Specific binding of perazine, a piperazine side-chain phenothiazine drug, to a serum protein (author's transl)]

A binding of perazine to a serum protein of 48 000 D was determined by gel filtration. The affinity constant of the perazine-protein complex was found to be 5.42 X 10(6) mol/l corresponding to a specific binding of 70 ng/ml serum. This result may gather clinical relevance with regard to the "CNS-bioavailability" and individual response to perazine, the average therapeutic serum concentrations having been shown to range between appr. 50 and 200 ng/ml serum. A specific binding of perazine to human or bovine albumine could not be detected.     

The metabolism of the piperazine-type phenothiazine neuroleptic perazine by the human cytochrome P-450 isoenzymes.

Identification of cytochrome P-450 isoenzymes (CYPs) involved in perazine 5-sulphoxidation and N-demethylation was carried out using human liver microsomes and cDNA-expressed human CYPs (Supersomes). In human liver microsomes, the formation of perazine metabolites correlated significantly with the level of CYP1A2 and ethoxyrezorufin O-deethylase activity, as well as with the level of CYP3A4 and cyclosporin A oxidase activity. Moreover, the formation of N-desmethylperazine also correlated well with S-mephenytoin 4'-hydroxylase activity (CYP2C19). alpha-Naphthoflavone (a CYP1A2 inhibitor) and ketoconazole (a CYP3A4 inhibitor) significantly decreased the rate of perazine 5-sulphoxidation, while ticlopidine (a CYP2C19 inhibitor) strongly reduced the rate of perazine N-demethylation in human liver microsomes. The cDNA-expressed human CYPs generated different amounts of perazine metabolites, but the preference of CYP isoforms to catalyze perazine metabolism was as follows (pmol of product/pmol of CYP isoform/min): 1A1>2D6>2C19>1A2>2B6>2E1>2A6 approximately 3A4>2C9 for 5-sulphoxidation and 2C19>2D6>1A1>1A2>2B6>3A4>2C9>2A6 for N-demethylation. In the light of the obtained results and regarding the contribution of each isoform to the total amount of CYP in human liver, it is concluded that CYP1A2 and CYP3A4 are the main isoenzymes catalyzing 5-sulphoxidation (32% and 30%, respectively), while CYP2C19 is the main isoform catalyzing perazine N-demethylation (68%). CYP2C9, CYP2E1 CYP2C19 and CYP2D6 are engaged to a lesser degree in 5-sulphoxidation, while CYP1A2, CYP3A4 and CYP2D6 in perazine N-demethylation (6-10%, depending on the isoform).     

Effects of cytochrome P-450 inducers on the perazine metabolism in a primary culture of human hepatocytes

The metabolism of perazine in a primary culture of human hepatocytes after treatment of cells with TCDD (a CYP1A1/2 inducer) or rifampicin (mainly a CYP3A4 inducer) were studied in vitro. The concentrations of perazine and its main metabolites (perazine 5-sulfoxide, N-desmethylperazine) formed in hepatocytes were assayed in the extracellular medium using the HPLC method. TCDD and rifampicin induced the formation of perazine 5-sulfoxide, however, such an effect was not observed in the case of N-desmethylperazine. The accumulation of perazine 5-sulfoxide in the extracellular medium was enhanced until up to 4 h by rifampicin, and until up to 8 h byTCDD. After 24 h, perazine and perazine 5-sulfoxide were not detected in the extracellular medium of the inducer-treated cultures, except for perazine 5-sulfoxide in the TCDD-treated cultures The obtained results indicate that CYP1A2 and CYP3A4 are involved in the perazine metabolism via 5-sulfoxidation pathway.     

Thin-layer and gas-liquid chromatographic procedures for the determination of perazine and its metabolites in human body fluids.

The quantitative determination of perazine, a neuroleptic drug, and its metabolites in body fluids is difficult in view of the low concentrations to be expected under therapeutic conditions as well as of the problem of convenient detectors. Different methods for extraction and measurement of perazine concentration in blood samples are discussed, with special consideration of partition coefficients and the properties of the chromatographic systems (thin-layer and gas-liquid chromatography). A new and simple method for rapid gas chromatographic determination of perazine is presented.     

Absorption and metabolism of the phenothiazine drug perazine in the rat intestinal loop

Jejunal loops of male rats were instilled with [³⁵S]perazine and venous Wood from the loops was collected. Plasma, erythrocytes, intestinal wall and intestinal contents were analysed for perazine and its metabolites by reverse isotope dilution; purification to constant specific radioactivity was carried out by thin-layer chromatography. Within 60 min, 57 per cent of the material appeared in blood, more than four-fifths in the form of unchanged perazine. The principal metabolites present in plasma were 3-hydroxyperazine glucuronide and perazine sulfoxide; besides the sulfoxide, red cells contained small quantities of desmethyl perazine. This metabolite was predominantly located in the intestinal wall which contained a total of 17 per cent of the administered radioactivity, mostly as unmetabolized perazine. Another 17 per cent was found in the intestinal contents and here the proportion of perazine sulfoxide -was one-third. Besides perazine as the major compound small amounts of hydroxyperazine glucuronide and desmethyl perazine and traces of perazine N-oxide were present in the intestinal lumen.     

Determination of the binding of perazine and perazine sulfoxide to human serum albumin

Determination of the Binding of Perazine and Perazine Sulfoxide to Human Serum Albumin The binding of Perazine and its sulfoxide to 4% human serum albumin (HSA) in buffered phosphate solution (pH 7.4) was investigated by means of ultrafiltration (flat chamber) and modified gel filtration. It was found that within the physiological concentration range the sulfoxide is riot bound by albumin, whereas perazine even in strongly diluted solutions (ng range) showed a high affinity to albumin. The results were compared with literature data concerning the binding of perazine dimalonate (PDM) and desmethylperazine to human serum albumin (HSA).     

Metabolism of the phenothiazine drug perazine by liver and lung microsomes from various species

The oxidative metabolism of perazine was studied in vitro, using solvent extraction and thin layer chromatography followed by spectrophotometry for determination of the metabolites. Liver microsomes from rats, rabbits, pigs, guinea-pigs and cats and lung microsomes from rats, rabbits and pigs served as the enzyme sources.

Kinetics of N-oxidation, N-demethylation, sulfoxidation and aromatic hydroxylation were measured with liver microsomes. Demethylation, sulfoxidation and aromatic hydroxylation underlie a substrate inhibition already at a perazine concentration of 1 mM, whereas N-oxidation usually is maximal with 2 mM perazine and starts to be inhibited at 4 mM perazine. In the concentration range tested (0·25-8 mM perazine) the N-oxide is always the major metabolite. Excessive N-oxidation has been observed in liver microsomes from individual pigs.

Lung microsomes form substantial quantities of perazine N-oxide only, while other metabolites are produced to a negligible extent. An extremely high capacity for perazine N-oxidation was observed with rabbit lung microsomes, whereas microsomal preparations from rat and pig lungs N-oxidize perazine at a slower rate.     

The effect of selective serotonin reuptake inhibitors (SSRIs) on the pharmacokinetics and metabolism of perazine in the rat

The aim of this study was to investigate the effect of three selective serotonin reuptake inhibitors (SSRIs), fluoxetine, fluvoxamine and sertraline, on the pharmacokinetics and metabolism of perazine in a steady state in rats. Perazine (10 mg kg(-1), i.p.) was administered twice daily for two weeks, alone or jointly with one of the SSRIs. Concentrations of perazine and its two main metabolites (N-desmethylperazine and 5-sulfoxide) in the plasma and brain were measured 30 min and 6 and 12 h after the last dose of the drugs. Of the investigated SSRIs, fluoxetine and fluvoxamine significantly increased plasma and brain concentrations of perazine (up to 900% and 760% of the control value, respectively), their effect being most pronounced after 30 min and 6 h. Moreover, simultaneous increases in perazine metabolites concentrations and in the perazine/metabolite concentration ratios were observed. Sertraline elevated plasma and brain concentrations of perazine after 30 min. In-vitro studies with liver microsomes of rats treated chronically with perazine, SSRIs ortheir combinations showed decreased concentrations of cytochrome P-450 after perazine and a combination of perazine and fluvoxamine (vs control), and increased concentration after a combination of perazine and fluoxetine (vs perazine-treated group). Prolonged treatment with perazine did not significantly change the rate of its own metabolism. Chronic administration of fluoxetine or sertraline, alone or in a combination with perazine, accelerated perazine N-demethylation (vs control or perazine group, respectively). Fluvoxamine had a similar effect. The 5-sulfoxidation of perazine was accelerated by fluvoxamine and sertraline treatment, but the process was inhibited by administration of a combination of perazine and fluoxetine or fluvoxamine (vs control). Kinetic studies using control liver microsomes, in the absence or presence of SSRIs added in-vitro, demonstrated competitive inhibition of both N-demethylation and sulfoxidation by the investigated SSRIs. Sertraline was the most potent inhibitor of perazine N-demethylation but the weakest inhibitor of sulfoxidation. Results of in-vivo and in-vitro studies indicate that the observed interaction between perazine and SSRIs mainly involves competition for an active site of perazine N-demethylase and sulfoxidase. Moreover, increases in the concentrations of both perazine and metabolites measured, produced by the investigated drug combinations in-vivo, suggest simultaneous inhibition of another, yet to be investigated, metabolic pathway of perazine (e.g. aromatic hydroxylation).     

On the mechanism of the synergistic effect of perazine and hexobarbital

Summary The synergistic effect of perazine and hexobarbital was studied by interference with drugs which alter the adrenergic, serotonergic and cholinergic systems. The determination of hexobarbital level in blood and brain was used as an indication of the rate of its biotransformation.According to the findings perazine, dihydroergotamine (DHE), and phentolamine (in low doses) as well as an elevation of dopamine level prolong the anaesthesia by way of a central mechanism. Phentolamine (in higher doses) and the anticholinergic drug biperidin slow down the metabolism of hexobarbital, probably by inhibiting the hepatic microsomal enzymes.An alteration of the overall level of catecholamines, serotonin and acetylcholine had no effect on the hexobarbital-induced anaesthesia.The mild hyperthermia caused by all drugs tested, did not influence the duration of hexobarbital sleeping time.The possibility that perazine, like other -receptor blocking agents, causes a preponderance of inhibitory dopaminergic neurons by obstructing the excitatory norepinephrine receptors in the central nervous system is discussed. This effect might also occur if the dopamin level in the brain is increased.     

Lysosomal trapping as an important mechanism involved in the cellular distribution of perazine and in pharmacokinetic interaction with antidepressants

Perazine, a piperazine-type phenothiazine neuroleptic, is the most frequently chosen drug for combination with antidepressants in the therapy of complex or ‘treatment-resistant’ psychiatric illnesses. The aim of the present study was to investigate the contribution of lysosomal trapping to the total tissue uptake of perazine, and the pharmacokinetic interaction between the neuroleptic and antidepressants. Experiments were carried out on slices of different rat organs regarded as a system with functional lysosomes. To distinguish between lysosomal trapping and tissue binding, the experiments were performed in the absence or presence of ‘lysosomal inhibitors’, i.e. the lysosomotropic compound ammonium chloride or [H⁺] ionophore monensin, which abolish the pH-gradient of lysosomes. Under steady-state conditions, the highest tissue uptake of perazine was observed for the adipose tissue, which descended in the following order: the adipose tissue>lungs>liver>heart=brain>kidneys>muscles. The contribution of lysosomal trapping to the total tissue uptake amounted to about 40% in the liver, brain and muscles, to 30% in the kidneys, and to 25% in the heart and lungs. In the adipose tissue, no lysosomotropism of perazine was observed. Of the psychotropics studied, perazine was the only drug showing such a high degree of lysosomal trapping in muscles and distinct lysosomotropic properties in the heart. Perazine and the antidepressants used, both tricyclic (imipramine, amitriptyline) and selective serotonin reuptake inhibitors (fluoxetine, sertraline), mutually decreased their tissue uptake. The potency of imipramine to decrease perazine uptake was similar to that of the ‘lysosomal inhibitors’. Other antidepressants seemed to exert a somewhat weaker effect. The above interactions between perazine and antidepressants were not observed in the presence of ammonium chloride, which indicates that they proceeded at the level of lysosomal trapping. The adipose tissue in which the drug uptake was not affected by the ‘lysosomal inhibitors’ was not the site of such an interaction. Ammonium chloride did not affect the drug metabolism in liver slices; other tissues displayed only a negligible biotransformation of the psychotropics studied. A parallel metabolic interaction between perazine and tricyclic antidepressants took part in liver slices (i.e. perazine and antidepressants mutually inhibited their metabolic pathways), but the influence of such an interaction on the lysosomal uptake of the parent compounds in liver slices did not seem to be great. A substantial decrease in concentrations of the drugs in lysosomes (depot form) observed in vitro may lead to an increase in the concentration in vivo of the neuroleptic and antidepressants at the site of action, which, in turn, may increase the risk of cardiotoxic and anticholinergic side-effects of tricyclic antidepressants and sedative and extrapyramidal effects of the neuroleptic.     

Perazine as a potent inhibitor of human CYP1A2 but not CYP3A4

The effects of perazine on the activities of CYP1A2 and CYP3A4 in a primary culture of human hepatocytes of one patient were studied in vitro. The CYPs activities were assessed by measuring the rate of acetanilide 4-hydroxylation (CYP1A2) and cyclosporine A oxidation (CYP3A4) after treatment with TCDD (a CYP1A subfamily inducer) or rifampicin (mainly a CYP3A4 inducer). The amounts of the metabolites formed in hepatocytes were assayed in the extracellular medium using the HPLC method. TCDD and rifampicin induced the formation of 4-hydroxyacetanilide and cyclosporine A metabolites (monohydroxycyclosporine A, dihydroxycyclosporine A, N-desmethylcyclosporine A), respectively. The formation of 4-hydroxyacetanilide was strongly inhibited by three different concentrations of perazine (10, 25 and 50 microM) reaching 8, 3 and 2% of the control value, respectively. In the case of CYP3A4 activity, no such an effect of perazine was observed. Perazine showed only a week inhibition of the activity of cyclosporine A oxidase (to 96-86% of the control value). The obtained results suggest a strong inhibitory effect of perazine on human CYP1A2 activity with predicted Ki value similar to those of the known for CYP1A2 inhibitors, such as furafylline and fluvoxamine.     

Clinical and pharmacological approach to the diagnosis of paranoid schizophrenia

The aim of the study was finding out the correlations between some clinical and biological variables in paranoid schizophrenia. The group of 16 patients with paranoid schizophrenia, chosen in careful diagnostic procedure was treated with perazine during 4 weeks time. Before and during the treatment, the following parameters: psychopathology, serum perazine and prolactin concentration, dopamine-beta-hydroxylase activity and simple reaction time were measured. We found changes of those parameters caused by the treatment. The simple reaction time retardation was side-dependent before the treatment and not side-dependent after the treatment.     

Identification of a urinary metabolite of perazine as a piperazine-2,5-dione derivative

A non-basic perazine metabolite has been isolated from the urine of schizophrenic patients ingesting perazine. Identification of this compound as 10-[3‘-(2“,5”-dioxo-4“-methyl-piperazinyl)-propyl]-phenothiazine sulphoxide was achieved using ultraviolet, infrared, nuclear magnetic resonance and mass spectroscopy.     

The effect of perazine on the CYP2D6 and CYP2C19 phenotypes as measured by the dextromethorphan and mephenytoin tests in psychiatric patients

There is evidence that the antipsychotic drug perazine is an inhibitor of CYP2D6. This study aimed at evaluating its effect on CYP2D6 and CYP2C19 activities in submitting psychiatric patients to phenotyping with dextromethorphan and mephenytoin, respectively, substrates of these enzymes, before and during a treatment with perazine. A total of 31 patients were phenotyped with dextromethorphan (CYP2D6) and mephenytoin (CYP2C19) before and after a 2‐week treatment with 450 ± 51 mg/day (mean ± sd) perazine. At baseline, five patients appeared to be poor metabolizers (PM) of dextromethorphan and two patients of mephenytoin. The metabolic ratio (MR) of dextromethorphan/dextrorphan as determined in collected urine increased significantly (Wilcoxon; P < .0001) from baseline (0.39 ± 1.38 [mean ± sd]) till day 14 (1.46 ± 2.22). In 19 out of 26 extensive metabolizers (EM) of dextromethorphan, the phenotype changed from EM to PM. This suggests an almost complete inhibition of CYP2D6 by perazine and/or its metabolites. On the other hand, perazine (or some of its metabolites) did seemingly not inhibit CYP2C19. In conclusion, this study suggests that in patients treated with perazine and co‐medicated with CYP2D6 substrates, there could be an increased risk of adverse effects as a consequence of a pharmacokinetic interaction.     

Perazine,chlorpromazine and imipramine as inducers of microsomal drug metabolism

Summary Male rats were treated orally for 7 days with perazine, chlorpromazine, imipramine or phenobarbital. Isolated liver microsomes were tested for their metabolic activities towards perazine, ethylmorphine and aniline. N-Demethylation of perazine and ethylmorphine was increased 2–3.5 fold by all pretreatments. Aromatic hydroxylation of perazine was decreased in microsomes from pretreated animals, whereas aniline hydroxylation was enhanced even more by perazine and imipramine than by phenobarbital. The yield of perazine sulfoxide was increased by phenobarbital treatment only. Perazine N-oxide formation was reduced by treatment with psychoactive drugs to 75–90% of control values and by phenobarbital to less than 50%. The microsomal cytochrome P-450 concentration was slightly elevated by perazine and substantially by chlorpromazine and imipramine treatment. The tricyclic drugs investigated are potent inducers of the drug-metabolizing enzyme system, the induction pattern differing in some respects from that seen after phenobarbital.     

Perazine at therapeutic drug concentrations inhibits human cytochrome P450 isoenzyme 1A2 (CYP1A2) and caffeine metabolism – an in vitro study

The aim of the present study was to estimate the inhibitory effect of perazine, a phenothiazine neuroleptic with piperazine structure in a side chain, on human CYP1A2 activity measured as a rate of caffeine 3-N- and 1-N-demethylation. Moreover, the influence of perazine on other caffeine metabolic pathways such as 7-N-demethylation (CYP1A2, CYP2C8/9, CYP3A4) and 8-hydroxylation (CYP3A4, CYP1A2, CYP2C8/9) was also determined. The Dixon analysis showed that in both human liver microsomes and Supersomes CYP1A2 perazine potently and to a similar degree inhibited caffeine 3-N-demethylation (K_i = 3.5 μM) and 1-N-demethylation (K_i = 5 μM). Perazine moderately diminished the rate of caffeine 7-N-demethylation in Supersomes CYP1A2 (K_i = 11.5 μM) and liver microsomes (K_i = 20 μM), and attenuated C-8-hydroxylation (K_i = 15.5μM) in Supersomes CYP1A2. On the other hand, perazine weakly inhibited caffeine C-8-hydroxylation in liver microsomes (K_i = 98 μM). About 80% of basal CYP1A2 activity was reduced by the therapeutic concentrations of perazine (5–10 μM).The obtained results show that perazine at its therapeutic concentrations is a potent inhibitor of human CYP1A2. Hence, taking account of CYP1A2 contribution to the metabolism of endogenous substances (steroids), drugs (xanthine derivatives, phenacetin, propranolol, imipramine, phenothiazine neuroleptics, clozapine) and carcinogenic compounds, the inhibition of CYP1A2 by perazine may be of physiological, pharmacological and toxicological importance.     

Measurement of plasma levels of tricyclic psychoactive drugs and their metabolites by UV reflectance photometry of thin layer chromatograms

Summary A method has been developed for the determination of perazine, clozapine, imipramine and amitriptyline and their demethylated metabolites in plasma. Other metabolites measured were perazine sulfoxide and the N-oxides of clozapine and perazine, the latter two following their reduction to the parent drugs with ascorbic acid. 10-Hydroxynortriptyline was identified as an amitriptyline metabolite in plasma. The general procedure included extraction of alkalinized plasma samples (3 – 6 g) with benzene or toluene and thin layer chromatography of the extracts, followed by reflectance photometry of the plates at appropriate wave lengths in ultraviolet light. Spots of questionable identity were further characterized by two-dimensional chromatography and by colour reactions. The recoveries of compounds added in therapeutic concentrations were between 70 and 98 %. The limits of detectability were 5 – 10 ng/g plasma.     

Improved high-performance liquid chromatographic method for the determination of tiotixene in human serum

The problem of accurate determination of tiotixene in body fluids is still challenging. Several methods have been published but most of them require a tedious, time-consuming sample preparation, are not specific enough and lack the necessary sensitivity or require highly sophisticated analytical devices. As carefully validated analytical methods represent the basis of conclusive clinical trials (e.g. evaluating bioavailability/bioequivalence), an assay was developed to fulfill these needs. The method present employs an HPLC system combined with a UV-detector and uses perazine as an internal standard. The achieved lower limit of detection in serum was 0.05 ng/ml and the calibration curves were linear in the range of 0.5-20 and 0.1-2.0 ng/ml, respectively. The chromatographic peaks were well resolved and the cis-/transisomers well separated. The imprecision and inaccuracy data typically ranged from 2 to 7%; the recovery from serum was always better than 80%. The assay has been successfully used for the determination of very low tiotixene serum levels during several clinical studies.     

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 16 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.     

Different effects of amitriptyline and imipramine on the pharmacokinetics and metabolism of perazine in rats

The aim of this study was to search for possible effects of imipramine and amitriptyline on the pharmacokinetics and metabolism of perazine at steady state in rats. Perazine (10 mg kg(-1), i.p.) was administered to rats twice daily for two weeks, alone or jointly with imipramine or amitriptyline (10 mg kg(-1) i.p.). Concentrations of perazine and its two main metabolites (5-sulphoxide and N-desmethylperazine) in the plasma and brain were measured at 30 min (Cmax), 6h and 12h (slow disposition phase) after the last dose of the drugs. Liver microsomes were prepared 24 h after withdrawal of the drugs. Amitriptyline increased the plasma and brain concentrations of perazine (up to 300% of the control) and N-desmethylperazine, while not affecting those of 5-sulphoxide. Imipramine only tended to increase the neuroleptic concentration in the plasma and brain. Studies with control liver microsomes showed that amitriptyline and imipramine added to the incubation mixture in-vitro, competitively inhibited N-demethylation (Ki (inhibition constant) = 16 microM and 164 microM, respectively) and 5-sulphoxidation (Ki = 57 microM and 86 microM, respectively) of perazine, amitriptyline being a more potent inhibitor of perazine metabolism, especially with respect to N-demethylation. Studies with microsomes of rats treated chronically with perazine or tricyclic antidepressants, or both, did not show significant differences in the rate of perazine metabolism between perazine- and perazine+antidepressant-treated rats. The data obtained were compared with the results of analogous experiments with promazine and thioridazine. It was concluded that elevations of perazine concentration were caused by direct inhibition of the neuroleptic metabolism by the antidepressants. Similar interactions, possibly leading to exacerbation of the pharmacological action of perazine, may be expected in man. Since the interactions between phenothiazines and tricyclic antidepressants may proceed in two directions, reduced doses of both the neuroleptic and the antidepressant are recommended when the drugs are administered jointly.