Steven M. Fogelman

Protease Inhibitors as Inhibitors of Human Cytochromes P450: High Risk Associated with Ritonavir

Four protease inhibitor antiviral agents (ritonavir, indinavir, nelfinavir, saquinavir) were evaluated as in vitro inhibitors of the activity of six human cytochromes using an in vitro model based on human liver microsomes. Ritonavir was a highly potent inhibitor of P450‐3A activity (triazolam hydroxylation), having inhibitory potency slightly less than ketoconazole. Indinavir was also a potent 3A inhibitor, while nelfinavir and saquinavir were less potent. Ritonavir had high inhibition potency against cytochrome P450‐2C9 (tolbutamide hydroxylation), −2C19 (S‐mephenytoin hydroxylation), and −2D6 (dextromethorphan O‐demethylation and desipramine hydroxylation), while the other protease inhibitors had one or more orders of magnitude lower inhibitory activity against these reactions. None of the protease inhibitors had important inhibitory potency against P450‐1A2 (phenacetin O‐deethylation) or −2E1 (chlorzoxazone hydroxylation). Thus, among available protease inhibitors, ritonavir carries the highest risk of incurring drug interactions due to inhibition of cytochrome P450 activity.

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, Km = 41 μM, and Ks 22901 μM (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 μM, and paroxetine (PX) was the most potent SSRI at inhibiting ODV formation with a mean Ki value of 0.17 μM. Studies using expressed cytochromes showed that ODV was formed by CYP2C9, −2C19, and −2D6. CYP2D6 was dominant with the lowest Km, 23.2 μM, and highest intrinsic clearance (Vmax/Km 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 Km = 2504 μM. 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 μM, 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.

Phenacetin O-deethylation by human liver microsomes in vitro: inhibition by chemical probes, SSRI antidepressants, nefazodone and venlafaxine

Abstract Biotransformation of phenacetin via O-deethylation to acetaminophen, an index reaction reflecting activity of Cytochrome P450-1A2, was studied in microsomal preparations from a series of human livers. Acetaminophen formation was consistent with a double Michaelis-Menten system, with low-Km (mean Km1 = 68 μM) and high-Km (mean Km2 = 7691 μM) components. The low-Km enzyme accounted for an average of 96% of estimated intrinsic clearance, and was predicted to contribute more than 50% of net reaction velocity at phenacetin concentrations less than 2000 μM. Among index inhibitor probes, α-naphthoflavone was a highly potent inhibitor of the low-Km enzyme (Ki1 = 0.013 μM); furafylline also was a moderately active inhibitor (Ki1 = 4.4 μM), but its inhibiting potency was increased by preincubation with microsomes. Ketoconazole was a relatively weak inhibitor (Ki1 = 32 μM); quinidine and cimetidine showed minimal inhibiting activity. Among six selective serotonin reuptake inhibitor (SSRI) antidepressants, fluvoxamine was a potent inhibitor of 1A2 (mean Ki1 = 0.24 μM). The other SSRIs were more than tenfold less potent. Mean Ki1 values were: fluoxetine, 4.4 μM; norfluoxetine, 15.9 μM; sertraline, 8.8 μM; desmethylsertraline, 9.5μM; paroxetine, 5.5 μM. The antidepressant nefazodone and four of its metabolites (meta-chloro-phenylpiperazine, two hydroxylated derivatives, and a triazoledione) were very weak inhibitors of P450-1A2. Venlafaxine and its O- and N-desmethyl metabolites showed minimal inhibitory activity.

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‐methoxy‐morphinan (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 μM). Kinetic parameters for the two O‐demethylation mediating enzymes predict an average relative intrinsic clearance (Vmax/Km 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, Km from 231 to 322 μM). However, ketoconazole, a CYP3A3/4 inhibitor, reduced N‐demethylation only by 60% and had a mean Ki an order of magnitude higher (0·37 μM) 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 μM. 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 μM between 33 and 43%. Chemical inhibitors of CYP2D6 (quinidine), CYP1A1/2 (α‐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.

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.

Short‐Term Exposure to Low‐Dose Ritonavir Impairs Clearance and Enhances Adverse Effects of Trazodone

Antiretroviral agents may participate in drug interactions that influence the efficacy and toxicity of other antiretrovirals, as well as pharmacologic treatments of coincident or complicating diseases. The viral protease inhibitor, ritonavir, may cause drug interactions by inhibiting the activity of cytochrome P450‐3A (CYP3A) isoforms. In a single‐dose, blinded, four‐way crossover study, 10 healthy volunteer subjects received 50 mg of trazodone hydrochloride or matching placebo concurrent with low‐dose ritonavir (four doses of 200 mg each) or with placebo. Compared to the control condition, ritonavir significantly reduced apparent oral clearance of trazodone (155 ± 23 vs. 75 ± 12 ml/min, p < 0.001), prolonged elimination half‐life (6.7 ± 0.7 vs. 14.9 ± 3.9 h, p < 0.05), and increased peak plasma concentrations (842 ± 64 vs. 1125 ± 111 ng/ml, p < 0.05) (mean ± SE). Coadministration of trazodone with ritonavir increased sedation, fatigue, and performance impairment compared to trazodone plus placebo; differences reached significance only for the digit‐symbol substitution test. Three subjects experienced nausea, dizziness, or hypotension when trazodone was given with ritonavir; 1 of these subjects also experienced syncope. Thus short‐term low‐dose administration of ritonavir impairs oral clearance of trazodone and increases the occurrence of adverse reactions. The findings are consistent with impairment of CYP3A‐mediated trazodone metabolism by ritonavir.

Venlafaxine and metabolites are very weak inhibitors of human cytochrome P450-3A isoforms

Cytochrome P450-3A isoforms mediate the metabolism of a large number of drugs used in clinical practice (Harvey and Preskom 1996; Ketter et al 1995; yon Moltke et al 1995b). The class of selective serotonin reuptake inhibitor (SSRI) antidepressants has the capacity to inhibit reversibly the activity of human cytochrome P450-3A isoforms, accounting for a number of pharmacokinetic drug interactions. Cotreatment with fluoxetine, for example, impairs metabolic clearance and/or elevates steadystate plasma concentrations of P450-3A substrates such as diazepam, alprazolam, amitriptyline, imipramine, and carbamazepine (Greenblatt et al in press). The inhibitory activity of fluoxetine appears to be attributable mainly to its metabolite, norfluoxetine. Fluvoxamine also is a P450-3A inhibitor, causing pharmacokinetic interactions with alprazolam (Fleishaker and Hulst 1994), diazepam (Perucca et al 1994), and haloperidol (Daniel et al 1994). The antidepressant nefazodone, which produces presynaptic serotonin and norepinephrine reuptake inhibition and postsynaptic serotonin (5-HT) 2 receptor blockade (Ellingrod and Perry 1995), is a reasonably potent 3A inhibitor (von Moltke et al 1996b), significantly elevating plasma concentrations of alprazolam (Greene et al 1995) and triazolam (Barbhaiya et al 1995) when coadministered with these drugs. The antidepressant venlafaxine (VF) acts by blocking reuptake

Gepirone and 1-(2-pyrimidinyl)-piperazine in vitro: human cytochromes mediating transformation and cytochrome inhibitory effects

Abstract Biotransformation of gepirone to its principal metabolite, 1-(2-pyrimidinyl)-piperazine (1-PP), was studied in human liver microsomes and in microsomes from cDNA-transfected human lymphoblastoid cells. Formation of 1-PP from gepirone in liver microsomes proceeded with a mean apparent Km ranging from 335 to 677 μM. Coincubation with 1 μM ketoconazole reduced reaction velocity to less than 5% of control values at a gepirone concentration of 250 μM. Three other metabolites, presumed to be hydroxylated products, were also formed from gepirone. Formation of all three products was reduced to approximately 20% of control values by 1 μM ketoconazole; quinidine at 1 μM produced a small reduction in formation (91–94% of control) of two of the metabolites. 1-PP was formed from gepirone exclusively by pure P450-3A4 with a Km of 849 μM; Km values for the other metabolites were 245, 240, and 415 μM. Two of the products were also formed by P450-2D6. The results indicate that 3A4 is the principal cytochrome mediating 1-PP formation, as well as formation of the other metabolites. The properties of gepirone and 1-PP themselves as cytochrome inhibitors were tested in human liver microsomes using index reactions representing activitiy of P450-1A2, -2C9, -2C19, -2D6, -2E1 and -3A. Gepirone and 1-PP produced negligible inhibition of all these reactions. Thus gepirone at therapeutic doses in humans has a low likelihood of inhibiting P450-mediated drug metabolism involving these cytochromes.

Ritonavir impairs clearance and enhances toxicity of trazodone

Clinical Pharmacology & Therapeutics (2003) 73, P18–P18; doi: