CYP3A is responsible for N-dealkylation of haloperidol and bromperidol and oxidation of their reduced forms by human liver microsomes

We studied the biotransformation of haloperidol, bromperidol and their reduced forms by human liver microsomes. Nifedipine oxidation (CYP3A) activity correlated significantly with N-dealkylation rates of haloperidol and bromperidol and oxidation rates of their reduced forms, while neither ethoxyresorufin O-deethylation (CYP1A2) activity nor dextromethorphan O-deethylation (CYP2D6) activity did. In chemical and immunoinhibition studies, only troleandomycin and anti-CYP3A4 serum inhibited both formation rates of 4-fluorobenzoylpropionic acid, a metabolite of haloperidol and bromperidol, and back oxidation rates. Among 10 recombinant isoforms examined, only CYP3A4 showed catalytic activity. The Vmax and Km values of N-dealkylation of bromperidol and reoxidation of reduced bromperidol were similar to those of haloperidol and reduced haloperidol, respectively. The present study indicates that CYP3A plays a major role in N-dealkylation of and oxidation back to bromperidol as well as haloperidol and suggests that modification of in vivo CYP3A activity by inhibition or induction may affect the pharmacokinetics and therapeutic effects of haloperidol and bromperidol.     

Liquid chromatographic-mass spectrometric determination of haloperidol and its metabolites in human plasma and urine

Haloperidol and its two metabolites, reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP) in human plasma and urine were analyzed by HPLC-MS using a new polymer column (MSpak GF-310), which enabled direct injection of crude biological samples without pretreatment. Recoveries of haloperidol and reduced haloperidol spiked into plasma were 64.4-76.1% and 46.8-50.2%, respectively; those for urine were 87.3-99.4% and 94.2-98.5%, respectively; those of CPHP for both samples were not less than 92.7%. The regression equations for haloperidol, reduced haloperidol and CPHP showed good linearity in the ranges of 10-800, 15-800 and 400-800 ng/ml, respectively, for both plasma and urine. Their detection limits were 5, 10 and 300 ng/ml, respectively, for both samples. Thus, the present method was sensitive enough for detection and determination of high therapeutic and toxic levels for haloperidol and its metabolites present in biological samples.     

Megabore capillary gas-liquid chromatographic method with nitrogen-phosphorus selective detection for the assay of haloperidol and reduced haloperidol in serum: results of therapeutic drug-monitoring during acute therapy of eight schizophrenics

A gas chromatographic method using a HP-5 megabore capillary and nitrogen-phosphorus selective detection for the quantitative analysis of haloperidol (H) and reduced haloperidol (RH) in human serum or plasma is described. A 3-step liquid-liquid extraction is applied. The extraction yield of this procedure is 63% for haloperidol at 20 ng/ml. The limits of detection are 0.4 ng/ml for haloperidol and 1.0 ng/ml for the metabolite if 2 ml of body fluid are applied. At 10 ng/ml the within-day precision is 4.5% for H and 8.3% for RH. Serum levels of eight schizophrenic patients have been monitored weekly over a therapeutic period of six weeks. Seven patients mainly had metabolite ratios RH/H < 1 over the entire period of investigation. They exhibited a linear correlation between dose and serum concentration of haloperidol. In contrast, one patient had metabolite ratios RH/H > 1 over the entire period of the study. Due to considerable increased serum concentrations this patient did not show a linear correlation between the dose and the serum level of haloperidol.     

The mechanism of action of the nitrosourea anti-tumor drugs on thioredoxin reductase, glutathione reductase and ribonucleotide reductase

The nitrosoureas BCNU, CCNU, ACNU, and Fotemustine covalently deactivate thioredoxin reductase, glutathione reductase and ribonucleotide reductase by alkylating their thiolate active sites. Since thioredoxin reductase and glutathione reductase function as alternative electron donors in the biosynthesis of deoxyribonucleotides, catalyzed by ribonucleotide reductase, the inhibition of these electron transfer systems by the nitrosoureas could determine the cytostatic property of this homologous series of drugs. A detailed study of the kinetics and mechanism for the inhibition of purified thioredoxin reductases from human metastatic melanotic and amelanotic melanomas by the nitrosoureas showed significantly different inhibitor constants. This difference is due to the regulation of these proteins by calcium. Calcium protects thioredoxin reductase from deactivation by the nitrosoureas. In addition, it has been shown that reduced thioredoxin displaces the nitrosourea-inhibitor complex from the active site of thioredoxin reductase to fully reactivate enzyme purified from human metastatic amelanotic melanoma. It has been possible to label the active sites of thioredoxin reductase and glutathione reductase by using chloro[14C]ethyl Fotemustine, resulting in the alkylation of the thiolate active sites to produce chloro[14C]ethyl ether-enzyme inhibitor complexes. These complexes can be reactivated via reduced thioredoxin and reduced glutathione, respectively, by a beta-elimination reaction yielding [14C]ethylene and chloride ions as reaction products.     

Reduction of mitochondrial protein mitoNEET [2Fe–2S] clusters by human glutathione reductase

The human mitochondrial outer membrane protein mitoNEET is a newly discovered target of the type 2 diabetes drug pioglitazone. Structurally, mitoNEET is a homodimer with each monomer containing an N-terminal transmembrane α helix tethered to the mitochondrial outer membrane and a C-terminal cytosolic domain hosting a redox-active [2Fe–2S] cluster. Genetic studies have shown that mitoNEET has a central role in regulating energy metabolism in mitochondria. However, the specific function of mitoNEET remains largely elusive. Here we find that the mitoNEET [2Fe–2S] clusters can be efficiently reduced by Escherichia coli thioredoxin reductase and glutathione reductase in an NADPH-dependent reaction. Purified human glutathione reductase has the same activity as E. coli thioredoxin reductase and glutathione reductase to reduce the mitoNEET [2Fe–2S] clusters. However, rat thioredoxin reductase, a human thioredoxin reductase homolog that contains selenocysteine in the catalytic center, has very little or no activity to reduce the mitoNEET [2Fe–2S] clusters. N-ethylmaleimide, a potent thiol modifier, completely inhibits human glutathione reductase from reducing the mitoNEET [2Fe–2S] clusters, indicating that the redox-active disulfide in the catalytic center of human glutathione reductase may be directly involved in reducing the mitoNEET [2Fe–2S] clusters. Additional studies reveal that the reduced mitoNEET [2Fe–2S] clusters in mouse heart cell extracts can be reversibly oxidized by hydrogen peroxide without disruption of the clusters, suggesting that the mitoNEET [2Fe–2S] clusters may undergo redox transition to regulate energy metabolism in mitochondria in response to oxidative signals.     

Characterization of a major form of human isatin reductase and the reduced metabolite.

Isatin, an endogenous indole, has been shown to inhibit monoamine oxidase, and exhibit various pharmacological actions. However, the metabolism of isatin in humans remains unknown. We have found high isatin reductase activity in the 105,000 g supernatants of human liver and kidney homogenates, and have purified and characterized a major form of the enzyme in the two tissues. The hepatic and renal enzymes showed the same properties, including an M(r) of 31 kDa, substrate specificity for carbonyl compounds and inhibitor sensitivity, which were also identical to those of recombinant human carbonyl reductase. The identity of the isatin reductase with carbonyl reductase was immunologically demonstrated with an antibody against the recombinant carbonyl reductase. About 90% of the soluble isatin reductase activity in the liver and kidney was immunoprecipitated by the antibody. The Km (10 microm) and k(cat)/K(m) (1.7 s(-1) x microm(-1)) values for isatin at pH 7.0 were comparable to those for phenanthrenequinone, the best xenobiotic substrate of carbonyl reductase. The reduced product of isatin was chemically identified with 3-hydroxy-2-oxoindole, which is also excreted in human urine. The inhibitory potency of the reduced product for monoamine oxidase A and B was significantly lower than that of isatin. The results indicate that the novel metabolic pathway of isatin in humans is mediated mainly by carbonyl reductase, which may play a critical role in controlling the biological activity of isatin.     

Effects of caerulein + haloperidol on amphetamine-induced locomotor stereotypy in rats

The combination of haloperidol + caerulein has been reported to produce a long-lasting reduction of amphetamine-induced hyperlocomotions in rats. This study was designed to replicate those findings and to determine whether haloperidol + caerulein produce any unique effect on amphetamine-induced locomotor stereotypy. In two experiments, haloperidol + caerulein failed to produce a long-lasting reduction in amphetamine-induced hyperlocomotions. Although haloperidol reduced the locomotor stereotypy produced by higher doses of amphetamine, caerulein had no effect, either alone or combined with haloperidol.     

Delineation of the catalytic components of the NADPH-dependent O2- generating oxidoreductase of human neutrophils

Four catalytic components of the NADPH-dependent O2- generating oxidoreductase of human neutrophils have been identified. DCIP reductase, cytochrome c reductase and a chromophore 450-455 reductase are present in phorbol myristate acetate stimulated neutrophils and absent in resting cells and phorbol myristate acetate stimulated chronic granulomatous disease cells. Quinol dehydrogenase activity has also been demonstrated in activated and resting cells. Furthermore, a chromophore absorbing in the reduced state at 450-455 nm participates in superoxide production. This chromophore is reduced by NADPH or duroquinol and is missing in cell lysates derived from a patient with chronic granulomatous disease.     

DIHYDROPTERIDINE REDUCTASE MAY FUNCTION IN TETRAHYDROFOLATE METABOLISM

Abstract— The tetrahydrofolate-dependent serine hydroxymethyl transferase ( l -serine: tetrahydrofolate 10-hydroxymethyl transferase, EC 2.1.2.1) reaction in rat or human brain homogenates incubated aerobically is dependent on added reducing agents for full activity in order to protect the readily oxidized substrate, tetrahydrofolate. In this role, 0.1 m m -NADH is as affective as 10m m -2-mercaptoethanol and it can be shown that the NADH prevents destruction of tetrahydrofolate incubated with brain homogenates. If the dihydropteridine reductase (NADPH:6,7-dihydropteridine oxidoreductase, EC 1.6.99.7) activity of the brain homogenate is inhibited by a specific antiserum, NADH, but not 2-mercaptoeth-anol, is no longer effective. Furthermore, an homogenate of a brain biopsy from a human lacking dihydropteridine reductase requires added dihydropteridine reductase for maximal stimulation by NADH of the serine hydroxymethyl transferase reaction. We conclude that dihydropteridine reductase mediates the NADH stimulation and can play a role in preserving tetrahydrofolate from oxidation. The rinding of greatly reduced folate levels in the brain biopsy from the human lacking dihydropteridine reductase supports this postulated role of dihydropteridine reductase in folate metabolism.     

Purification and characterization of chlordecone reductase from human liver

The toxic organochlorine pesticide, chlordecone (Kepone), is excreted in human bile primarily as a stable, reduced monoalcohol metabolite. This bioreduction is catalyzed by a hepatic cytosolic enzyme activity termed chlordecone reductase. We purified this enzyme from human liver and found that chlordecone reductase resembles the family of xenobiotic metabolizing enzymes referred to as the aldo-keto reductases based on its biochemical characteristics, including its ability to catalyze the reduction of a carbonyl-containing substrate. However, analyses of liver cytosolic samples on immunoblots developed with anti-chlordecone reductase antibodies revealed that immunoreactive proteins were present only in those mammalian species that convert chlordecone to chlordecone alcohol in vivo (man, gerbil, and rabbit) and not in those species unable to reduce chlordecone (rat, mouse, and hamster). Hence, chlordecone reductase is unique among aldo-keto reductases in being species-specific. Quantitative immunoblot analyses of seven human liver specimens disclosed two immunoreactive proteins whose total concentration varied over a 6-fold range. Moreover, the amount of immunoreactive protein was directly proportional to chlordecone reductase activity in each sample. We conclude that chlordecone reductase is a unique aldo-keto reductase of potential clinical importance whose expression varies markedly among individuals.     

Radioimmunoassay of haloperidol in human serum

Immunization of rabbits with a haloperidol hydrazone-bovine serum albumin conjugate elicited the formation of antibody to haloperidol. With this antiserum, concentrations of haloperidol as low as 1 ng/ml can be easily measured by radioimmunoassay of unextracted human serum. None of the known major metabolites of haloperidol displayed any significant cross-reactivity. Psychiatric patients receiving daily oral doses of 20 to 200 mg haloperidol had serum levels ranging between 5.8 and 245 ng/ml.     

A gas chromatographic mass spectrometric chemical ionization assay for haloperidol with selected ion monitoring

A new gas chromatographic mass spectrometric chemical ionization assay for haloperidol with selectedion monitoring is presented which provides for better combined selectivity and sensitivity than previous assays. Levels of haloperidol in 2 ml of human plasma were reproducibly measured down to subnanogram levels. Both methane and methane--ammonia chemical ionization spectra are presented for haloperidol and the internal standard trifluperidol.     

Tetrahydrobiopterin is synthesized from 6-pyruvoyl-tetrahydropterin by the human aldo-keto reductase AKR1 family members

Tetrahydrobiopterin (BH(4)) is a cofactor for aromatic amino acid hydroxylases and nitric oxide synthase. The biosynthesis includes two reduction steps catalyzed by sepiapterin reductase. An intermediate, 6-pyruvoyltetrahydropterin (PPH(4)) is reduced to 1(')-oxo-2(')-hydroxypropyl-tetrahydropterin (1(')-OXPH(4)) or 1(')-hydroxy-2(')-oxopropyl-tetrahydropterin (2(')-OXPH(4)), which is further converted to BH(4). However, patients with sepiapterin reductase deficiency show normal urinary excretion of pterins without hyperphenylalaninemia, suggesting that other enzymes catalyze the two reduction steps. In this study, the reductase activities for the tetrahydropterin intermediates were examined using several human recombinant enzymes belonging to the aldo-keto reductase (AKR) family and short-chain dehydrogenase/reductase (SDR) family. In the reduction of PPH(4) by AKR family enzymes, 2(')-OXPH(4) was formed by 3 alpha-hydroxysteroid dehydrogenase type 2, whereas 1(')-OXPH(4) was produced by aldose reductase, aldehyde reductase, and 20 alpha-hydroxysteroid dehydrogenase, and both 1(')-OXPH(4) and 2(')-OXPH(4) were detected as the major and minor products by 3 alpha-hydroxysteroid dehydrogenases (types 1 and 3). The activities of aldose reductase and 3 alpha-hydroxysteroid dehydrogenase type 2 (106 and 35 nmol/mg/min, respectively) were higher than those of the other enzymes (0.2-4.0 nmol/mg/min). Among the SDR family enzymes, monomeric carbonyl reductase exhibited low 1(')-OXPH(4)-forming activity of 5.0 nmol/mg/min, but L-xylulose reductase and peroxisomal tetrameric carbonyl reductase did not form any reduced product from PPH(4). Aldose reductase reduced 2(')-OXPH(4) to BH(4), but the other enzymes were inactive towards both 2(')-OXPH(4) and 1(')-OXPH(4). These results indicate that the tetrahydropterin intermediates are natural substrates of the human AKR family enzymes and suggest a novel alternative pathway from PPH(4) to BH(4), in which 3 alpha-hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert.     

Cyclo (Leu-Gly) + haloperidol: effects on dopamine receptors and conditioned avoidance responding

Behavioral effects of cyclo (Leu-Gly) (cLG), administered either acutely or chronically, were assessed in combination with haloperidol in the rat. cLG administered chronically, produced a significant reduction in the increase in apomorphine-induced stereotypy produced by chronic haloperidol infusion. On the other hand, the same dose of cLG which reduced this induction of dopamine receptor supersensitivity due to chronic haloperidol treatment, failed to produce a change in the potency of haloperidol in blocking conditioned avoidance responding in the rat. Furthermore, degeneration-induced supersensitivity of dopamine neurons, produced by unilateral destruction of the nigrostriatal pathway, was not reduced by acute or chronic treatment with cLG as measured by apomorphine-induced rotation. These data suggest that cLG may decrease motor system side effects thought to be caused by chronic antipsychotic administration without affecting the therapeutic efficacy of the antipsychotic agent.     

Haloperidol-induced suppression of carotid chemoreception in vitro

Effects of antagonism of endogenous dopamine with haloperidol on single-unit frequency, interspike interval distribution, and interval serial dependency of the cat sinus nerve were tested using an in vitro carotid body-sinus nerve superfusion technique. A dose dependency of inhibition by haloperidol (0.05-2.0 microgram/ml) was observed. Superfusion with 1-2 microgram/ml haloperidol significantly reduced frequency within 5 min (P less than 0.05) and caused a complete cessation of firing within 25 min in 5 of 10 chemoreceptor units. Frequency recovered to control during drug washout. Acetylcholine (10-micrograms/ml superfusion or 500-micrograms bolus) increased sinus nerve activity under control conditions but not during superfusion with haloperidol. No effect of haloperidol on impulse serial dependency was detected. However, interval distribution was significantly altered by haloperidol in five of six chemoreceptor units. Our results suggest an excitatory role for dopamine in carotid chemoreception.     

Cytoplasmic Determinants of Piperidine Blocking Affinity for N-type Calcium Channels

Piperidines are a relatively novel class of calcium channel blockers which act at a unique receptor site associated with the calcium channel α₁ subunit. Calcium channel blocking affinities ranging from subnanomolar to several hundred micromolar have been reported in the literature, suggesting that piperidine block is highly sensitive to the cellular environment experienced by the channel. Here, I have investigated some of the cytoplasmic determinants of haloperidol block of N-type calcium channels expressed in human embryonic kidney cells. In perforated patch clamp recordings, haloperidol blocks N-type calcium channels with an inhibition constant of 120 μM. Upon internal dialysis with chloride containing pipette solution, the blocking affinity increases by 40-fold. This effect could be attributed in part to the presence of internal chloride ions, as replacement of intracellular chloride with methanesulfonate reduced haloperidol blocking affinity by almost one order of magnitude. Tonic inhibition of N-type channels by G_βγ subunits further enhanced the blocking effects of haloperidol, suggesting the possibility of direct effects of G_βγ binding on the local environment of the piperidine receptor site. Overall, depending on the cytoplasmic environment experienced by the channel, the blocking affinity of N-type calcium channels for haloperidol may vary by more than two orders of magnitude. Thus, absolute blocking affinities at the piperidine receptor site must be interpreted cautiously and in the context of the particular experimental setting. Received: 23 July 1998/Revised: 19 October 1998     

Isolation and sequence studies of cysteinyl peptides from Spirulina glutathione reductase: comparison of active site cysteine peptides with those of other flavoprotein disulfide oxidoreductases

The amino acid sequences of the cysteinyl peptides of Spirulina sp. glutathione reductase were determined. Spirulina glutathione reductase was covalently bound to Thiopropyl-Sepharose 6B in the presence of 8M urea through thiol-disulfide exchange. After tryptic digestion, 4 distinct cysteinyl peptides were finally isolated from NADPH-reduced glutathione reductase and 2 from oxidized glutathione reductase. The amino acid sequences of the two cysteinyl peptides which could not be isolated from the oxidized glutathione reductase were very similar to those around the active site disulfide of the other flavoprotein disulfide oxidoreductases and a unique replacement of asparagine and valine by isoleucine and arginine between the two cysteine residues was found. The other two peptides isolated from both oxidized and reduced glutathione reductase also show considerable homology to the corresponding parts of human and Escherichia coli glutathione reductases.     

Epicatechin Inhibits Human Plasma Lipid Peroxidation Caused by Haloperidol In Vitro

Epicatechin belongs to flavonoids protecting cells against oxidative/nitrative stress. Oxidative/nitrative stress observed in schizophrenia may be caused partially by the treatment of patients with various antipsychotics. The aim of our study was to establish the effects of epicatechin and antipsychotics action (the first generation antipsychotic (FGA)—haloperidol and the second generation antipsychotic (SGA)—amisulpride) on peroxidation of plasma lipids in vitro. Lipid peroxidation in human plasma was measured by the level of thiobarbituric acid reactive species (TBARS). The properties of epicatechin were also compared with the action of a well characterized antioxidative commercial polyphenol—resveratrol (3,4′,5-trihydroxystilbene) and quercetin (3,5,7,3′,4′-pentahydroxyflavone). Amisulpride, contrary to haloperidol (after 1 and 24 h) does not significantly influence the increase of plasma TBARS level in comparison with control samples (P > 0.05). After incubation (1 and 24 h) of plasma with haloperidol in the presence of epicatechin we observed a significantly decreases the level of TBARS (P < 0.001, P < 0.001, respectively). In our other experiments, we found that epicatechin also decreased the amount of TBARS in human plasma treated with amisulpride. In conclusion, the presented results indicate that epicatechin—the major polyphenolic component of green tea reduced significantly human plasma lipid peroxidation caused by haloperidol. Moreover, epicatechin was found to be a more effective antioxidant, than the solution of pure resveratrol or quercetin.     

The effects of some antipsychotic drugs, D-amphetamine, and reserpine on the concentration and rate of accumulation of tryptamine and 5-hydroxytryptamine in the mouse striatum

The mouse striatum contains about 2 ng/g of tryptamine and 600 ng/g of 5-hydroxytryptamine. No significant changes in mouse striatal tryptamine were observed after the administration of chlorpromazine, haloperidol, spiperone, or alpha-flupenthixol. The levels of 5-hydroxytryptamine were moderately reduced by chlorpromazine, spiperone, and alpha-flupenthixol but not by haloperidol. The administration of antipsychotic drugs to mice pretreated with a monoamine oxidase inhibitor (pargyline) produced an increase in the rate of accumulation of striatal tryptamine compared with that of pargyline-treated mice. In contrast, the rate of accumulation of 5-hydroxytryptamine after monoamine oxidase inhibition was reduced by chlorpromazine, spiperone, and alpha-flupenthixol but not haloperidol. D-Amphetamine administration did not change either tryptamine or its 5-hydroxyderivative while reserpine increased tryptamine and reduced 5-hydroxytryptamine. The results suggest that changes in striatal tryptamine may be controlled by the availability of tryptophan, the amino acid precursor of tryptamine.     

Sensitivity and resistance in human metastatic melanoma to the new chloroethylnitrosourea anti-tumor drug Fotemustine

Fotemustine is a novel chloroethylnitrosourea derivative currently used in Phase III clinical trials for disseminated metastatic melanoma. This drug has been shown to inhibit enzymes in the ribonucleotide reduction pathway (i.e., thioredoxin reductase, glutathione reductase and ribonucleotide reductase). 14C chloroethyl-labelled Fotemustine covalently labels the thiolate active sites of thioredoxin reductase and glutathione reductase yielding 14C chloroethyl-thioether enzyme-inhibitor complexes. Enzyme activities can be restored by a reduced thioredoxin or reduced glutathione mediated beta-elimination of the chloroethyl group. 14C Fotemustine has been used to determine its reactivity and metabolism in drug sensitive and resistant melanoma metastases and in cultures of sensitive and resistant clones of human melanoma cells. Melanoma metastases from four different patients who were treated with Fotemustine could be labelled with radioactive drug only under reducing conditions with NADPH as electron donor and DTNB as substrate. FPLC analysis of these extracts revealed two radioactive proteins (I) glutathione reductase and (II) an unidentified protein with 95 and 50 kDa subunits. A similar labelling pattern was also found in extracts of Fotemustine sensitive melanoma cells (Cal 1). Fotemustine resistant tumors were melanotic and contained more glutathione reductase than thioredoxin reductase, whereas sensitive tumors were clinically amelanotic with more thioredoxin reductase than glutathione reductase. Fotemustine resistant melanoma cells (Cal 7) showed a slower uptake of 14C-label with 34% less isotope intracellularly in 1 h compared to sensitive melanoma cells (Cal 1). These results strongly indicate (I) the induction of alternate electron donors thioredoxin reductase or glutathione reductase for ribonucleotide reduction determines tumor and melanoma cell responses to the drug and (II) Fotemustine transport and the intracellular redox status seems to regulate resistance in melanoma cells and tissues.