Brian W. Granda

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.

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.

Multiple human cytochromes contribute to biotransformation of dextromethorphan in-vitro : role of CYP2C9, CYP2C19, CYP2D6, and CYP3A

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3‐methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA‐transfected human lymphoblastoid cells.

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.

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 para-hydroxy-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.

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.

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.

Analysis of ritonavir in plasma/serum and tissues by high-performance liquid chromatography.

A method has been developed to quantify ritonavir concentrations in human plasma and in mouse serum, liver, and brain using high-performance liquid chromatography. Extraction recoveries for ritonavir and its internal standard averaged greater than 95%. Within-day variability, expressed as a coefficient of variation, averaged 6% over the concentration range 0.5 microg/mL to 15 microg/mL ritonavir, and between-day variability averaged 5.6% over 5 microg/mL to 15 microg/mL ritonavir. The method was applied to quantitation of ritonavir in mouse serum and tissue. Measured values deviated less than 5% from the actual values in mouse serum, liver, and brain samples containing 5 microg/mL ritonavir. The slopes of calibration curves for extracted calf serum, mouse serum, mouse liver and mouse brain standards were nearly identical to the calibration slope of standards which were not extracted. All curves were linear through zero, and r2 was no less than 0.998 for any form of calibration. In addition, there was no chromatographic interference from commonly prescribed medications.

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.

Appetite suppressant drugs as inhibitors of human cytochromes P450: in vitro inhibition of P450-2D6 by D- and L-fenfluramine, but not phentermine.

The activity of D-fenfluramine, L-fenfluramine, and phentermine as inhibitors of five human cytochromes P450 was evaluated using human liver microsomes in vitro. All three compounds produced negligible inhibition of P450-1A2, -2C9, -2E1, and -3A. Phentermine also did not inhibit P450-2D6. However, D- and L-fenfluramine significantly inhibited P450-2D6 activity as measured by dextromethorphan O-demethylation, with mean 50% inhibitory concentrations (15.1 microM) within one order of magnitude of that for fluoxetine (2.7 microM). Findings from the in vitro assay are consistent with clinical studies showing significant inhibition of desipramine clearance by coadministration of fenfluramine.