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J S Harmatz

Publications and source records attributed to J S Harmatz.

At least 19 recordsLinked to original sources
PubMed2001

Molecular and pharmacokinetic evaluation of rat hepatic and gastrointestinal cytochrome p450 induction by tamoxifen.

Tamoxifen (TAM) is a first-line endocrine treatment for all stages of postmenopausal breast cancer. The cytochrome P450 (CYP) enzymes catalyze the majority of TAM's primary metabolism, producing N-desmethyltamoxifen (DMT) and 4-hydroxytamoxifen (4-OH-TAM) in both humans and rats. CYP 3A isoforms are the predominant subfamily involved in the formation of DMT and recent studies have shown that TAM induces hepatic forms of these enzymes. TAM's inductive effect on gastrointestinal CYP 3A has not been previously reported. The current studies investigated TAM's induction of CYP isoforms (3A and 2B) in female rat gastrointestinal and hepatic tissue at the mRNA, protein, and catalytic level. Since previous studies have not addressed whether TAM induction causes changes to the overall pharmacokinetics (PKs), a rat PK model was used to determine if TAM induced its own metabolism, and/or the metabolism of a CYP 3A substrate, midazolam (MDZ). Phenobarbital (PB) and/or dexamethasone (DEX) were used as positive controls for all studies. TAM significantly induced, or caused a trend towards induction of all studied parameters for hepatic CYP 3A and 2B, whereas intestinal CYP 3A and 2B analysis did not show significant induction by TAM at any level. A study evaluating time-dependent alterations in the PK profile of TAM showed no change in apparent oral clearance (Cl(app)) during two weeks of chronic dosing with TAM. However, the Cl(app) for MDZ was shown to trend towards an increase after two weeks of dosing with TAM, in a second PK study. These combined investigations suggest that TAM is an inducer of rat hepatic CYP 3A and 2B isoforms, and this agent has the potential of influencing the PK of coadministered 3A substrates.

Analysis of Variance↗
PubMed2001

Inhibition of human cytochrome P450 isoforms by nonnucleoside reverse transcriptase inhibitors.

The capacity of three clinically available nonnucleoside reverse transcriptase inhibitors (NNRTIs) to inhibit the activity of human cytochromes P450 (CYPs) was studied in vitro using human liver microsomes. Delavirdine, nevirapine, and efavirenz produced negligible inhibition of phenacetin O-deethylation (CYP1A2) or dextromethorphan O-demethylation (CYP2D6). Nevirapine did not inhibit hydroxylation of tolbutamide (CYP2C9) or S-mephenytoin (CYP2C19), but these CYP isoforms were importantly inhibited by delavirdine and efavirenz. This indicates the likelihood of significantly impaired clearance of CYP2C substrate drugs (such as phenytoin, tolbutamide, and warfarin) upon initial exposure to these two NNRTIs. Delavirdine and efavirenz (but not nevirapine) also were strong inhibitors of CYP3A, consistent with clinical hazards of initial cotreatment with either of these drugs and substrates of CYP3A. The in vitro microsomal model provides relevant predictive data on probable drug interactions with NNRTIs when the mechanism is inhibition of CYP-mediated drug biotransformation. However, the model does not incorporate interactions attributable to enzyme induction.

Alkynes↗
PubMed2001

Relative contribution of CYP3A to amitriptyline clearance in humans: in vitro and in vivo studies.

The relative contribution of cytochrome P450 3A (CYP3A) to the oral clearance of amitriptyline in humans has been assessed using a combination of in vitro approaches together with a clinical pharmacokinetic interaction study using the CYP3A-selective inhibitor ketoconazole. Lymphoblast-expressed CYPs were used to study amitriptyline N-demethylation and E-10 hydroxylation in vitro. The relative activity factor (RAF) approach was used to predict the relative contribution of each CYP isoform to the net hepatic intrinsic clearance (sum of N-demethylation and E-10 hydroxylation). Assuming no extrahepatic metabolism, the model-predicted contribution of CYP3A to net intrinsic clearance should equal the fractional decrement in apparent oral clearance of amitriptyline upon complete inhibition of the enzyme. This hypothesis was tested in a clinical study of amitriptyline (50 mg, p.o.) with ketoconazole (three 200 mg doses spaced 12 hours apart) in 8 healthy volunteers. The RAF approach predicted CYP2C19 to be the dominant contributor (34%), with a mean 21% contribution of CYP3A (range: 8%-42% in a panel of 12 human livers). The mean apparent oral clearance of amitriptyline in 8 human volunteers was decreased from 2791 ml/min in the control condition to 2069 ml/min with ketoconazole. The average 21% decrement (range: 2%-40%) was identical to the mean value predicted in vitro using the RAF approach. The central nervous system (CNS) sedative effects of amitriptyline were slightly greater when ketoconazole was coadministered, but the differences were not statistically significant. In conclusion, CYP3A plays a relatively minor role in amitriptyline clearance in vivo, which is consistent with in vitro predictions using the RAF approach.

Adult↗
PubMed2001

Escitalopram (S-citalopram) and its metabolites in vitro: cytochromes mediating biotransformation, inhibitory effects, and comparison to R-citalopram.

Transformation of escitalopram (S-CT), the pharmacologically active S-enantiometer of citalopram, to S-desmethyl-CT (S-DCT), and of S-DCT to S-didesmethyl-CT (S-DDCT), was studied in human liver microsomes and in expressed cytochromes (CYPs). Biotransformation of the R-enantiomer (R-CT) was studied in parallel. S-CT was transformed to S-DCT by CYP2C19 (K(m) = 69 microM), CYP2D6 (K(m) = 29 microM), and CYP3A4 (K(m) = 588 microM). After normalization for hepatic abundance, relative contributions to net intrinsic clearance were 37% for CYP2C19, 28% for CYP2D6, and 35% for CYP3A4. At 10 microM S-CT in liver microsomes, S-DCT formation was reduced to 60% of control by 1 microM ketoconazole, and to 80 to 85% of control by 5 microM quinidine or 25 microM omeprazole. S-DDCT was formed from S-DCT only by CYP2D6; incomplete inhibition by quinidine in liver microsomes indicated participation of a non-CYP pathway. Based on established index reactions, S-CT and S-DCT were negligible inhibitors (IC(50) > 100 microM) of CYP1A2, -2C9, -2C19, -2E1, and -3A, and weakly inhibited CYP2D6 (IC(50) = 70-80 microM). R-CT and its metabolites, studied using the same procedures, had properties very similar to those of the corresponding S-enantiomers. Thus S-CT, biotransformed by three CYP isoforms in parallel, is unlikely to be affected by drug interactions or genetic polymorphisms. S-CT and S-DCT are also unlikely to cause clinically important drug interactions via CYP inhibition.

Algorithms↗
PubMed2000

Differential impairment of triazolam and zolpidem clearance by ritonavir.

BACKGROUND: The viral protease inhibitor ritonavir has the capacity to inhibit and induce the activity of cytochrome P450-3A (CYP3A) isoforms, leading to drug interactions that may influence the efficacy and toxicity of other antiretroviral therapies, as well as pharmacologic treatments of coincident or complicating diseases. METHODS: The inhibitory effect of ritonavir on the biotransformation of the hypnotic agents triazolam and zolpidem was tested in vitro using human liver microsomes. In a double-blind clinical study, volunteer study subjects received 0.125 mg triazolam or 5.0 mg zolpidem concurrent with low-dose ritonavir (four doses of 200 mg), or with placebo. RESULTS: Ritonavir was a potent in vitro inhibitor of triazolam hydroxylation but was less potent as an inhibitor of zolpidem hydroxylation. In the clinical study, ritonavir reduced triazolam clearance to < 4% of control values (p < .005), prolonged elimination half-life (41 versus 3 hours; p < .005), and magnified benzodiazepine agonist effects such as sedation and performance impairment. In contrast, ritonavir reduced zolpidem clearance to 78% of control values (p < .08), and slightly prolonged elimination half-life (2.4 versus 2.0 hours; NS). Benzodiazepine agonist effects of zolpidem were not altered by ritonavir. CONCLUSION: Short-term low-dose administration of ritonavir produces a large and significant impairment of triazolam clearance and enhancement of clinical effects. In contrast, ritonavir produced small and clinically unimportant reductions in zolpidem clearance. The findings are consistent with the complete dependence of triazolam clearance on CYP3A activity, compared with the partial dependence of zolpidem clearance on CYP3A.

Adult↗
PubMed2000

In vitro metabolism of trazodone by CYP3A: inhibition by ketoconazole and human immunodeficiency viral protease inhibitors.

BACKGROUND: Pharmacologic treatment of emotional disorders in HIV-infected patients can be more easily optimized by understanding of potential interactions of psychotropic drugs with medications used to treat HIV infection and its sequelae. METHODS: Biotransformation of the antidepressant trazodone to its principal metabolite, meta-chlorophenylpiperazine (mCPP), was studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes. Interactions of trazodone with the azole antifungal agent, ketoconazole, and with human immunodeficiency virus protease inhibitors (HIVPIs) were studied in the same system. RESULTS: Formation of mCPP from trazodone in liver microsomes had a mean (+/- SE) K(m) value of 163 (+/- 21) micromol/L. Ketoconazole, a relatively specific CYP3A inhibitor, impaired mCPP formation consistent with a competitive mechanism, having an inhibition constant (K(i)) of 0.12 (+/- 0.01) micromol/L. Among heterologously expressed human cytochromes, only CYP3A4 mediated formation of mCPP from trazodone; the K(m) was 180 micromol/L, consistent with the value in microsomes. The HIVPI ritonavir was a potent inhibitor of mCPP formation in liver microsomes (K(i) = 0.14 +/- 0.04 micromol/L). The HIVPI indinavir was also a strong inhibitor, whereas saquinavir and nelfinavir were weaker inhibitors. CONCLUSIONS: CYP3A-mediated clearance of trazodone is inhibited by ketoconazole, ritonavir and indinavir, and indicates the likelihood of pharmacokinetic interactions in vivo.

Antidepressive Agents, Second-Generation↗
PubMed2000

Alprazolam-ritonavir interaction: implications for product labeling.

BACKGROUND: Pharmacokinetic interactions involving antiretroviral therapies may critically influence the efficacy and toxicity of these drugs, as well as pharmacologic treatments of coincident or complicating diseases. The viral protease inhibitor ritonavir is of particular concern since it both inhibits and induces the activity of cytochrome P450 3A (CYP3A) isoforms. METHODS: The inhibitory effect of ritonavir on the metabolism of alprazolam, a CYP3A-mediated reaction in humans, was tested in vitro using human liver microsomes. In a double-blind clinical study, volunteer subjects received 1.0 mg of alprazolam concurrent with low-dose ritonavir (four doses of 200 mg) or with placebo. RESULTS: Ritonavir was a potent in vitro inhibitor of alprazolam hydroxylation. The 50% inhibitory concentration was 0.11 micromol/L (0.08 microg/mL); this is below the usual therapeutic plasma concentration range (generally exceeding 2 microg/mL). In the clinical study, ritonavir reduced alprazolam clearance to 41% of control values (P < .001), prolonged elimination half-life (mean values, 30 versus 13 hours; P < .005), and magnified benzodiazepine agonist effects such as sedation and performance impairment. CONCLUSION: Consistent with in vitro results, administration of low doses of ritonavir for a short duration of time resulted in large impairment of alprazolam clearance and enhancement of clinical effects. Removal from product labeling of a warning against coadministration of ritonavir and alprazolam was based on a previous study only of extended exposure to ritonavir, in which CYP3A induction offset inhibition. Kinetic interactions involving antiretroviral therapies may be complex and time dependent. Product labeling should reflect this complexity.

Adult↗
PubMed2000

Kinetics and dynamics of lorazepam during and after continuous intravenous infusion.

OBJECTIVE: To evaluate the kinetics and dynamics of lorazepam during administration as a bolus plus an infusion, using electroencephalography as a pharmacodynamic end point. METHODS: Nine volunteers received a 2-mg bolus loading dose of lorazepam, coincident with the start of a 2 microg/kg/hr zero-order infusion. The infusion was stopped after 4 hrs. Plasma lorazepam concentrations and electroencephalographic activity in the 13- to 30-Hz range were monitored for 24 hrs. RESULTS: The bolus-plus-infusion scheme rapidly produced plasma lorazepam concentrations that were close to those predicted to be achieved at true steady state. Mean kinetic values for lorazepam were as follows: volume of distribution, 126 L; elimination half-life, 13.8 hrs; and clearance, 109 mL/min. Electroencephalographic effects were maximal 0.5 hr after the loading dose, were maintained essentially constant during infusion, and then declined in parallel with plasma concentrations after the infusion was terminated. There was no evidence of tolerance. Plots of pharmacodynamic electroencephalographic effect vs. plasma lorazepam concentration demonstrated counterclockwise hysteresis, consistent with an effect-site equilibration delay. This was incorporated into a kinetic-dynamic model in which hypothetical effect-site concentration was related to pharmacodynamic electroencephalographic effect via the sigmoid Emax model. The analysis yielded the following mean estimates: maximum electroencephalographic effect, 12.7% over baseline; 50% effective concentration, 13.1 ng/mL; and effect-site equilibration half-life, 8.8 mins. CONCLUSION: Despite the delay in effect onset, continuous infusion of lorazepam, preceded by a bolus loading dose, produces a relatively constant sedative effect on the central nervous system, which can be utilized in the context of critical care medicine.

Adult↗
PubMed2000

Differences in pharmacodynamics but not pharmacokinetics between subjects with panic disorder and healthy subjects after treatment with a single dose of alprazolam.

The pharmacokinetics and pharmacodynamics of the benzodiazepine alprazolam (1 mg, administered orally) were compared between eight patients with panic disorder and eight age- and sex-matched healthy volunteers. Subjects received orally administered placebo and alprazolam in a randomized, double-blind, single-dose crossover study. The elimination half-life, time of maximum plasma concentration, maximum concentration, volume of distribution, and clearance of alprazolam were similar for both groups. For each cohort, alprazolam treatment (vs. placebo) produced significant changes in typical benzodiazepine agonist effects, such as increased sedation and impaired cognitive performance on the digit-symbol substitution test. For the panic disorder group only, there was a significant increase in the subjective rating of"contented" and a reduction in the rating of "easily irritated." For the healthy volunteer group, alprazolam produced increases in ratings of "fatigued" and "slowed thinking," but also increases in ratings of "relaxed." In each group, alprazolam significantly increased the electroencephalographic (EEG) measure of relative beta amplitude (range, 13-30 Hz) compared with placebo. Concentration-EEG response curves fit a sigmoid E(max) model, and there was greater sensitivity to EEG effects, as measured by a 28% reduction in the EC50 value, in the panic disorder group compared with healthy control subjects. After alprazolam treatment, there was increased sensitivity to EEG and mood effects and fewer aversive effects in the panic disorder group compared with healthy subjects. There were no differences in the pharmacodynamic measures of sedation and cognition or differences in pharmacokinetics between the two groups.

Adult↗
PubMed2000

Effects of age on in vitro midazolam biotransformation in male CD-1 mouse liver microsomes.

To study age-related changes in drug metabolism, we examined the in vitro biotransformation of midazolam (MDZ), a human cytochrome P-450 (CYP) 3A substrate, using liver microsomes from three age groups of male CD-1 mice ranging from 6 weeks to 2 years old. MDZ was metabolized to two major products, alpha-OH- and 4-OH-MDZ, which were quantified by HPLC. For both metabolites, V(max) values were reduced in old livers (P <.05), while K(m) values did not change with age. The net intrinsic clearance (the sum of V(max)/K(m) for both pathways) also was reduced in the old animals (P <.05). The capacity of ketoconazole, a CYP3A inhibitor in humans, to inhibit the biotransformation of MDZ and of alprazolam, another human CYP3A substrate, did not differ significantly with age. At 100 microM alprazolam, 0.5 microM ketoconazole inhibited metabolite formation by >80%. At 30 microM MDZ, 2.5 microM ketoconazole impaired 4-OH-MDZ formation by 88%, whereas it reduced alpha-OH-MDZ formation by only 46%. Immunoinhibition studies with polyclonal anti-rat CYP3A1/2 and CYP2C11 antibodies confirmed that 4-OH-MDZ formation was largely CYP3A-dependent, while alpha-OH-MDZ formation was mediated by CYP3A and -2C isoforms. Western blot analysis revealed decreased microsomal content of CYP3A in old livers. Net intrinsic clearance of MDZ was correlated with total CYP3A content (P <.001). These results demonstrate a reduction in MDZ biotransformation in old male mice, which may be attributable, in part, to decreased CYP3A content in old livers. Changes in expression and activity of CYP2C isoforms also may contribute to age-related changes in MDZ biotransformation, but this requires more investigation.

Age Factors↗
PubMed2000

Comparative kinetics and response to the benzodiazepine agonists triazolam and zolpidem: evaluation of sex-dependent differences.

Eighteen healthy volunteers (10 men and 8 women) participated in a single-dose, double-blind, three-way crossover pharmacokinetic and pharmacodynamic study. Treatment conditions were 0.25 mg of triazolam, a full-agonist benzodiazepine ligand; 10 mg of zolpidem, an imidazopyridine having relative selectivity for the type 1 benzodiazepine receptor subtype; and placebo. Weight-normalized clearance of triazolam was higher in women than in men (8.7 versus 5. 5 ml/min/kg), but the difference was not significant. In contrast, zolpidem clearance was lower in women than in men (3.5 versus 6.7 ml/min/kg, P <.06). Compared to placebo, both active medications produced significant benzodiazepine agonist-like pharmacodynamic effects: sedation, impaired psychomotor performance, impaired information recall, and increased electroencephalographic beta-amplitude. Effects of triazolam and zolpidem in general were comparable and less than 8 h in duration. There was no evidence of a substantial or consistent sex difference in pharmacodynamic effects or in the kinetic-dynamic relationship, although subtle differences could not be ruled out due to low statistical power. The complete dependence of triazolam clearance on CYP3A activity, as opposed to the mixed CYP participation in zolpidem clearance, may explain the differing sex effects on clearance of the two compounds.

Adult↗
PubMed2000

Comparison between cytochrome P450 (CYP) content and relative activity approaches to scaling from cDNA-expressed CYPs to human liver microsomes: ratios of accessory proteins as sources of discrepancies between the approaches.

Relative activity factors (RAFs) and immunoquantified levels of cytochrome P450 (CYP) isoforms both have been proposed as scaling factors for the prediction of hepatic drug metabolism from studies using cDNA-expressed CYPs. However, a systematic comparison of the two approaches, including possible mechanisms underlying differences, is not available. In this study, RAFs determined for CYPs 1A2, 2B6, 2C19, 2D6, and 3A4 in 12 human livers using lymphoblast-expressed enzymes were compared to immunoquantified protein levels. 2C19, 2D6, and 3A4 RAFs were similar to immunoquantified enzyme levels. In contrast, 1A2 RAFs were 5- to 20-fold higher than CYP1A2 content, and the RAF:content ratio was positively correlated with the molar ratio of NADPH:CYP oxidoreductase (OR) to CYP1A2. The OR:CYP1A2 ratio in lymphoblast microsomes was 92-fold lower than in human liver microsomes. Reconstitution experiments demonstrated a 10- to 20-fold lower activity at OR:CYP1A2 ratios similar to those in lymphoblasts, compared with those in human livers. CYP2B6-containing lymphoblast microsomes had 29- and 13-fold lower OR:CYP and cytochrome b(5):CYP ratios, respectively, than did liver microsomes and yielded RAFs that were 6-fold higher than CYP2B6 content. Use of metabolic rates from cDNA-expressed CYPs containing nonphysiologic concentrations of electron-transfer proteins (relative to human liver microsomes) in conjunction with hepatic CYP contents may lead to incorrect predictions of liver microsomal rates and relative contributions of individual isoforms. Scaling factors used in bridging the gap between expression systems and liver microsomes should not only incorporate relative hepatic abundance of individual CYPs but also account for differences in activity per unit enzyme in the two systems.

Adolescent↗
PubMed1999

Citalopram and desmethylcitalopram in vitro: human cytochromes mediating transformation, and cytochrome inhibitory effects.

BACKGROUND: Biotransformation of citalopram (CT), a newly available selective serotonin reuptake inhibitor antidepressant, to its principal metabolite, desmethycitalopram (DCT), and the capacity of CT and DCT to inhibit human cytochromes P450, were studied in vitro. METHODS: Formation of DCT from CT was evaluated using human liver microsomes and microsomes from cDNA-transfected human lymphoblastoid cells. Cytochrome inhibition by CT and DCT in liver microsomes was studied using isoform-specific index reactions. RESULTS: Formation of DCT from CT in liver microsomes had a mean apparent K(m) of 174 mumol/L. Coincubation with 1 mumol/L ketoconazole reduced reaction velocity to 46 to 58% of control values, while omeprazole, 10 mumol/L, reduced velocity to 80% of control. Quinidine produced minimal inhibition. DCT was formed from CT by heterologously expressed human P450-2D6, -2C19, -3A4. After accounting for the relative abundance of individual cytochromes, 3A4 and 2C19 were estimated to make major contributions to net reaction velocity, with a possible contribution of 2D6 at therapeutic CT concentrations. CT and DCT themselves produced negligible inhibition of 2C9, 2E1, and 3A, and only weak inhibition of 1A2, 2C19, and 2D6. CONCLUSIONS: Formation of DCT from CT is mediated mainly by P450-3A4 and 2C19, with an additional contribution of 2D6. CT at therapeutic doses in humans may produce a small degree of inhibition of P450-1A2, -2C19, and -2D6, but negligible inhibition of P450-2C9, -2E1, and -3A.

Biotransformation↗
PubMed1999

Nefazodone, meta-chlorophenylpiperazine, and their metabolites in vitro: cytochromes mediating transformation, and P450-3A4 inhibitory actions.

RATIONALE: Understanding of the mechanisms of biotransformation of antidepressant drugs, and of their capacity to interact with other medications, is of direct relevance to rational clinical psychopharmacology. OBJECTIVES: To determine the human cytochromes P450 mediating the metabolism of nefazodone, and the inhibitory activity of nefazodone and metabolites versus human P450-3A. METHODS: Biotransformation of nefazodone to its metabolic products, and of meta-chlorophenylpiperazine (mCPP) to para-hydroxy-mCPP, was studied in vitro using human liver microsomes and heterologously expressed human cytochromes. Nefazodone and metabolites were also tested as inhibitors of alprazolam hydroxylation, reflecting activity of cytochrome P450-3A isoforms. RESULTS: mCPP and two hydroxylated derivatives were the principal metabolites formed from nefazodone by liver microsomes. Metabolite production was strongly inhibited by ketoconazole or troleandomycin (relatively specific P450-3A inhibitors), and by an anti-P450-3A antibody. Only heterologously expressed human P450-3A4 mediated formation of nefazodone metabolites from the parent compound. Nefazodone, hydroxy-nefazodone, and para-hydroxy-nefazodone were strong 3A inhibitors, being more potent than norfluoxetine and fluvoxamine, but less potent than ketoconazole. The triazoledione metabolite and mCPP had weak or negligible 3A-inhibiting activity. Formation of parahydroxy-mCPP from mCPP was mediated by heterologously expressed P450-2D6; in liver microsomes, the reaction was strongly inhibitable by quinidine, a relatively specific 2D6 inhibitor. CONCLUSION: The complex parallel biotransformation pathways of nefazodone are mediated mainly by human cytochrome P450-3A, whereas clearance of mCPP is mediated by P450-2D6. Nefazodone and two of its hydroxylated metabolites are potent 3A inhibitors, accounting for pharmacokinetic drug interactions of nefazodone with 3A substrate drugs such as triazolam and alprazolam.

Alprazolam↗
PubMed1999

O- and N-demethylation of venlafaxine in vitro by human liver microsomes and by microsomes from cDNA-transfected cells: effect of metabolic inhibitors and SSRI antidepressants.

The biotransformation of venlafaxine (VF) into its two major metabolites, O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) was studied in vitro with human liver microsomes and with microsomes containing individual human cytochromes from cDNA-transfected human lymphoblastoid cells. VF was coincubated with selective cytochrome P450 (CYP) inhibitors and several selective serotonin reuptake inhibitors (SSRIs) to assess their inhibitory effect on VF metabolism. Formation rates for ODV incubated with human microsomes were consistent with Michaelis-Menten kinetics for a single-enzyme mediated reaction with substrate inhibition. Mean parameters determined by non-linear regression were: Vmax = 0.36 nmol/min/mg protein, K(m) = 41 microM, and Ks 22901 microM (Ks represents a constant which reflects the degree of substrate inhibition). Quinidine (QUI) was a potent inhibitor of ODV formation with a Ki of 0.04 microM, and paroxetine (PX) was the most potent SSRI at inhibiting ODV formation with a mean Ki value of 0.17 microM. Studies using expressed cytochromes showed that ODV was formed by CYP2C9, -2C19, and -2D6. CYP2D6 was dominant with the lowest K(m), 23.2 microM, and highest intrinsic clearance (Vmax/K(m) ratio). No unique model was applicable to the formation of NDV for all four livers tested. Parameters determined by applying a single-enzyme model were Vmax = 2.14 nmol/min/mg protein, and K(m) = 2504 microM. Ketoconazole was a potent inhibitor of NDV production, although its inhibitory activity was not as great as observed with pure 3A substrates. NDV formation was also reduced by 42% by a polyclonal rabbit antibody against rat liver CYP3A1. Studies using expressed cytochromes showed that NDV was formed by CYP2C9, -2C19, and -3A4. The highest intrinsic clearance was attributable to CYP2C19 and the lowest to CYP3A4. However the high in vivo abundance of 3A isoforms will magnify the importance of this cytochrome. Fluvoxamine (FX), at a concentration of 20 microM, decreased NDV production by 46% consistent with the capacity of FX to inhibit CYP3A, 2C9, and 2C19. These results are consistent with previous studies that show CYP2D6 and -3A4 play important roles in the formation of ODV and NDV, respectively. In addition we have shown that several other CYPs have important roles in the biotransformation of VF.

Animals↗
PubMed1999

Zolpidem metabolism in vitro: responsible cytochromes, chemical inhibitors, and in vivo correlations.

AIMS: To determine the human cytochromes mediating biotransformation of the imidazopyridine hypnotic, zolpidem, and the clinical correlates of the findings. METHODS: Kinetic properties of zolpidem biotransformation to its three hydroxylated metabolites were studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes. RESULTS: The metabolic product termed M-3 accounted for more than 80% of net intrinsic clearance by liver microsomes in vitro. Microsomes containing human cytochromes CYP1A2, 2C9, 2C19, 2D6, and 3 A4 expressed by cDNA-transfected human lymphoblastoid cells mediated zolpidem metabolism in vitro. The kinetic profile for zolpidem metabolite formation by each individual cytochrome was combined with estimated relative abundances based on immunological quantification, yielding projected contributions to net intrinsic clearance of: 61% for 3 A4, 22% for 2C9, 14% for 1A2, and less than 3% for 2D6 and 2C19. These values were consistent with inhibitory effects of ketoconazole and sulfaphenazole on zolpidem biotransformation by liver microsomes. Ketoconazole had a 50% inhibitory concentration (IC50 ) of 0.61 microm vs formation of the M-3 metabolite of zolpidem in vitro; in a clinical study, ketoconazole coadministration reduced zolpidem oral clearance by approximately 40%, somewhat less than anticipated based on the IC50 value and total plasma ketoconazole levels, but much more than predicted based on unbound plasma ketoconazole levels. CONCLUSIONS: The incomplete dependence of zolpidem clearance on CYP3A activity has clinical implications for susceptibility to metabolic inhibition.

Biotransformation↗
PubMed1999

Extrapolating in vitro data on drug metabolism to in vivo pharmacokinetics: evaluation of the pharmacokinetic interaction between amitriptyline and fluoxetine.

Recently, models have been proposed to extrapolate in vitro data on the influence of inhibitors on drug metabolism to in vivo decrement in drug clearance. Many factors influence drug clearance such as age, gender, habits, diet, environment, liver disease, heredity, and other drugs. In vitro investigation of hepatic cytochrome P450 activity has generally centered on genetic influences and interactions with other drugs. This group of enzymes is involved in many, although not all, drug interactions. The interaction of amitriptyline and fluoxetine is an example. Of the different in vitro paradigms, interaction studies utilizing human liver microsomal preparations have proved to be the most generally applicable for in vitro scaling models. Assuming Michaelis-Menten conditions and applying nonlinear regression, a hybrid inhibition constant (Ki) can be generated that allows classification of the inhibitory potency of an inhibitor toward a specific reaction. This constant is largely independent of the substrate concentration, but in vivo relevance is critically dependent on the inhibitor concentration in the site of metabolic activity, the liver cell cytosol. Many lipophilic drugs are extensively bound to plasma protein but, nonetheless, demonstrate extensive partitioning into liver tissue. This is not compatible with diffusion only of the unbound drug fraction into liver cells. The introduction of a partition factor, based on data from a number of possible sources, provided a reasonable basis for the scaling of in vitro data to in vivo conditions. Many interactions could be reconstructed or predicted with greater accuracy and clinical relevance for interactions such as terfenadine or midazolam and ketoconazole. Even for less marked interactions such as amitriptyline and fluoxetine, this model provides a forecast consistent with the clinically observed range of 22-45% reduction in oral clearance, although this interaction is complicated by the presence of two inhibitors, fluoxetine and norfluoxetine. The concept of in vitro-in vivo scaling is promising and might ultimately yield a fast and more cost-effective screening for drug interactions with reduced human drug exposure and risk.

Amitriptyline↗