Inhibition of peripheral dopamine metabolism and the ventilatory response to hypoxia in the rat.

Dopamine (DA) is a putative neurotransmitter in the carotid body engaged in the generation of the hypoxic ventilatory response (HVR). However, the action of endogenous DA is unsettled. This study seeks to determine the ventilatory effects of increased availability of endogenous DA caused by inhibition of DA enzymatic breakdown. The peripheral inhibitor of MAO - debrisoquine, or COMT - entacapone, or both combined were injected to conscious rats. Ventilation and its responses to acute 8 % O(2) in N(2) were investigated in a whole body plethysmograph. We found that inhibition of MAO augmented the hyperventilatory response to hypoxia. Inhibition of COMT failed to influence the hypoxic response. However, simultaneous inhibition of both enzymes, the case in which endogenous availability of DA should increase the most, reversed the hypoxic augmentation of ventilation induced by MAO-inhibition. The inference is that when MAO alone is blocked, COMT takes over DA degradation in a compensatory way, which lowers the availability of DA, resulting in a higher intensity of the HVR. We conclude that MAO is the enzyme predominantly engaged in the chemoventilatory effects of DA. Furthermore, the findings imply that endogenous DA is inhibitory, rather than stimulatory, for hypoxic ventilation.

Extrapolating in vitro metabolic interactions to isolated perfused liver: predictions of metabolic interactions between R-bufuralol, bunitrolol, and debrisoquine.

Drug-drug interactions (DDIs) are a great concern to the selection of new drug candidates. While in vitro screening assays for DDI are a routine procedure in preclinical research, their interpretation and relevance for the in vivo situation still represent a major challenge. The objective of the present study was to develop a novel mechanistic modeling approach to quantitatively predict DDI solely based upon in vitro data. The overall strategy consisted of developing a model of the liver with physiological details on three subcompartments: the sinusoidal space, the space of Disse, and the cellular matrix. The substrate and inhibitor concentrations available to the metabolizing enzyme were modeled with respect to time and were used to relate the in vitro inhibition constant (K(i)) to the in vivo situation. The development of the liver model was supported by experimental studies in a stepwise fashion: (i) characterizing the interactions between the three selected drugs (R-bufuralol (BUF), bunitrolol (BUN), and debrisoquine (DBQ)) in microsomal incubations, (ii) modeling DDI based on binary mixtures model for all the possible pairs of interactions (BUF-BUN, BUF-DBQ, BUN-DBQ) describing a mutual competitive inhibition between the compounds, (iii) incorporating in the binary mixtures model the related constants determined in vitro for the inhibition, metabolism, transport, and partition coefficients of each compound, and (iv) validating the overall liver model for the prediction of the perfusate kinetics of each drug determined in isolated perfused rat liver (IPRL) for the single and paired compounds. Results from microsomal coincubations showed that competitive inhibition was the mechanism of interactions between all three compounds, as expected since those compounds are all substrates of rat CYP2D2. For each drug, the K(i) values estimated were similar to their K(m) values for CYP2D2 indicative of a competition for the same substrate-binding site. Comparison of the performance between the novel liver physiologically based pharmacokinetic (PBPK) model and published empirical models in simulating the perfusate concentration-time profile was based on the area under the curve (AUC) and the shape of the curve of the perfusate time course. The present liver PBPK model was able to quantitatively predict the metabolic interactions determined during the perfusions of mixtures of BUF-DBQ and BUN-DBQ. However, a lower degree of accuracy was obtained for the mixtures of BUF-BUN, potentially due to some interindividual variability in the relative proportion of CYP2D1 and CYP2D2 isoenzymes, both involved in BUF metabolism. Overall, in this metabolic interaction prediction exercise, the PBPK model clearly showed to be the best predictor of perfusate kinetics compared to more empirical models. The present study demonstrated the potential of the mechanistic liver model to enable predictions of metabolic DDI under in vivo condition solely from in vitro information.

Validation of incorporating flurbiprofen into the Pittsburgh cocktail.

BACKGROUND: We have previously shown that flurbiprofen metabolism to 4'-hydroxyflurbiprofen provides an in vivo measure of cytochrome P450 (CYP) 2C9 activity. This study evaluated the possibility of incorporating flurbiprofen into the current 5-drug Pittsburgh cocktail. METHODS: In a randomized, 3-way, Latin-square, crossover-design study, 24 healthy subjects (mean age [+/-SD], 47.8 +/- 15.1 years) received flurbiprofen (50 mg) and the Pittsburgh 5-drug cocktail (100 mg caffeine, 100 mg mephenytoin, 10 mg debrisoquin [INN, debrisoquine], 250 mg chlorzoxazone, and 100 mg dapsone) separately and in combination on 3 occasions over a period of 5 weeks. Urine was collected from 0 to 8 hours, and plasma was obtained at 4 and 8 hours after drug administration. Parent drug and metabolite concentrations were measured to determine phenotypic indices for each of the metabolizing enzymes. RESULTS: The geometric mean ratio and 90% confidence interval of the phenotypic indices were included within the 80% to 125% bioequivalence range for each of the probe drugs. There were no statistically significant differences between the phenotypic indices determined after administration of the 5-drug and 6-drug cocktails. However, there was a small but statistically significant increase (7.5%, P = .03) in the 8-hour urinary flurbiprofen recovery ratio after administration of the 6-drug cocktail compared with that after administration of flurbiprofen alone. The 6-drug cocktail was well tolerated. CONCLUSION: The results of this study show that caffeine (CYP1A2), chlorzoxazone (CYP2E1), dapsone (N-acetyltransferase 2), debrisoquin (CYP2D6), flurbiprofen (CYP2C9), and mephenytoin (CYP2C19) can be simultaneously administered in low doses without metabolic interaction.

Assessment of in vivo CYP2D6 activity: differential sensitivity of commonly used probes to urine pH.

Drug/metabolite ratios (MRs) are used as in vivo markers of enzyme activity. The ratios are potentially confounded by the renal clearance of the drug (urine-based MRs) or metabolite (plasma-based MRs). The authors have investigated the relative sensitivity of urinary MR of 3 in vivo probe substrates of CYP2D6 debrisoquine (DB), dextromethorphan (DM), and metoprolol (MP) to changes in urine pH. Three groups of healthy volunteers each comprising 12 individuals were given DB (10 mg), DM (25 mg), or MP (100 mg) on 3 occasions. In 1 study arm, urine was acidified by the oral intake of ammonium chloride; in another, it was alkalinized by intake of sodium bicarbonate; and in the third, urine pH was uncontrolled. Urinary MP/alpha-hydroxy-MP, DM/dextrorphan, and DB/4-hydroxy-DB ratios were calculated. The mean(geo) MR for DB was not significantly different in any of the study arms, whereas those for MP and DM were significantly different under acidified and alkalinized urine conditions compared to uncontrolled urine pH (P < .01) and were correlated with urine pH (P < .001). Without control of urine pH, in vivo estimates of CYP2D6 metabolic activity are likely to be less precise using DM or MP as probe substrates compared to DB. Although this is unlikely to cause any problem in distinguishing the large functional differences in CYP2D6 in poor metabolizer (PM) and extensive metabolizer (EM) phenotypes, this may contribute to difficulties in differentiating in vivo metabolic activity among allelic variants within the overall CYP2D6 EM phenotype using MP or DM. However, because DB is not available in many countries (eg, United States), alternative in vivo markers of CYP2D6 with low sensitivity to urine pH should be sought.

Pharmacokinetic and pharmacodynamic assessment of a five-probe metabolic cocktail for CYPs 1A2, 3A4, 2C9, 2D6 and 2E1.

AIMS: The primary objectives of the present study were to establish whether there was a pharmacokinetic or pharmacodynamic interaction between the probe drugs caffeine (CYP1A2), tolbutamide (CYP2C9), debrisoquine (CYP2D6), chlorzoxazone (CYP2E1) and midazolam (CYP3A4), when administered in combination as a cocktail. Furthermore, the tolerability of these probe drugs, both alone and in combination as a cocktail was assessed. METHODS: Twelve healthy volunteer subjects (age range 22-48 years) were entered into an open, fixed sequence, 6-limb, single centre study. The randomization was such that all drugs were given individually followed by the full "cocktail" as the last treatment limb. The phenotypic index used to assess the intrinsic activity of the CYP isoforms included metabolite/parent ratios in plasma and urine (CYPs 1A2, 2E1 & 2C9), parent/metabolite ratios in urine (CYP2D6) and plasma AUClast (CYP3A4). Blood pressure and blood glucose measurements were used to assess pharmacodynamic interactions. Tolerability was assessed through reporting of adverse events RESULTS: Overall, there was little evidence that the probe drugs interacted metabolically when co-administered as the cocktail. The ratio of the geometric mean (and 90% confidence interval) of the phenotypic index, obtained after administration of the probe as part of the cocktail and when given alone were: caffeine, 0.86 (0.67-1.10), midazolam, 0.96 (0.74-1.24), tolbutamide, 0.86 (0.72-1.03), debrisoquine 1.04 (0.97-1.12) and chlorzoxazone, 0.95 (0.86-1.05). There was no difference in blood pressure and blood glucose concentrations following the cocktail and dosing of the individual probes. There was no effect on ECG recordings at any time-point. The adverse events reported for individual drug administrations were mild, transient and expected. Overall no more adverse events were reported on the cocktail study days than on the days when the drugs were administered alone. CONCLUSIONS: The five probe drugs when coadministered, in this dosing regimen, demonstrated no evidence of either a metabolic or pharmacodynamic interaction that might confound the conclusions drawn during a cocktail study. The present cocktail methodology has the potential to become a useful tool to aid the detection of clinically important drug-drug interactions during drug development.

Effect of chronic disulfiram administration on the activities of CYP1A2, CYP2C19, CYP2D6, CYP2E1, and N-acetyltransferase in healthy human subjects.

AIMS: Short-term disulfiram administration has been shown to selectively inhibit CYP2E1 activity but the effects of chronic disulfiram administration on the activities of drug metabolizing enzymes is unclear. The purpose of this study was to evaluate the effects of disulfiram given for 11 days on selected drug metabolizing enzyme activities. METHODS: Seven healthy volunteers were given disulfiram 250 mg daily for 11 days. Activities of the drug metabolizing enzymes CYP1A2, CYP2C19, CYP2D6, CYP2E1 and N-acetyltransferase were determined using the probe drugs caffeine, mephenytoin, debrisoquine, chlorzoxazone, and dapsone, respectively. Chlorzoxazone was administered before disulfiram administration and after the second and eleventh doses of disulfiram, while the other probe drugs were given before disulfiram administration and after the eleventh disulfiram dose. RESULTS: Disulfiram administration markedly inhibited chlorzoxazone 6-hydroxylation by more than 95%, but did not affect metabolism of debrisoquine or mephenytoin. Caffeine N3-demethylation was decreased by 34% (P < 0.05). Monoacetyldapsone concentrations were markedly elevated by disulfiram administration resulting in a nearly 16-fold increase in the dapsone acetylation index, calculated as the plasma concentration ratio of monoacetyldapsone to dapsone. CYP-mediated dapsone N-hydroxylation was not significantly altered. CONCLUSIONS: These data suggest that disulfiram-mediated inhibition is predominantly selective for CYP2E1. The magnitude of CYP2E1 inhibition was similar after both acute and chronic disulfiram administration. The effects on caffeine N3-demethylation (CYP1A2) and dapsone metabolism suggest that chronic disulfiram administration may affect multiple drug metabolizing enzymes, which could potentially complicate the use of chronically administered disulfiram as a diagnostic inhibitor of CYP2E1.

In vivo modulation of CYP enzymes by quinidine and rifampin.

BACKGROUND: The metabolism of drugs and other xenobiotics is mediated by enzymes whose activities can be modulated by different compounds. The activities of these modulators have the potential to be used to optimize drug action, prevent toxicity, or identify the enzymes involved in a reaction. This approach requires that selective agents be used for specific enzymes. However, selectivity of action has been poorly characterized in vivo. METHODS: This study investigated the effect of 3 and 28 days of treatment with quinidine (200 mg daily) and rifampin (INN, rifampicin) (600 mg daily) on the activities of four cytochrome P450 enzymes and N-acetyltransferase in 28 healthy young male volunteers divided into three groups with a cocktail of drug probes used, including caffeine, mephenytoin, debrisoquin (INN, debrisoquine), and dapsone. RESULTS: Quinidine selectively and almost completely inhibited the activity of CYP2D6 from day 3 through day 28 without affecting any other enzymes. Rifampin showed evidence of time-dependent induction of the activities of all measured oxidative routes of metabolism but decreased the acetylation ratio in fast acetylators. The quinidine/rifampin combination resulted in selective CYP2D6 inhibition and induction of all other enzymes evaluated over this time period, suggesting that predictable complex interactions occur with the drug combination. CONCLUSIONS: These observations illustrate the value of simultaneous assessment of the effect of modulators on the activities of multiple specific enzymes with the drug cocktail approach.

Inhibition of debrisoquine hydroxylation with quinidine in subjects with three or more functional CYP2D6 genes.

AIMS: To study whether the CYP2D6 capacity in ultrarapid metabolizers of debrisoquine due to duplication/multiduplication of a functional CYP2D6 gene, can be 'normalised' by low doses of the CYP2D6 inhibitor quinidine and whether this is dose-dependent. METHODS: Five ultrarapid metabolizers of debrisoquine with 3, 4 or 13 functional CYP2D6 genes were given single oral doses of 5, 10, 20, 40, 80 and 160 mg quinidine. Four hours after quinidine intake, 10 mg debrisoquine was given. Urine was collected for 6 h after debrisoquine administration. Debrisoquine and its 4-hydroxymetabolite were analysed by h.p.l.c. and the debrisoquine metabolic ratio (MR) was calculated. RESULTS: Without quinidine the MR in the ultrarapid metabolizers ranged between 0.01 and 0.07. A dose-effect relationship could be established for quinidine with regard to the inhibitory effect on CYP2D6 activity. To reach an MR of 1-2, subjects with 3 or 4 functional genes required a quinidine dose of about 40 mg, while the sister and brother with 13 functional genes required about 80 mg quinidine. After 160 mg quinidine, the MRs, in the subjects with 3, 3, 4, 13 and 13 functional genes, were 12.6, 10.1, 9.2, 2.4 and 2.2, respectively. CONCLUSIONS: A dose-effect relationship could be established for quinidine inhibition of CYP2D6 in ultrarapid metabolizers. The clinical use of low doses of quinidine as an inhibitor of CYP2D6 might be considered in ultrarapid metabolizers taking CYP2D6 metabolized drugs rather than giving increased doses of the drug. Normalizing the metabolic capacity of CYP2D6, by giving a low dose of quinidine, may solve the problem of 'treatment resistance' caused by ultrarapid metabolism.

In-vitro metabolic interaction of bunitrolol enantiomers in rabbit liver microsomes.

We have examined the 4-hydroxylation of bunitrolol in rabbit and rat liver microsomes. Significant species differences (rabbit < rat of both sexes) and sex (male > female of both species) were observed in the formation of 4-hydroxybunitrolol from racemic bunitrolol (10 microM). The 4-hydroxylation of bunitrolol racemate and enantiomers showed biphasic kinetics, a low-Km system and a high-Km system, in liver microsomes from rabbits of both sexes. There were significant differences in Km and Vmax values [(+) > (-)] for 4-hydroxylations of (+)-bunitrolol and (-)-bunitrolol in the low-K(m) system. Furthermore, the rate of clearance (Vmax/Km) was 20- to 200-fold for the low-Km system compared with the high-Km system, indicating that enzymes in the low-Km system play a major part in the rabbit liver microsomal bunitrolol metabolism. Inhibition studies using cytochrome P450 inhibitors such as quinidine, quinine, and alpha-naphthoflavone or polyclonal antibodies raised against rat P450-2D and -1A enzymes did not make clear which P450 enzymes are involved in bunitrolol 4-hydroxylation in rabbit liver microsomes. The 4-hydroxylase activity of (+)-bunitrolol was slightly higher than that of (-)-bunitrolol in separated incubations containing male rabbit liver microsomes and an enantiomer concentration of 10 microM. However, the 4-hydroxylation of (+)-bunitrolol (10 microM) was markedly suppressed in the presence of its antipode (10 microM), whereas (-)-bunitrolol 4-hydroxylation was not affected by the presence of its antipode, resulting in a change of the stereoselectivity from (+) > (-) for enantiomer to (+) < (-) for racemate. The difference in the Michaelis constants in the low-Km system, where the Km value of (-)-bunitrolol is one-eighth that of (+)-bunitrolol, is thought to cause the change in the stereoselectivity in rabbit liver microsome-mediated bunitrolol 4-hydroxylation.

A three-dimensional protein model for human cytochrome P450 2D6 based on the crystal structures of P450 101, P450 102, and P450 108.

Cytochromes P450 (P450s) constitute a superfamily of phase I enzymes capable of oxidizing and reducing various substrates. P450 2D6 is a polymorphic enzyme, which is absent in 5-9% of the Caucasian population as a result of a recessive inheritance of gene mutations. This deficiency leads to impaired metabolism of a variety of drugs. All drugs metabolized by P450 2D6 contain a basic nitrogen atom, and a flat hydrophobic region coplanar to the oxidation site which is either 5 or 7 A away from the basic nitrogen atom. The aim of this study was to build a three-dimensional structure for the protein and more specifically for the active site of P450 2D6 in order to determine the amino acid residues possibly responsible for binding and/ or catalytic activity. Furthermore, the structural features of the active site can be implemented into the existing small molecule substrate model, thus enhancing its predictive value with respect to possible metabolism by P450 2D6. As no crystal structures are yet available for membrane-bound P450s (such as P450 2D6), the crystal structures of bacterial (soluble) P450 101 (P450cam), P450 102 (P450BM3), and P450 108 (P450terp) have been used to build a three-dimensional model for P450 2D6 with molecular modeling techniques. Several important P450 2D6 substrates were consecutively docked into the active site of the protein model. The energy optimized positions of the substrates in the protein agreed well with the original relative positions of the substrates within the substrate model. This confirms the usefulness of small molecule models in the absence of structural protein data. Furthermore, the derived protein model indicates new leads for experimental validation and extension of the substrate model.

Amezinium and debrisoquine are substrates of uptake1 and potent inhibitors of monoamine oxidase in perfused lungs of rats.

Previous studies have resulted in the classification of amezinium as a selective inhibitor of neuronal monoamine oxidase (MAO), because it is a much more potent MAO inhibitor in intact tissues, in which it is accumulated in noradrenergic neurones by uptake1, than in tissue homogenates. In the present study, the effects of amezinium on the deamination of noradrenaline were investigated in intact lungs of rats, since the pulmonary endothelial cells are a site where the catecholamine transporter is non-neuronal uptake1. In addition, another drug that is both a substrate of uptake1 and a MAO inhibitor, debrisoquine, was investigated in the study. The first aim of the study was to show whether amezinium and debrisoquine are substrates of uptake1 in rat lungs. After loading of isolated perfused lungs with 3H-noradrenaline (MAO and catechol-O-methyltransferase (COMT) inhibited), the efflux of 3H-noradrenaline was measured for 30 min. When 1 mumol/l amezinium or 15 mumol/l debrisoquine was added for the last 15 min of efflux, there was a rapid and marked increase in the fractional rate of loss of 3H-noradrenaline, which was reduced by about 70% when 1 mumol/l desipramine was present throughout the efflux period. These results showed that both drugs were substrates for uptake1 in rat lungs. In lungs perfused with 1 nmol/l 3H-noradrenaline (COMT inhibited), 10, 30 and 300 nmol/l amezinium caused 58%, 76% and 74% inhibition of noradrenaline deamination, respectively, and 30, 300 and 3000 nmol/l debrisoquine caused 56%, 89% and 96% inhibition of noradrenaline deamination, respectively. When MAO-B was also inhibited, 10 nmol/l amezinium caused 84% inhibition of the deamination of noradrenaline by MAO-A in the lungs. In contrast, in hearts perfused with 10 nmol/l 3H-noradrenaline under conditions where the amine was accumulated by uptake2 (COMT, uptake1 and vesicular transport inhibited), 10 nmol/l amezinium had no effect and 300 nmol/l amezinium caused only 36% inhibition of deamination of noradrenaline. The results when considered with previous reports in the literature show that amezinium is about 1000 times more potent and debrisoquine is about 20 times more potent for MAO inhibition in rat lungs than in tissue homogenates, and the reason for their high potencies in the intact lungs is transport and accumulation of the drugs in the pulmonary endothelial cells by uptake1. Amezinium is much less potent as a MAO inhibitor in cells with the uptake2 transporter, such as the myocardial cells of the heart. The results also confirmed previous reports that amezinium is highly selective for MAO-A.

Development of a high-performance liquid chromatographic method for the analysis of enatiomer/enantiomer interaction in oxidative metabolism of bunitrolol in rat liver microsomes.

A high-performance liquid chromatographic method for the assay of enantiomeric 4-hydroxybunitrolol (4-OH-BTL) formed from racemic bunitrolol (BTL) in rat liver microsomes was developed. Racemic bunitrolol was incubated with rat liver microsomes fortified with an NADPH-generating system. Metabolites extracted with ethyl acetate were converted to acetyl derivatives with acetic anhydride in pyridine. The derivatives of 4-OH-BTL were well separated by the liquid chromatography equipped with a chiral column. Using this method, the metabolic interaction of BTL enantiomers was examined. The 4-OH-BTL-forming activities from enantiomeric BTL were higher than those from racemic BTL in rat liver microsomes, while the formation of ratios of 4-OH-BTL enantiomer to its antipode were the same under the two conditions. The Ki values obtained from kinetic studies using each BTL enantiomer as an inhibitor of its antipode were almost the same (ca 0.9 microM), which were close to their Michaelis constants (Km values). Oxidative activities of enantiomeric and racemic BTL were almost equally inhibited by debrisoquine and quinidine, a typical substrate and a selective inhibitor of the CYP2D subfamily, respectively. These results indicate that a BTL enantiomer is a mutual metabolic inhibitor of its antipode and BTL enantiomers compete for the same CYPD2D isozyme in rat liver microsomes.

Acute effects of neuroleptics on unmedicated schizophrenic patients and controls.

Acute administration of haloperidol (0.2 mg/kg) produced many more side effects in normal controls than in unmedicated schizophrenic patients. Prior to the neuroleptic challenge, both groups were on the peripheral monoamine oxidase inhibitor, debrisoquin, for at least 1 week, in order to enhance the relative contribution of CNS catecholamine metabolites to those measured in both plasma and urine. The patient group had higher plasma levels of methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) and higher urinary MHPG output than controls, but there were no effects of haloperidol challenge, compared to placebo challenge. In both groups there were significant declines in plasma HVA levels from 8:30 AM to 12 NOON. These declines were unaffected by the haloperidol challenge. Explanations for the marked differences in behavioral effects of haloperidol on patients and controls include the possibility that dopamine receptor numbers were increased in the brains of the schizophrenic patients.

Cytochrome P-450-dependent hydroxylation in migraine.

The hypothesis was tested that an acute oxidation deficiency related to potential dietary trigger factors plays a role in the migraine attack. Migraine sufferers (14F and 4M), fulfilling the criteria for migraine with and without aura according to the classification of the International Headache Society, were coadministered oral mephenytoin (100 mg) and debrisoquine (10 mg) during the initial phase of a typical migraine attack. This was repeated during a period without migraine. The hydroxylation of mephenytoin and debrisoquine hydroxylation did not differ during and without the migraine attack. We conclude that hydroxylation, via cytochrome P-450 (2D6, 2C8 and 9), is not reduced during the migraine attack. The results do not support the hypothesis that oxidation deficiency is involved in the pathophysiology of migraine.

Cytochrome-P450-dependent hydroxylation in cluster headache.

Two isozymes of the cytochrome-P450-dependent drug oxidizing system exhibit polymorphism. Five to 10% of a Caucasian population are deficient in debrisoquine-hydroxylase activity and about 3% in mephenytoin-hydroxylase activity (poor metabolizers). We tested the hypothesis of a possible over-representation of poor metabolizers among patients with cluster headache. The individual metabolic capacity was determined in 30 cluster headache patients after administration of a test dose of 10 mg of debrisoquine and 100 mg of mephenytoin. Two patients (6.7%) were poor metabolizers of debrisoquine and one (3.3%) a poor metabolizer of mephenytoin. This was no different from the rate of poor metabolizers, 7.1% and 3.3% respectively, in a reference panel of healthy Swedish volunteers.

Inhibition of debrisoquin clearance in perfused rat livers and inhibition of dextromethorphan metabolism in human liver microsomes by 4-hydroxydebrisoquin or other metabolites of debrisoquin.

Debrisoquin undergoes oxidative metabolism to 4-hydroxydebrisoquin, catalyzed by cytochrome CYP2D1 in rats and CYP2D6 in humans. Cytochrome CYP2D6 also plays a major role in dextromethorphan O-demethylation. In preliminary studies in perfused Lewis rat livers, we observed a difference in repeat clearance experiments using debrisoquin, but not dextromethorphan. To determine whether this change in clearance with time was due to the accumulation of 4-hydroxydebrisoquin, we sequentially used a recirculating and nonrecirculating perfusion system in the same liver perfusion experiment. We also studied the kinetics of dextromethorphan O-demethylation in microsomes prepared from human and rat livers in the presence and absence of 4-hydroxydebrisoquin. Results from the perfused rat liver experiments showed a drop in clearance from 3.27 +/- 0.57 ml/min (clearance 1) to 1.61 +/- 0.27 ml/min (clearance 2) (p less than 0.05 vs. clearance 1) during recirculation, but clearance returned to 3.21 +/- 0.46 ml/min (clearance 3, no significance vs. clearance 1) after a 30-min period of liver perfusion using a nonrecirculating system. There was significant accumulation of 4-hydroxydebrisoquin in the liver perfusate during recirculation, and concentrations fell when the nonrecirculating system was used. In microsomal studies, 4-hydroxydebrisoquin competitively inhibited dextromethorphan metabolism in human microsomes was 600 microM. These data suggest that: (a) 4-hydroxydebrisoquin and/or other metabolites of debrisoquin have an inhibitory effect on CYP2D1 and CYP2D6; (b) the active site of human CYP2D6 has different substrate specificity than the rat isozyme (CYP2D1) and/or that the pathways of metabolism of dextromethorphan are different in the Lewis rat and not primarily dependent on the activity of CYP2D1.

Cytochrome P450 isozymes catalyzing 4-hydroxylation of parkinsonism-related compound 1,2,3,4-tetrahydroisoquinoline in rat liver microsomes.

Microsomal 4-hydroxylase of 1,2,3,4-tetrahydroisoquinoline (TIQ), a possible candidate for causing Parkinson disease, was characterized by using rat hepatic microsomes and purified P450 isozymes. Kinetic analysis revealed that Km and Vmax values (mean +/- SE) for hepatic microsomal TIQ 4-hydroxylase of male Wistar rats were 319.6 +/- 26.8 microM and 12.13 +/- 1.43 pmol.min-1.mg-1 protein, respectively. When TIQ 4-hydroxylase activity was compared in Wistar (an animal model of extensive debrisoquine metabolizers) and Dark Agouti (an animal model of poor debrisoquine metabolizers) rats, significant strain (Wistar greater than Dark Agouti) and sex (male greater than female) differences were observed. The microsomal activity toward TIQ 4-hydroxylation was increased by pretreatment of male Wistar rats with P448 inducers (beta-naphthoflavone and sudan I), but not with phenobarbital. Pretreatment with propranolol, an inhibitor of P450 isozymes belonging to the P450 IID gene subfamily, decreased TIQ 4-hydroxylase activity. P450 BTL, a P450 isozyme belonging to the IID subfamily, showed TIQ 4-hydroxylase activity of 64.1 pmol.min-1.nmol P450(-1), which was 3.2-fold that of microsomes (20.9 pmol.min-1.nmol P450(-1)). Antibody (IgG) against this isozyme suppressed microsomal TIQ 4-hydroxylase activity concentration-dependently. A male-specific P450 ml (P450IIC11) catalyzed this reaction to a much lesser extent (10.0 pmol.min-1.nmol P450(-1)), and its antibody did not affect the microsomal activity. These results suggest that TIQ 4-hydroxylation in hepatic microsomes are catalyzed predominantly by a P450 isozyme (or isozymes) belonging to the IID gene subfamily in non-treated rats and its immunochemically related P450 isozyme (or isozymes), and that a P450 isozyme (or isozymes) belonging to the IA subfamily also participates in TIQ 4-hydroxylation in rats pretreated with P448-inducers.

Influence of amineptine on changes of blood pressure evoked by norepinephrine and dopamine.

The influence of amineptine, an antidepressant currently employed having mainly selective dopaminergic neurochemical activity, on the pressor responses evoked by norepinephrine (NE) and dopamine (DA) was studied in anesthetized whole rats. Amineptine at doses of 0.5, 1.5, and 5.0 mg/kg/30 min infused into the femoral vein of the rat caused a dose-related inhibition of the pressor responses of NE and DA. The hypertensive responses of NE and DA augmented by pretreatment with reserpine, a catecholamine depletor, were also clearly depressed following the infusion of amineptine with a rate of 1.5 mg/kg/30 min. Furthermore, the pressor responses of NE and DA potentiated by pretreatment with debrisoquin, a sympathetic neuron blocker, were markedly diminished after pretreatment with the infusion of amineptine at the above same rate (1.5 mg/kg/30 min). These experimental results demonstrate that amineptine causes an inhibitory effect on the pressor responses evoked by NE and DA. It is thought that the amineptine effect may be due to the blockade of the peripheral adrenergic alpha-receptors in addition to the previously described uptake inhibition of dopamine.

Effect of ceruletide on plasma monoamine metabolites in the rabbit.

Ceruletide, a cholecystokinin octapeptide-like substance, has been shown to have some effect on tardive dyskinesia. We, too, previously examined the effect of ceruletide on various types of involuntary movement, and found that responders tended to have high plasma homovanillic acid (HVA) levels. It is generally accepted that both central and peripheral sources make a contribution of plasma HVA. In this study, the response of plasma HVA in rabbits to ceruletide was investigated after pretreatment with debrisoquin sulfate, a drug which selectively blocks peripheral HVA production by inhibition of MAO. As a result, 8 and 50 micrograms/kg ceruletide treatment showed a tendency to decrease plasma HVA levels, but showed no significant differences; however, 140 and 200 micrograms/kg ceruletide showed a significant reduction of plasma HVA. These results are important to the understanding of the mechanism of ceruletide's effect on the brain, as well as to predict the effect of ceruletide on involuntary movements.