And N-Demethylation of Venlafaxine in Vitro by Human Liver Microsomes

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 Steven M. Fogelman, Jürgen Schmider, Karthik Venkatakrishnan, Lisa L. von Moltke, Jerold S. Harmatz, Richard I. Shader, and David J. Greenblatt The biotransformation of venlafaxine (VF) into its two to the formation of NDV for all four livers tested. major metabolites, O-desmethylvenlafaxine (ODV) and Parameters determined by applying a single-enzyme model 5 / / 5 N-desmethylvenlafaxine (NDV) was studied in vitro with were Vmax 2.14 nmol min mg protein, and Km 2504 human liver microsomes and with microsomes containing mM. Ketoconazole was a potent inhibitor of NDV individual human cytochromes from cDNA-transfected production, although its inhibitory activity was not as great human lymphoblastoid cells. VF was coincubated with as observed with pure 3A substrates. NDV formation was selective cytochrome P450 (CYP) inhibitors and several also reduced by 42% by a polyclonal rabbit antibody against selective serotonin reuptake inhibitors (SSRIs) to assess rat liver CYP3A1. Studies using expressed cytochromes their inhibitory effect on VF metabolism. Formation rates showed that NDV was formed by CYP2C9, 22C19, and for ODV incubated with human microsomes were 23A4. The highest intrinsic clearance was attributable to consistent with Michaelis-Menten kinetics for a single- CYP2C19 and the lowest to CYP3A4. However the high in enzyme mediated reaction with substrate inhibition. Mean vivo abundance of 3A isoforms will magnify the 5 parameters determined by non-linear regression were: Vmax importance of this cytochrome. Fluvoxamine (FX), at a / / 5 m m 0.36 nmol min mg protein, Km 41 M, and Ks 22901 concentration of 20 M, decreased NDV production by m M (Ks represents a constant which reflects the degree of 46% consistent with the capacity of FX to inhibit CYP3A, substrate inhibition). Quinidine (QUI) was a potent 2C9, and 2C19. These results are consistent with previous m 2 inhibitor of ODV formation with a Ki of 0.04 M, and studies that show CYP2D6 and 3A4 play important roles paroxetine (PX) was the most potent SSRI at inhibiting in the formation of ODV and NDV, respectively. In m ODV formation with a mean Ki value of 0.17 M. Studies addition we have shown that several other CYPs have using expressed cytochromes showed that ODV was formed important roles in the biotransformation of VF. by CYP2C9, 22C19, and 22D6. CYP2D6 was dominant [Neuropsychopharmacology 20:480–490, 1999] m with the lowest Km, 23.2 M, and highest intrinsic © 1999 American College of Neuropsychopharmacology. / clearance (Vmax Km ratio). No unique model was applicable Published by Elsevier Science Inc. From the Department of Pharmacology and Experimental Thera- Address correspondence to: David J. Greenblatt, Department of peutics, Tufts University School of Medicine, and the Division of Pharmacology and Experimental Therapeutics, Tufts University Clinical Pharmacology, New England Medical Center Hospital, Bos- School of Medicine, 136 Harrison Ave., Boston, MA 02111. ton, MA. Received January 16, 1998; accepted November 3, 1998. NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 5 © 1999 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/99/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(98)00113-4 NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 5 O- and N-demethylation of Venlafaxine 481 KEY WORDS: Venlafaxine; Cytochromes P-450; Selective biotransformation of VF to its principal metabolites serotonin reuptake inhibitors ODV and NDV over a large range of substrate concentrations. We also assessed the inhibitory potencies of The last several years has seen a dramatic increase in the currently available SSRIs and their metabolites. This our understanding of drug metabolism and drug-drug was done to evaluate the possibility of pharmacokinetic interactions that are mediated through the inhibition or interactions of VF with SSRIs, and to evaluate the con- induction of cytochrome P-450 (CYP) enzymes. This is sistency across substrates of impaired CYP2D6 medi- due in part to advances in techniques collectively called ated metabolism caused by SSRIs. “reaction phenotyping,” and includes the use of cDNA- expressed enzymes, and human liver microsomal preparations in combination with chemical inhibitors and antibodies directed against specific CYPs. METHODS One of the most frequently prescribed classes of Chemicals medications is the antidepressants, many of which are either substrates of CYP2D6 or CYP3A3/4, or are inhibi- VF, ODV, and NDV were generously provided by Wyeth- tors of these two CYP systems. With the large amount Ayerst Laboratories (Philadelphia, PA). Ketoconazole of data being produced, clinicians have been looking (KET) was provided by Janssen Research Foundation for a way to generalize information regarding the ca- (Beerse, Belgium). Dextrorphan D-tartrate was purchased pacity of antidepressants to inhibit various CYPs. from Research Biochemicals International (Natick, MA). a At least seven other studies have examined the rela- Quinidine (QUI), -naphthoflavone (ANA), and sulfin- tive inhibitory potencies of the SSRIs and their metabo- pyrazone (SFZ) were purchased from Sigma Chemical lites in relation to 2D6 mediated reactions. These reac- (St. Louis, MO). Sertraline (SER) and desmethylsertraline tions include sparteine to 2-dehydrosparteine (Crewe et (DES) were gifts from Pfizar (Groton, CT), and fluoxetine al. 1992), dextromethorphan to dextrorphan (Otton et (FLU) and norfluoxetine (NOR) were gifts from Eli Lilly al. 1993), desipramine to 2-hydroxydesipramine (von (Indianapolis, IN). Paroxetine (PX) was generously pro- Moltke et al. 1995), imipramine to 2-hydroxyimipramine vided by SmithKline Beecham (Philadelphia, PA), and (Skjelbo and Brøsen 1992; Ball et al. 1997), clomipramine fluvoxamine (FX) from Solvay (Marietta, GA). The co- 6 to 8-hydroxyclomipramine (Nielsen et al. 1996), desme- factors, including NADP, ( )-isocitric acid, MgCl2, and thylclomipramine to 8-hydroxydesmethylclomipramine isocitrate dehydrogenase, as well as the potassium phos- (Nielsen et al. 1996), and metoprolol to a-hydroxymeto- phate salt used in the buffer solution were purchased prolol and O-demethylmetoprolol (Belpaire et al. 1998). from Sigma. All organic solvents were reagent grade. One of the newer antidepressants, venlafaxine (VF), is a structurally novel compound of the phenylethy- Liver Samples and Preparation of Microsomes lamine class. VF inhibits the presynaptic reuptake of serotonin, norepinephrine, and to a lesser extent dopam- Liver samples were obtained from the International In- ine (Muth et al. 1986). It has little to no affinity for a-1 stitute for the Advancement of Medicine (Exton, PA), or adrenergic, muscarinic, or histaminergic receptors, and the Liver Tissue Procurement and Distribution Service, does not inhibit monoamine oxidase (Muth et al. 1986). University of Minnesota (Minneapolis, MN). VF is effective as an antidepressant (Schweizer et al. Donors had no known liver disease. The tissue was 1994; Cunningham et al. 1994), and may have utility in partitioned and stored at 2808 until microsome prepa- the treatment of panic disorder (Geracioti 1995), and at- ration. Microsomes were prepared as previously de- tention-deficit disorder (Findling et al. 1996). scribed by von Moltke et al. (1993; 1994), and protein VF is biotransformed in the liver to O-desmethylven- concentrations of the microsome samples was deter- lafaxine (ODV), N-desmethylvenlafaxine (NDV), and mined using the Bicinchoninic Acid Protein Assay N,O-didesmethylvenlafaxine (N,O-DV) (Muth et al. (BCA-Pierce Rockford IL). Bovine serum albumin was 1991). ODV has a receptor affinity profile similar to its used as the standard. parent compound VF, while the latter two metabolites Microsomes containing human cytochromes P4501A2, have little if any affinity for the above receptor sites 22C9, 22C19, 22D6, 22E1, and 23A4, expressed in (Muth et al. 1991). Previous studies have shown that the cDNA-transfected human lymphoblastoid cells (Crespi biotransformation of VF to ODV is mediated by 1995) were obtained from Gentest (Woburn, MA). CYP2D6, and NDV via CYP3A3/4, and possibly a sec- ond enzyme (Otton et al. 1996). Antibody We used an in vitro model of human liver microsomal preparations and of microsomes containing indi- Serum containing polyclonal antibody against rabbit rat vidual human cytochromes expressed by cDNA-trans- liver CYP3A1 and goat antibody against rat CYP2C were fected human lymphoblastoid cells, to evaluate the purchased from Human Biologics, Inc. (Phoenix, AZ). 482 S.M. Fogelman et al. NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 5 Calibration Standards Immunoinhibition Studies Mixtures used to generate calibration curves contained Using the same incubation preparations as above, mi- ODV (0–10 nmoles) or NDV (0–10 nmoles) which were crosomes and either antibody or control serum were evaporated to dryness prior to the addition of 50 mM preincubated at 378 for 20 minutes with a fixed VF con- potassium phosphate buffer (pH adjusted to 7.5 at centration (750 mM). Antibody concentrations varied 258C), and dextrorphan as internal standard. from 1:5 to 1:15 microsomal:antibody mass ratios. Reac- tions were initiated by the addition of cofactors, and incubated for 40 minutes at 378. Final reaction volumes were 250 mL. The mixtures were processed as above

Load more