were also explored, and chain length showed no evidence of affecting drug uptake by liposomes. Liposomes compete favorably with other binding targets for drugs, and pharmacokinetic considerations suggest that liposomes could reduce toxicity by transporting drugs from fast-equilibrating organs such as the heart to slow-equilibrating organs such as the fat, muscle, and skin.

Fatal intoxication due to dothiepin.

An ingestion of an unknown quantity of Harmomed (dothiepin and diazepam) capsules in a suicide is described. The authors report a new and fast method of analysing and determining the dothiepin concentration in postmortem specimens. Quantitation of dothiepin, and its metabolite desmethyldothiepin was performed by ethyl acetate extraction from alkalinized body fluids before GC-MS analysis. The analyses were performed without any complex sample clean-up steps and with little sample material. Postmortem concentrations of dothiepin, desmethyldothiepin, diazepam and desmethyldiazepam in body fluids are given. The proposed method is a rapid procedure for analysis in cases of deliberate poisoning with the antidepressant drug dothiepin.

The excretion of dothiepin and its primary metabolites in breast milk.

1. The excretion of dothiepin, nordothiepin, dothiepin-S-oxide and nordothiepin-S-oxide into breast milk was studied in eight women. Exposure to drug was measured in five of their infants, and possible drug-related effects were assessed in all eight infants. 2. Using pre-feed milk samples mean (+/- s.e. mean) milk:plasma (M:P) ratios were 0.78 +/- 0.12, 0.85 +/- 0.16, 1.18 +/- 0.29 and 1.86 +/- 0.29 for dothiepin, nordothiepin, dothiepin-S-oxide and nordothiepin-S-oxide, respectively. In post-feed milk samples, the mean M:P ratio for dothiepin (1.59 +/- 0.32) was significantly greater (P less than 0.05) but M:P ratios for the metabolites were similar. 3. Mean total calculated infant daily doses, (in dothiepin equivalents and as a percent of the maternal dose) were 0.58% for dothiepin, 0.23% for nordothiepin, 2.47% for dothiepin-S-oxide, and 1.17% for nordothiepin-S-oxide. 4. Plasma samples were obtained from five infants. In one, both dothiepin and nordothiepin were below their minimum quantifiable levels (2 micrograms l-1) while in four others both dothiepin-S-oxide and nordothiepin-S-oxide were below their minimum quantifiable levels (10 micrograms l-1). No adverse effects were found in any of the eight infants. 5. Use of dothiepin by depressed mothers is unlikely to be a significant hazard to their breast-feeding infants.

Determination of dosulepin and its metabolite: application of high-performance liquid chromatography with electrochemical detection.

Dosulepin, 11-(3-dimethylaminopropylidene)-6,11-dihydrodibenzo [b,e]thiepin hydrochloride, is a thio analogue of amitriptyline and is used for the treatment of anxiety and affective disorders. The present study developed a simple and sensitive procedure for the determination of this compound and its metabolite, northiaden, by a combination of high-performance liquid chromatography with electrochemical detection. Hydrodynamic voltammograms demonstrated an optimal applied potential at 1300 mV for both dosulepin and northiaden. A mobile phase consisting of 0.1 M acetate buffer-acetonitrile-perchloric acid-trichloroacetic acid (50:50:2:1.5) provided the best separation of the drugs. The extraction procedure, which used a heptane-isoamyl alcohol (99:1) mixture, was successfully applied with a recovery of over 90%. A preliminary pharmacokinetic study was performed by the proposed method.

Pharmacokinetics of dothiepin in humans: a single dose dose-proportionality study.

Dothiepin hydrochloride (N,N-dimethyldibenzo[b,e]thiepin-delta 11(6 H), gamma-propylamine hydrochloride) is a tricyclic antidepressant which is structurally similar to amitriptyline. Twenty-seven healthy men received three single oral doses of 50-, 100-, and 150-mg dothiepin hydrochloride capsules in a three-way randomized, crossover dose-proportionality study. Plasma concentration-time profiles of dothiepin (1) were described by both one- and two-compartment models with first-order absorption. The total intrinsic clearance of dothiepin decreased from 165.5 to 121.1 L/h as the dose was increased from 50 to 150 mg, but there was no significant effect on the terminal half-life (approximately 20 h). Plasma concentration-time profiles of the three major metabolites of dothiepin, the S-oxide derivative of dothiepin, N,N-dimethyl[b,e]thiepin-delta 11(6 H), gamma-propylamine 5-oxide (2), the demethyl derivative, N-methyldibenzo[b,e]thiepin-delta 11(6 H), gamma-propylamine (3) and the demethyl S-oxide derivative N-methyldibenzo[b,e]thiepin-delta 11(6 H), gamma-propylamine 5-oxide (4), were described by a one-compartment model with apparent first-order formation. The AUC infinity values of the S-oxide 2 and the demethyl S-oxide 4 increased proportionally with dose. The dose proportionality of the demethyl metabolite 3 may not be ascertained from the data in this study. The corresponding half-lives of the three metabolites, which are dose independent, were approximately 24, 28, and 40 h, respectively.

Urinary excretion of conjugates of dothiepin and northiaden (mono-N-demethyl-dothiepin) after an oral dose of dothiepin to humans.

Only small amounts of unconjugated dothiepin (unchanged drug) and northiaden were excreted in urine over a 72 hr period. More than 10% of the dose was excreted as conjugated dothiepin and less than 0.8% of the dose as conjugated northiaden. Conjugated dothiepin was thus found to be an important metabolite of dothiepin. Conjugated dothiepin and northiaden were hydrolyzed with beta-glucuronidase, and their hydrolysis inhibited with 1,4 saccharolactone. Conjugated dothiepin and northiaden were found to be a quaternary ammonium-linked glucuronide and a tertiary N-glucuronide, respectively.

Clinical pharmacokinetics of dothiepin. Single-dose kinetics in patients and prediction of steady-state concentrations.

The pharmacokinetics of dothiepin were evaluated in 9 depressed patients following a single oral dose of 75 mg. Blood and plasma concentrations of dothiepin and 2 major metabolites, northiaden and dothiepin S-oxide, were measured by gas chromatography/mass fragmentography. The mean (+/-SD) peak plasma concentrations of dothiepin were 49 +/- 27 micrograms/L at 3 +/- 1.2h. Mean (+/-SD) estimates of other parameters were as follows: absorption half-life 1.1 +/- 1.1h; distribution half-life 2.2 +/- 0.8 h; elimination half-life 25 +/- 7h; apparent volume of distribution 70 +/- 62 L/kg; and oral clearance 2.1 +/- 1.6 L/kg/h. The mean (+/-SD) peak plasma concentration of dothiepin S-oxide was 125 +/- 43 micrograms/L at 3.5 +/- 1.3h with an elimination half-life of 22 +/- 12 h. The mean peak plasma concentration of northiaden was 6 +/- 3 micrograms/L at 4.5 +/- 1.1h, with an elimination half-life of 31 +/- 12 h. No significant differences were found in pharmacokinetic parameters compared with a previous study in 7 healthy volunteers. When data for the patients and healthy volunteers were combined (n = 16), pharmacokinetic parameters were not found to be affected by age. However, a significant difference was found between males and females for the elimination half-lives of dothiepin and northiaden, and for the apparent volume of distribution of dothiepin. The 24-hour blood/plasma concentrations of dothiepin and dothiepin S-oxide accurately predicted the steady-state concentrations obtained following 4 weeks' treatment with dothiepin 150 mg nocte.

Age differences in effects on blood pressure, flicker fusion frequency, salivation and pharmacokinetics of single oral doses of dothiepin and amitriptyline.

Blood pressure, critical flicker fusion frequency (CFF), salivary flow rate and pharmacokinetics were compared in 7 young healthy volunteers (average age: 22.7 years) and in 7 elderly healthy volunteers (average age: 70.6 years) after single oral doses of the antidepressants dothiepin (DP) 25 mg and amitriptyline (AMP) 25 mg. Systolic blood pressure fell further and the reduction lasted longer in the elderly than in the young after both drugs. The decrease in CFF after AMP 25 mg, and the reduction in salivary flow rate after either DP 25 mg or AMP 25 mg were larger in the elderly than in the young. Plasma levels, T1/2 and Cl of both drugs in the elderly were also higher, longer and smaller, respectively, in the elderly. Clearance was found to be reduced in the elderly. More cautions dosage regimens of these drug should be considered for elderly patients.

Metabolism and pharmacokinetics of dothiepin.

1 Seven healthy volunteers received a single oral dose of 75 mg dothiepin. Plasma concentrations of dothiepin were measured by gas chromatography-mass fragmentography. 2 The plasma concentrations obtained were fitted to the equation Ct = Ae-a(t-tau) + Be-beta(t-tau) - Ce-ka(t-tau). The mean peak concentration was 47(33-71) microgram/l at 3(2-5) h. Mean estimates were as follows: absorption half life 1.2(0.07-3.0) h, distribution half-life 2.6(1.1-3.8) h, elimination half-life 22(14-40) h, apparent volume of distribution 45(20-92) l/kg, and oral clearance 1.36(0.88-1.8) l kg-1 h-1. 3 Blood concentrations of dothiepin were measured in comparison in five of the volunteers. The mean blood/plasma ratio was 0.7(0.6-0.8). 4 Plasma and blood concentrations of northiaden and blood concentrations of dothiepin S-oxide, two metabolites of dothiepin, were also measured. Dothiepin S-oxide was the major metabolic reaching a peak level of 81(34-150) microgram/l at 5(4-6) h. In comparison, northiaden reached a peak concentration of only 10 (3-21) microgram/l at 5 (4-9) h. The mean half-life of elimination of dothiepin S-oxide was 19 (13-35) h while that for northiaden was 33 (22-60) h.

High performance liquid chromatographic determination of dothiepin and northiaden in human plasma and serum.

A method has been developed for the separation and measurement of dothiepin and the N-demethyl metabolites, northiaden, in human plasma or serum by high performance liquid chromatography. The method uses a structurally-related drug, amitriptyline, as an internal standard and provides a limit of detection of about 10 ng/ml for each component. At a concentration of 20 ng/ml, northiaden and dothiepin could be measured with +/-11% and +/- 6% of the mean respectively and at 200 ng/ml within +/-3% and 1% of the mean. The method has been applied to the analysis of serum from patients undergoing dothiepin therapy.

Steady-state serum concentrations of dothiepin and northiaden after two dosage regimens of dothiepin hydrochloride (Prothiaden).

Five healthy volunteers took part in a crossover study which examined the serum concentrations of dothiepin and northiaden after a 25 mg three times a day and a 75 mg once a day dosage regimen of Prothiaden. The inter-individual variation of serum levels was large after either schedule which is to be expected with this group of drugs. The minimum steady-state level of dothiepin tended to be lower after the single daily dose, but the differences were small and not statistically significant. The approximate maximum steady-state levels of dothiepin showed large intra-and inter-subject variation and no obvious trend. The values of the desmethylated metabolite, norhiaden, tended to follow the dothiepin concentrations but were lower than the parent drug. Average steady-state levels tended, with one exception, to be very similar after both regimens with no evidence with no evidence fo any trend when comparing the two regiments. The study showed that the two regimens yielded similar steady-state serum concentrations both of drug and metabolite but inter-individual differences were large.

An evaluation of a once daily dosage régime of dothiepin hydrochloride (prothiaden).

This paper reports on the use of a single daily dose of dothiepin hydrochloride (Prothiaden). The results from a metabolism study using [14C] dothiepin were used to calculate the daily variations in blood levels after once daily and thrice daily dose regimes. A clinical study on the use of a single daily dose of 75 mg of dothiepin showed this type of regime to be well tolerated and efficacious in the treatment of 105 depressed patients in general practice. Compared to a thrice daily regime the once daily dose appeared to be more beneficial on symptomatic insomnia during the early treatment period.