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PubMed2002

CYP2D6 inhibition by selective serotonin reuptake inhibitors: analysis of achievable steady-state plasma concentrations and the effect of ultrarapid metabolism at CYP2D6.

STUDY OBJECTIVE: To assess the correlation between plasma concentrations of four commonly administered selective serotonin reuptake inhibitors (SSRIs) and the magnitude of cytochrome P450 (CYP) 2D6 inhibition. DESIGN: Prospective analysis. SETTING: University-affiliated research laboratory. PATIENTS: Thirty-two healthy, drug-free volunteers. INTERVENTION: Subjects were randomized to four groups and received daily administration of either fluoxetine 60 mg (as a loading dose), fluvoxamine 100 mg, paroxetine 20 mg, or sertraline 100 mg for 8 days. MEASUREMENTS AND MAIN RESULTS: The urinary concentration ratio of dextromethorphan:dextrorphan (interpreted as an in vivo index of CYP2D6 activity) was determined for each subject before and after the 8 days of receiving SSRIs. Plasma SSRI trough concentrations were measured on days 6-9. The CYP2D6 genotype was determined in a subject with an undetectable paroxetine concentration. Inhibition of CYP2D6 correlated significantly with plasma concentrations of paroxetine and fluoxetine. In contrast, no significant correlations emerged between CYP2D6 inhibition and plasma concentrations of sertraline or fluvoxamine. The subject with an undetectable paroxetine concentration was found to carry at least three functional CYP2D6 genes. CONCLUSIONS: For paroxetine and fluoxetine, plasma concentrations and dosage strongly influence the magnitude of enzyme inhibition. The potential of paroxetine (a CYP2D6 substrate) as an inhibitor may be affected by the genotypes and metabolic capacities of individual subjects.

Cyclooxygenase Inhibitors↗

PubMed1999

Characterization of cytochrome P-450 2D1 activity in rat brain: high-affinity kinetics for dextromethorphan.

We investigated the enzymatic function, stability, and regional distribution of rat brain cytochrome P-450 (CYP) 2D1 activity. CYP2D1 is the homolog of human CYP2D6, a genetically variable enzyme that activates or inactivates many clinical drugs acting on the central nervous system (e.g., antidepressants, monoamine oxidase inhibitors, serotonin uptake inhibitors, and neuroleptics), drugs of abuse (e.g., amphetamine and codeine), neurotoxins (e.g., 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3, 4-tetrahydroquinoline), and endogenous neurochemicals (e.g., tryptamine). The CYP2D family has been identified in rodent, canine, and primate brain. Conversion of dextromethorphan to dextrorphan by rat brain membranes was assayed by HPLC and was dependent on NADPH, protein concentration, and incubation time. Significant loss of activity was observed in some homogenizing buffers and after freezing of whole tissues or membrane preparations. Dextromethorphan (0.5-640 microM) metabolism was mediated by high- and low-affinity enzyme systems; K(m1) was 2.7 +/- 2.6 and K(m2) was 757 +/- 156 microM (n = 3 rats, mean +/- S.E.). The enzyme activity was significantly (p <.01) and stereoselectively inhibited by CYP2D1 inhibitors quinine and quinidine (not by CYP2C or CYP3A inhibitors), and by anti-CYP2D6 peptide antiserum (not by anti-CYP2C, -CYP2B, or -CYP3A antibodies). The enzymatic activity demonstrated significant brain regional variation (n = 10 regions, p <.001). These data characterize CYP2D1-mediated dextromethorphan metabolism in rat brain and suggest that localized metabolism of other CYP2D1 substrates (drugs, neurotoxins, and possibly endogenous compounds) within the brain will occur. In humans, CYP2D6 is genetically polymorphic; the variable expression of brain CYP2D6 may result in interindividual differences in central drug and neurotoxin metabolism, possibly contributing to interindividual differences in drug effects and neurotoxicity.

Alcohol Oxidoreductases↗

PubMed2001

Comparative contribution to dextromethorphan metabolism by cytochrome P450 isoforms in vitro: can dextromethorphan be used as a dual probe for both CTP2D6 and CYP3A activities?

Dextromethorphan (DXM) is a widely used probe drug for human CYP2D6 activity both in vitro and in vivo. In humans, DXM is metabolized to dextrorphan (DXO), as well as 3-methoxymorphinan (MEM) and 3-hydroxymorphinan (HYM). The formation of MEM has been attributed primarily to CYP3A4, and the use of DXM has been debated as a simultaneous probe for CYP3A4 and CYP2D6 activities. Recently, we found that highly purified CYP2D6 has significant DXM N-demethylase activity in addition to its well known DXM O-demethylase activity. Therefore, we desired to further compare the contribution to DXM metabolism by individual human cDNA-expressed cytochromes P450, including 2C8, 2C9, 2C18, 2C19, 2D6, 2B6, and 3A4. Metabolites were quantified following separation by high-pressure liquid chromatography and apparent Michaelis-Menten constants determined for the appearance of DXO and MEM. Intrinsic clearance values were estimated for each P450 and normalized using the average percentage content and relative activity factor approaches for comparison. Simplified kinetic models (when [S] << K(m), V(max)/K(m) = V(o)/[S]) were used at fixed DXM concentrations of 20 (for DXM N-demethylation) and 0.2 microM (for DXM O-demethylation), as well as 2 microM to mimic plasma DXM concentrations in human extensive metabolizers. The results confirm that CYP2D6 contributes at least 80% to the formation of DXO, and CYP3A4 contributes more than 90% to the formation of MEM. All of our in vitro results are consistent and indicate that DXM as a marker for monitoring both CYP2D6 and CYP3A activities is practical in an average human or human liver microsomal preparation.

Antitussive Agents↗

PubMed1991

Effects of phencyclidine on contractile forces of isolated rabbit papillary muscles.

Phencyclidine (PCP) (0.01-50 mumol.L-1) and its analogue, TCP (0.01-50 mumol.L-1) exhibited positive inotropic effects on electrically stimulated rabbit papillary muscle preparations. Dextrorphan (5 or 10 mumol.L-1) antagonized the actions of PCP in non-competitive manner (pD'2 = 5.25). This demonstrated the involvement of PCP receptors in the positive inotropic effects of PCP. By using high performance liquid chromatography with electrochemical detector (HPLC-ECD), an increase of DOPAC content was found in bath medium after PCP addition. Each of the dopamine receptor antagonists SCH23390, haloperidol and sulpiride (1 mumol.L-1) attenuated the maximal inotropic effects of PCP. These results suggest that PCP induces positive inotropic effects by increasing the release and/or blocking the uptake of dopamine.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1979

Acute effects of opiate agonists, partial agonists and antagonists on brain monoamines in the rat.

Single doses of 4.5 mg/kg of the opiate agonist, morphine, the partial agonists, cyclazocine, pentazocine, levallorphan and nalorphine, the antagonists, naloxone and naltrexone and dextrorphan were injected i.p. in groups of rats. Behaviour was noted for 30 min following injection and at this time, brains were removed and levels of noradrenaline (NA), dopamine (DA), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were determined spectrophotofluorimetrically. Only morphine and cyclazocine altered behaviour and 5-HIAA was the only substance which was consistently elevated by all the compounds tested. These results indicate that a causal relationship between the acute effects of opiate agonists, partial agonists and antagonists on brain 5-HT metabolism and their pharmacological actions is unlikely.

Animals↗

PubMed1981

Stimulation of hypothalamic arcuate nucleus inhibits locus coeruleus unit activity: evidence for endorphin mediation.

We have investigated the effect of arcuate nucleus stimulation on locus coeruleus unit activity. Extracellular LC unit activity was recorded in anesthetized cats and rats. Brief conditioning trains (25-300 microA, three shocks, 100 Hz) delivered to the arcuate nucleus produced profound inhibition (average 1000 msec, n = 30) of spontaneous coerulear cell discharges. Intravenously administered naloxone (2.5-5.0 mg/kg) or iontophoretically applied naloxone eliminated arcuate-elicited inhibition. Additionally, naloxone often increased spontaneous cell activity. In contrast to naloxone, systemically administered morphine inhibited spontaneous LC unit activity in a dose-related fashion and augmented the hypothalamic-derived inhibition. Naloxone, 5 mg/,g, reversed morphine activity to beyond predrug levels. To characterized the stereospecificity of the response, dextrorphan was administered. This inactive enantiomer failed to mimic morphine actions on LC cells. The results suggest that LC may be influenced by the beta-endorphine system originating from the arcuate nucleus.

Action Potentials↗

PubMed1995

Prediction of phenotype for dextromethorphan O-demethylation by using polymerase chain reaction in healthy volunteers.

The polymorphism of dextromethorphan (CAS 125-71-3) metabolism is dependent on hepatic cytochrom P4502D6 (CYP2D6) activity. The relationship between the CYP2D6 genotype and the dextromethorphan phenotype was studied in 83 healthy unrelated subjects. Genotype was determined by allele-specific polymerase chain reaction (PCR). Phenotyping was performed by administration of 25 mg dextromethorphan hydrobromide and determination of the urinary metabolic ratio of dextromethorphan and its O-demethylated metabolite dextrorphan (DEM/DOR). Six subjects (7.2%) were homozygous for mutant alleles (95% confidence interval 2.7%-14.7%), 18 subjects (22%) were heterozygous carriers, and 59 were homozygous for the wild-type allele. Six subjects were classified as poor metabolizers (PM) of dextromethorphan, 77 as extensive metabolizers (EM). Genotyping correctly predicted all PMs and EMs. The CYP2D6-B mutation was most frequently found, being present in 83% of PM and 8% of EM alleles. Heterozygous EMs (22% of the total population studied) were significantly underrepresented compared to the expected genotype frequency of 31% (p < 0.05). The extensive metabolizers who were heterozygous for the wild-type allele had a significantly higher metabolic ratio, compared to the homozygous EMs (log10DEM/DOR [95% = -1.99 [(-2.30)-(-1.69)] vs. -2.55 [(-2.67)-(-2.43)]; p < 0.001), indicating a gene-dose effect for CYP2D6.

Adult↗

PubMed1993

Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides inhibit Na(+)-Ca2+ exchange in cardiac sarcolemma vesicles.

The molluscan cardioexcitatory tetrapeptide FMRF-amide (Phe-Met-Arg-Phe-NH2) and related peptides inhibit Na(+)-Ca2+ exchange in calf cardiac sarcolemma vesicles. FMRFa itself has a low inhibitory potency (IC50 = 750 microM) which completely resides in its COOH-terminal RFa portion. The physiologically active analog FLRFa is 10-fold more potent (IC50 = 60 microM). Two other substitutions of the Met2 in FMRFa, by either Ile or Lys increase inhibitory potency 7- and 50-fold, respectively. The inhibitory potency increases 300-500-fold if the NH2-terminal Phe1 in FMRFa is substituted by either Val or His (IC50 = 1-2 microM). The inhibitory activity of WnLRFa (IC50 = 40 microM) is lost when either the NH2-terminal amino group is acylated or the NH2-terminal Trp1 is deleted. These data suggest that the COOH-terminal portion is essential for the basic low potency inhibition of Na(+)-Ca2+ exchange, whereas the NH2-terminal portion is important for the potentiation of the inhibitory activity. Although the IC50 values of various peptides range widely (10(-6)-10(-3) M), all of them induce a complete inhibition. The dose-response pattern of the peptide-induced inhibition is identical for the Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange. The inhibitory effect is reversible and affects both Nai(or Cai)-dependent 45Ca uptake and Nao-dependent 45Ca efflux, suggesting that the bidirectional movements of ions are altered. A mild pretreatment of vesicles with trypsin augments the Na(+)-Ca2+ exchange 1.5-fold but diminishes the inhibitory potency 3-4-fold, suggesting that the inhibition is mediated by an extravesicular membrane protein. The characteristics of the peptide-induced inhibition resemble the effect of opiates on Na(+)-Ca2+ exchange. FLRFa and dextrorphan (a non-opioid stereoisomer of an opiate agonist) are mutually exclusive inhibitors, suggesting that they may bind to the same site. This putative site lacks the pharmacological properties of opiate receptors and may be located either on the Na(+)-Ca2+ exchanger or at its vicinity. Endogenous analogs of FMRFa may regulate intracellular calcium via Na(+)-Ca2+ exchange.

Amino Acid Sequence↗

PubMed1994

Neurophysiology of opioid poorly responsive pain.

This review has covered the five potential causes for opioid poorly responsive pain, namely (a) a loss of opioid receptors on the spinal terminals of C-fibres as a result of peripheral nerve damage, (b) an accumulation of morphine-3-glucuronide, (c) changes in the non-opioid peptides, F8Fa or CCK, either spinally or supraspinally, (d) actions of the opioid peptide dynorphin and (e) spinally generated hypersensitive states via activation of the NMDA receptor. The loss of opioid receptors is likely to be important where peripheral nerve pathology or compression occurs, but the evidence suggests that increasing the dose will overcome the reduced opioid response. Morphine-3-glucuronide is unlikely to be a factor, nor is dynorphin, but the endogenous peptides CCK and F8Fa may be important. Finally, there is an association between the NMDA receptor and hyperalgesia/allodynia and reduced opioid sensitivity. Dextrorphan and ketamine reduce NMDA mediated events and so are available to test this hypothesis.

Analgesics, Opioid↗

PubMed1998

Novel single-point plasma or saliva dextromethorphan method for determining CYP2D6 activity.

O-Demethylation of dextromethorphan co-segregates with 4-hydroxylation of debrisoquin and is used for CYP2D6 phenotyping. In most previous studies, 8-h urinary samples were collected for determining the dextromethorphan metabolic ratio (dextromethorphan/dextrorphan molar ratio). In addition, a salivary sampling at 3 h had been suggested for the phenotyping. To evaluate the repeatability and validity of previously reported and other potential phenotyping methods, we determined the metabolic ratios from urine samples (for various intervals), or from plasma or saliva (at varying time points) after repetitive single doses of immediate-release or repetitive multiple doses of controlled-release dextromethorphan preparations. For the single-dose study, each of 12 subjects received 15 mg of immediate-release dextromethorphan in period I and period II, respectively, with a 1-week washout period. For the multiple-dose study, each of 16 subjects received 60 mg controlled-release dextromethorphan twice daily for 5 days in period I and period II, respectively, with a 2-week washout period. Dextromethorphan and dextrorphan were assayed by high-performance liquid chromatography. In the single-dose study, most metabolic ratios revealed good repeatabilities for the two periods (paired t test). The metabolic ratio from urine collected for 4 h, 6 h, 8 h or 12 h from plasma at any time between 1 h and 5 h or at 8 h, or from saliva at 2 h or 6 h, could reflect that from 0- to 24-h urine or AUCinfinity. In the multiple-dose study, all metabolic ratios revealed good repeatabilities. The plasma metabolic ratio at any time between 0.5 h and 10 h or the saliva metabolic ratio at any time between 3 h and 12 h, but not the urine metabolic ratio from any interval, could predict the metabolic ratio from ACUSStau. The 2 h, 3 h, 4 h or 5 h plasma metabolic ratio and 6 h saliva metabolic ratios after a single dose correlated significantly with their corresponding multiple-dose metabolic ratio (r > 0.8, P < .05). In conclusion, the plasma sample at 2 h, 3 h, 4 h or 5 h or the saliva sample at 6 h in either the single immediate-release (15 mg) or the multiple controlled-release dose (60 mg) procedure could be used for determining the dextromethorphan metabolic ratio.

Adult↗

PubMed2003

Effect of St John's wort on drug metabolism by induction of cytochrome P450 3A4 enzyme.

CONTEXT: St John's wort is a popular herbal product used to treat depression but it has been implicated in drug interactions. OBJECTIVE: To assess the potential of St John's wort administration to alter the activity of the cytochrome P450 (CYP) enzymes extensively involved in drug metabolism. DESIGN, SETTING, AND PARTICIPANTS: Open-label crossover study with fixed treatment order conducted March 2002 to February 2003 in a US general clinical research center involving 12 healthy volunteers (6 men and 6 women) aged 22 to 38 years before and after 14 days of administration of St John's wort. INTERVENTION: Participants were given probe drugs (30 mg of dextromethorphan and 2 mg of alprazolam) to establish baseline CYP 3A4 and CYP 2D6 activity. After a minimum 7-day washout period, participants began taking one 300-mg tablet 3 times per day. After 14 days of St John's wort administration, participants were given the probe drugs along with 1 St John's wort tablet to establish postadministration CYP activity; the St John's wort dosing regimen was continued for 48 hours. MAIN OUTCOME MEASURES: Changes in plasma pharmacokinetics of alprazolam as a probe for CYP 3A4 activity and the ratio of dextromethorphan to its metabolite, dextrorphan, in urine as a probe for CYP 2D6 activity. RESULTS: A 2-fold decrease in the area under the curve for alprazolam plasma concentration vs time (P<.001) and a 2-fold increase in alprazolam clearance (P<.001) were observed following St John's wort administration. Alprazolam elimination half-life was shortened from a mean (SD) of 12.4 (3.9) hours to 6.0 (2.4) hours (P<.001). The mean (SD) urinary ratio of dextromethorphan to its metabolite was 0.006 (0.010) at baseline and 0.014 (0.025) after St John's wort administration (P =.26). CONCLUSIONS: A 14-day course of St John's wort administration significantly induced the activity of CYP 3A4 as measured by changes in alprazolam pharmacokinetics. This suggests that long-term administration of St John's wort may result in diminished clinical effectiveness or increased dosage requirements for all CYP 3A4 substrates, which represent at least 50% of all marketed medications.

Adult↗

PubMed1997

Metabolism of dextromethorphan in vitro: involvement of cytochromes P450 2D6 and 3A3/4, with a possible role of 2E1.

Dextromethorphan (DMO), a cough suppressing synthetic analog of codeine, undergoes parallel O-demethylation to dextrorphan (DOP), and N-demethylation to 3-methoxymorphinan (MEM), in humans. 3-hydroxymorphinan, a didemethylated metabolite, is formed secondarily. O-demethylation activity is well established as an index reaction for CYP2D6. However, this pathway appears to be mediated by at least two different enzymes in vitro. N-demethylation activity has recently been proposed to reflect CYP3A3/4 activity. We investigated both pathways in vitro with microsomal preparations from three human livers to assess the value of DMO as a probe drug for CYP2D6 and CYP3A3/4, DMO O-demethylation displayed a biphasic pattern with a high-affinity site reflecting CYP2D6 activity (mean Ki for quinidine, 0.1 +/- 0.13 microM). Kinetic parameters for the two O-demethylation mediating enzymes predict an average relative intrinsic clearance (Vmax/K(m) ratio) of 96% of total O-demethylation mediated via the high-affinity enzyme. Thus, in vitro data confirms the usefulness of DMO O-demethylation as an index reaction to monitor CYP2D6 activity. The Eadie-Hofstee plot of DMO N-demethylation was consistent with single-enzyme Michaelis-Menten kinetics (Vmax varying from 3.3 to 6.8 nmol mg-1 min-1, K(m) from 231 to 322 microM). However, ketoconazole, a CYP3A3/4 inhibitor, reduced N-demethylation only by 60% and had a mean Ki an order of magnitude higher (0.37 microM) compared to other pure CYP3A3/4 mediated reactions. Troleandomycin, a mechanism based CYP3A3/4 inhibitor, inhibited MEM formation by an average maximum of 46%, with an IC50 varying from 1 to 2.6 microM. A polyclonal rat liver CYP3A1 antibody inhibited MEM formation only by approximately 50%. Diethyldithiocarbamate (DDC), a mechanism based CYP2E1 inhibitor, reduced MEM formation at concentrations up to 150 microM between 33 and 43%. Chemical inhibitors of CYP2d6 (quinidine), CYP1A1/2 (alpha-naphthoflavone), and CYP2C9 (sulfaphenazole), as well as a goat rat liver CYP2C11 polyclonal antibody (inhibitory against human CYP2C9 and CYP2C19), had minimal effect on MEM formation rate, thus excluding an involvement of any of these enzymes. DMO N-demethylation is only partly mediated by CYP3A3/4, and therefore is not a reliable index reaction for CYP3A3/4 activity either in vitro or probably in vivo.

Animals↗

PubMed2000

High-throughput cytochrome P450 inhibition screening via cassette probe-dosing strategy. IV. Validation of a direct injection on-line guard cartridge extraction/tandem mass spectrometry method for simultaneous CYP3A4, 2D6 and 2E1 inhibition assessment.

A highly efficient direct injection on-line guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 3A4, 2D6 and 2E1 inhibition potential via cassette dosing of midazolam, dextromethorphan and chlorzoxazone using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI-GCE/MS/MS. Due to the novel use of an extremely short C(18) guard cartridge (4 mm in length), this method exhibits several advantages such as no sample preparation, excellent on-line extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI-GCE/MS/MS method demonstrates acceptable accuracy and precision for the simultaneous quantification of 1'-hydroxymidazolam, dextrorphan and 6-hydroxychlorzoxazone in microsomal incubations. The inhibition potential of CYP3A4, 2D6 and 2E1 has been evaluated using their known selective inhibitors. The IC(50) values measured by the cassette dosing approach (high-throughput) are consistent with those observed by an individual dosing regimen (conventional) and are all in good agreement with the literature values. The results suggest that the cassette probe-dosing strategy may provide an in vitro approach to minimize cost while maximizing throughput of CYP inhibition evaluation of new chemical entities in support of drug discovery and development.

Calibration↗

PubMed1989

N-methyl-D-aspartate antagonists: ready for clinical trial in brain ischemia?

Antagonists of the N-methyl-D-aspartate (NMDA) subclass of glutamate receptors may offer a new approach for the treatment of ischemic brain injury. This strategy is supported by a well-developed scientific foundation and encouraging results in a variety of in vivo and in vitro experimental models. Several specific antagonists, including MK-801, dextrorphan, dextromethorphan, and ketamine, have already been used at low doses in humans for other indications and are potential candidates for Phase I clinical trials.

Aspartic Acid↗

PubMed2002

Development of a rapid and sensitive high-performance liquid chromatographic method to determine CYP2D6 phenotype in human liver microsomes.

Dextromethorphan is a probe substrate to determine CYP2D6 phenotype. The conversion of dextromethorphan to dextrorphan by CYP2D6 accounts for approximately 60% of total metabolism. Most analytical methods utilize complicated labor- and time-intensive sample processing methods with several liquid-liquid extraction (LLE) steps. Our goal was to develop a non-LLE based rapid and sensitive HPLC method, to measure dextromethorphan metabolism in human liver microsomes. A solid-phase filtration based reverse-phase HPLC method with fluorescence detection was developed and validated. Human liver (n = 6) microsomal incubations were carried out with dextromethorphan, under optimum conditions. The analytes were separated by one-step centrifugal filtration with Nanosep separation units. The filtrate was injected ( 50 microL) into a Waters Alliance 2690 HPLC system. Metabolic incubations were also conducted to determine levels using LLE for comparisons. The Nanosep separation step reduced the extraction time from 3h to 40 min. The limit of quantitation was 23.8 nM (9.7 ng/mL), recovery was approximately 98%, the mean precision values were <10% RSD for the controls (80, 320 and 640 nM) and mean percentage error was <5%. Michaelis-Menten parameters were determined to distinguish CYP2D6 phenotypes. A rapid and sensitive HPLC method is reported, which may be suitable for automation and allows phenotyping of human liver microsomes.

Antitussive Agents↗

PubMed1987

Bioavailability evaluation of a controlled-release dextromethorphan liquid.

A randomized, two-way, steady-state crossover study was performed in 24 healthy male volunteers to evaluate the bioavailability of a controlled-release (CR) dextromethorphan (DM) suspension. Only slow and intermediate DM metabolizers were allowed to participate in the study; determination of metabolizer status was performed before study enrollment. Each volunteer was administered 30 mg of an immediate-release (IR) DM solution qid or 60 mg DM as a CR suspension bid for two weeks, for a total daily dose of 120 mg. After a two-week washout period, the subjects were administered the alternate treatment. Blood samples were collected over a 12-hour dosing period on the last day of each treatment and analyzed for DM and its active metabolite, dextrorphan (DP). In addition, urine was collected over the 12-hour steady-state dosing interval and measured for DM and two metabolites. Pharmacokinetic determinations were made from plasma DM and DP data, and total urinary excretion was determined. All comparisons made between the two formulations indicated that the CR DM suspension was bioequivalent to the IR DM solution at steady state, while producing a prolonged release of the drug over time.

Adolescent↗

PubMed2001

High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1.

The inhibition potential of drugs towards five major human hepatic cytochrome P450 (CYP) isozymes (CYP2A6, 3A4, 2C9, 2D6, and 2E1) was investigated via cassette dosing of the five probe substrates (coumarin, midazolam, tolbutamide, dextromethorphan, and chlorzoxazone) in human liver microsomes using a 96-well plate format. After microsomal incubations had been terminated with formic acid, the five marker metabolites (7-hydroxycoumarin, 1'-hydroxymidazolam, 4-hydroxytolbutamide, dextrorphan, and 6-hydroxychlorzoxazone) were simultaneously quantified using direct injection/online guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS). Several advantages resulted from the use of a short C(18) guard cartridge (4 mm in length) for DI-GCE/MS/MS, including minimal sample preparation, fast online extraction, short analysis time (2.5 min), and minimal source contamination. In addition, this method demonstrated an inter-day accuracy range from -8.7 - 7.4% with a precision less than 8.3% for the quantification of all the marker metabolites. The inhibition assay for the five CYP isozymes was evaluated using their known selective inhibitors via individual and cassette dosing of the probe substrates. The IC(50) values measured via cassette dosing were consistent with those observed via individual dosing, which were all in agreement with the reported values. In addition, the validated assay was used to evaluate the inhibitory potential of 23 generic drugs (randomly selected) towards the five CYP isozymes. The results suggest the integration of the cassette dosing strategy and the DI-GCE/MS/MS method can provide a reliable in vitro approach to screening the inhibitory potential of new chemical entities, with maximal throughput and cost-effectiveness, in support of drug discovery and development.

Aryl Hydrocarbon Hydroxylases↗

PubMed1999

Molecular cloning, expression, and characterization of CYP2D17 from cynomolgus monkey liver.

The cynomolgus monkey is a species used in drug-safety evaluation and biotransformation studies by the pharmaceutical industry. Relatively little is known, however, about the catalytic activities and specificities of cytochromes P450 (CYP) in this species. As a first step in characterizing monkey CYPs, a cDNA was cloned by reverse-transcriptase PCR from cynomolgus monkey liver mRNA using oligonucleotide primers based on the human CYP2D6 sequence. The full-length cDNA (called CYP2D17) encoded a 497-amino-acid protein that is 93% identical to human CYP2D6 and 90% identical to marmoset CYP2D19. The CYP2D17 cDNA was cloned into a baculovirus expression vector, and microsomes prepared from CYP2D17-infected insect cells were used to determine the catalytic properties of the recombinant enzyme. The recombinant CYP2D17 results were compared to data generated with monkey liver microsomes, human liver microsomes, and recombinant CYP2D6 and demonstrated catalytic similarity using probe substrates and inhibitors. Recombinant CYP2D17 catalyzed the oxidation of bufuralol to 1'-hydroxybufuralol and dextromethorphan to dextrorphan, reactions shown to be mediated by CYP2D6 in humans; the apparent K(m) values for bufuralol and dextromethorphan were 1 and 0.8 microM, respectively. Moreover, both of these reactions were more strongly inhibited by quinidine than by quinine. A more complete understanding of the substrate specificities and activities of monkey CYPs will be advantageous in delineating species differences in metabolite profiles and metabolic activation of new chemical entities in the pharmaceutical industry.

Amino Acid Sequence↗