Veronique Michaud

The Dual Role of Pharmacogenetics in HIV Treatment: Mutations and Polymorphisms Regulating Antiretroviral Drug Resistance and Disposition

Significant intra- and interindividual variability has been observed in response to use of pharmacological agents in treatment of HIV infection. Treatment of HIV infection is limited by high rates of adverse drug reactions and development of resistance in a significant proportion of patients as a result of suboptimal drug concentrations. The efficacy of antiretroviral therapy is challenged by the emergence of resistant HIV-1 mutants with reduced susceptibility to antiretroviral drugs. Moreover, pharmacotherapy of patients infected with HIV is challenging because a great number of comorbidities increase polypharmacy and the risk for drug-drug interactions. Drug-metabolizing enzymes and drug transporters regulate drug access to the systemic circulation, target cells, and sanctuary sites. These factors, which determine drug exposure, along with the emergence of mutations conferring resistance to HIV medications, could explain variability in efficacy and adverse drug reactions associated with antiretroviral drugs. In this review, the major factors affecting the disposition of antiretroviral drugs, including key drug-metabolizing enzymes and membrane drug transporters, are outlined. Genetic polymorphisms affecting the activity and/or the expression of cytochromes P450 or UGT isozymes and membrane drug transport proteins are highlighted and include such examples as the association of neurotoxicity with efavirenz, nephrotoxicity with tenofovir, hepatotoxicity with nevirapine, and hyperbilirubinemia with indinavir and atazanavir. Mechanisms of drug resistance conferred by specific viral mutations are also reviewed, with particular attention to replicative viral fitness and transmitted HIV drug resistance with the objectives of providing a better understanding of mechanisms involved in HIV drug resistance and helping health care providers to better manage interpatient variability in drug efficacy and toxicity.

Factors predisposing to coma and delirium: fentanyl and midazolam exposure; CYP3A5, ABCB1, and ABCG2 genetic polymorphisms; and inflammatory factors.

Background:Delirium and sedative-induced coma are described as incremental manifestations of cerebral dysfunction. Both may be associated with sedative or opiate doses and pharmacokinetic or pharmacogenetic variables, such as drug plasma levels (exposure), drug metabolism, and/or their transport across the blood-brain barrier. Objectives:To compare biological and drug treatment characteristics in patients with coma and/or delirium while in the ICU. Patients and Measurements:In 99 patients receiving IV fentanyl, midazolam, or both, we evaluated drug doses, covariates likely to influence drug effects (age, body mass index, and renal and hepatic dysfunction); delirium risk factors; concomitant administration of CYP3A and P-glycoprotein substrates/inhibitors; ABCB1, ABCG2, and CYP3A5 genetic polymorphisms; and fentanyl and midazolam plasma levels. Delirium and coma were evaluated daily. In patients with only coma (n = 15), only delirium (n = 7), and neither ever (n = 14), we measured plasma levels of tumor necrosis factor-&agr;, interleukin (IL)-1&bgr;, IL-1RA, IL-6, IL-8, IL-10, IL-17,macrophage inflammatory protein-1&bgr;, and monocyte chemotactic protein-1. Results:Time to first coma was associated with fentanyl and midazolam doses (p = 0.03 and p = 0.01, respectively). The number of days in coma was associated with the number of days of coadministration of CYP3A inhibitors (r = 0.30; p = 0.006). Plasma levels of fentanyl were higher in patients with clinical coma (3.7 ± 4.7 vs. 2.0 ± 1.8 ng/mL, p = 0.0001) as were midazolam plasma levels (1050 ± 2232 vs. 168 ± 249 ng/mL, p = 0.0001). Delirium occurrence was unrelated to midazolam administration, cumulative doses, or serum levels. Days with delirium were associated with days of coadministration of P-glycoprotein inhibitor (r = 0.35; p = 0.0004). Delirious patients had higher levels of the inflammatory mediator IL-6 than comatose patients (129.3 vs. 35.0 pg/mL, p = 0.05). Conclusions:Coma is associated with fentanyl and midazolam exposure; delirium is unrelated to midazolam and may be linked to inflammatory status. These data suggest that iatrogenic coma and delirium are not mechanistically linked.

HIV-1 subtype B and C integrase enzymes exhibit differential patterns of resistance to integrase inhibitors in biochemical assays.

Background:Because of high intersubtype HIV-1 genetic variability, it has been shown that subtype-specific patterns of resistance to antiretroviral drugs exist. We wished to ascertain whether this might be true for integrase inhibitors. Methods:We compared the susceptibility of subtype B and C HIV-1 integrase enzymes, harboring the previously reported resistance mutations E92Q, N155H, and E92Q/N155H, to clinically relevant integrase inhibitors. This was performed biochemically using a microtiter plate system. Results:Subtype C integrase enzymes bearing the resistance mutations E92Q/N155H were approximately 10-fold more susceptible to each of two integrase inhibitors, raltegravir and elvitegravir, than were subtype B recombinant integrase containing the same mutations. Conclusion:Polymorphic differences within the subtype B and C integrase genes likely cause variations in the contribution of N155H alone or in combination with E92Q to drug resistance. It is possible that different viral subtypes may favor different mutational pathways, potentially leading to varying levels of drug resistance among different subtypes.

Combination of Phenotype Assessments and CYP2C9‐VKORC1 Polymorphisms in the Determination of Warfarin Dose Requirements in Heavily Medicated Patients

The relative contribution of phenotypic measures and CYP2C9‐vitamin K epoxide reductase complex subunit 1 (VKORC1) polymorphisms to warfarin dose requirements at day 14 was determined in 132 hospitalized, heavily medicated patients. Phenotypic measures were (1) the urinary losartan metabolic ratio before the first dose of warfarin, (2) the S:R‐warfarin ratio at day 1, and (3) a dose‐adjusted international normalized ratio (INR) at day 4. CYP2C9 and VKORC1 genotypes were determined by gene chip analysis. In multivariate analyses, the dose‐adjusted INR at day 4 explained 31% of variability observed in warfarin doses at day 14, whereas genotypic measures (CYP2C9‐VKORC1) contributed 6.5%. When S:R‐warfarin ratio was used, genotypes contributed more significantly (23.5%). Finally, urinary losartan metabolic ratio was of low predictive value. The best models obtained explained 51% of intersubject variability in warfarin dose requirements. Thus, combination of a phenotypic measure to CYP2C9‐VKORC1 genotypes represents a useful strategy to predict warfarin doses in patients receiving multiple drugs (11±4 drugs/day).

Metabolic Activity and mRNA Levels of Human Cardiac CYP450s Involved in Drug Metabolism

Background Tissue-specific expression of CYP450s can regulate the intracellular concentration of drugs and explain inter-subject variability in drug action. The overall objective of our study was to determine in a large cohort of samples, mRNA levels and CYP450 activity expressed in the human heart. Methodology CYP450 mRNA levels were determined by RTPCR in left ventricular samples (n = 68) of explanted hearts from patients with end-stage heart failure. Samples were obtained from ischemic and non-ischemic hearts. In some instances (n = 7), samples were available from both the left and right ventricles. A technique for the preparation of microsomes from human heart tissue was developed and CYP450-dependent activity was determined using verapamil enantiomers as probe-drug substrates. Principal Findings Our results show that CYP2J2 mRNA was the most abundant isoform in all human heart left ventricular samples tested. Other CYP450 mRNAs of importance were CYP4A11, CYP2E1, CYP1A1 and CYP2C8 mRNAs while CYP2B6 and CYP2C9 mRNAs were present at low levels in only some of the hearts analyzed. CYP450 mRNAs did not differ between ischemic and non-ischemic hearts and appeared to be present at similar levels in the left and right ventricles. Incubation of verapamil with heart microsomes led to the formation of nine CYP450-dependent metabolites: a major finding was the observation that stereoselectivity was reversed compared to human liver microsomes, in which the R-enantiomer is metabolized to a greater extent. Conclusions This study determined cardiac mRNA levels of various CYP450 isozymes involved in drug metabolism and demonstrated the prevalent expression of CYP2J2 mRNA. It revealed that cardiomyocytes can efficiently metabolize drugs and that cardiac CYP450s are highly relevant with regard to clearance of drugs in the heart. Our results support the claim that drug metabolism in the vicinity of a drug effector site can modulate drug effects.

Assessing drug distribution in tissues expressing P-glycoprotein through physiologically based pharmacokinetic modeling: model structure and parameters determination

BackgroundThe expression and activity of P-glycoproteins due to genetic or environmental factors may have a significant impact on drug disposition, drug effectiveness or drug toxicity. Hence, characterization of drug disposition over a wide range of conditions of these membrane transporters activities is required to better characterize drug pharmacokinetics and pharmacodynamics. This work aims to improve our understanding of the impact of P-gp activity modulation on tissue distribution of P-gp substrate.MethodsA PBPK model was developed in order to examine activity and expression of P-gp transporters in mouse brain and heart. Drug distribution in these tissues was first represented by a well-stirred (WS) model and then refined by a mechanistic transport-based (MTB) model that includes P-gp mediated transport of the drug. To estimate transport-related parameters, we developed an original three-step procedure that allowed extrapolation of in vitro measurements of drug permeability to the in vivo situation. The model simulations were compared to a limited set of data in order to assess the model ability to reproduce the important information of drug distributions in the considered tissues.ResultsThis PBPK model brings insights into the mechanism of drug distribution in non eliminating tissues expressing P-gp. The MTB model accounts for the main transport mechanisms involved in drug distribution in heart and brain. It points out to the protective role of P-gp at the blood-brain barrier and represents thus a noticeable improvement over the WS model.ConclusionBeing built prior to in vivo data, this approach brings an interesting alternative to fitting procedures, and could be adapted to different drugs and transporters.The physiological based model is novel and unique and brought effective information on drug transporters.

Clinical Practice Recommendations on Genetic Testing of CYP2C9 and VKORC1 Variants in Warfarin Therapy.

Objective: To systematically review evidence on genetic variants influencing outcomes during warfarin therapy and provide practice recommendations addressing the key questions: (1) Should genetic testing be performed in patients with an indication for warfarin therapy to improve achievement of stable anticoagulation and reduce adverse effects? (2) Are there subgroups of patients who may benefit more from genetic testing compared with others? (3) How should patients with an indication for warfarin therapy be managed based on their genetic test results? Methods: A systematic literature search was performed for VKORC1 and CYP2C9 and their association with warfarin therapy. Evidence was critically appraised, and clinical practice recommendations were developed based on expert group consensus. Results: Testing of VKORC1 (−1639G>A), CYP2C9*2, and CYP2C9*3 should be considered for all patients, including pediatric patients, within the first 2 weeks of therapy or after a bleeding event. Testing for CYP2C9*5, *6, *8, or *11 and CYP4F2 (V433M) is currently not recommended. Testing should also be considered for all patients who are at increased risk of bleeding complications, who consistently show out-of-range international normalized ratios, or suffer adverse events while receiving warfarin. Genotyping results should be interpreted using a pharmacogenetic dosing algorithm to estimate the required dose. Significance: This review provides the latest update on genetic markers for warfarin therapy, clinical practice recommendations as a basis for informed decision making regarding the use of genotype-guided dosing in patients with an indication for warfarin therapy, and identifies knowledge gaps to guide future research.

Domperidone and sudden cardiac death: How much longer should we wait?

In this issue of the Journal, Luc M. Hondeghem reports on the risk of sudden cardiac death with the use of the dopamine receptor blocker, domperidone. His article contains 3 following parts: (1) confirmatory studies to demonstrate block of the rapid component of the delayed rectifier potassium current (IKr; HERG) by domperidone; (2) reports on 5 population-based studies showing an increase in the odds ratio for sudden cardiac death in patients treated with domperidone; and (3) comments on studies suggesting poor gastrointestinal benefits associated with the use of domperidone. Block of IKr (HERG) by domperidone was first demonstrated in 2000, when the drug was considered to be “the” alternative to cisapride for treating gastrointestinal disorders. Indeed, at the time, several regulatory agencies had just restricted the use of cisapride or removed it from the market because of drug-induced long QT syndrome (LQTS), torsades de pointes, and sudden cardiac death. Domperidone was a proposed alternative to cisapride even though several reports had already seemed indicating the same potential side effects and risk with it. Using in vitro systems, we showed that domperidone has cardiac electrophysiological effects similar to those of cisapride, although the inhibitory constant for block of IKr was 10-fold lower for cisapride (14 nM) than for domperidone (150 nM). 1,9 The recent studies of Hondeghem—with longer equilibration times, longer cycle lengths, and a different animal model—suggest that the IC50 for block of IKr by domperidone is even lower in the range of 57 nM. Hondeghem rightly notes in his article that an IC50 for block of IKr becomes relevant if plasma concentrations can reach or approach this concentration. For instance, pharmacokinetic studies with domperidone suggest that plasma levels reach 50–120 nM after a 10 or 20 mg oral dose of domperidone. Hence, the safety margin with this drug looks rather small and would predict significant QT prolongation and proarrhythmic events in almost all patients treated with domperidone. The same is also true for other QT prolonging drugs with similar safety margins such as terfenadine, astemizole, cisapride, or moxifloxacin. In reality, however, this is not observed clinically. Indeed, an estimate of 1:120,000 patients experience drug-induced torsades de pointes and observed QT prolongation is in the range of 5–10 ms, a value almost undetectable in clinical practice. In this light, other factors should also be considered when estimating the risk of LQTS with orally administered drugs. The first factor is drug bioavailability. Domperidone has a bioavailability of 17%, indicating that only a fraction of the dose reaches the systemic circulation. But, under conditions of complete inhibition of drug metabolism during the first pass (intestine and liver), plasma concentrations (Cmax) of domperidone could rise up to 6-fold. Under these conditions, Cmax could be as high as 300–600 nM after a 10 or 20 mg dose. The second factor is domperidone metabolism. We and others conducted drug metabolism studies and demonstrated that CYP3A4 and CYP3A5 are the major enzymes involved in the metabolism of domperidone in the liver. These enzymes control access of the drug to the systemic circulation and determine its residence time. Coadministration of potent CYP3A inhibitors such as clarithromycine or ketoconazole have been shown to increase domperidone plasma Cmax, thus increasing the risk of LQTS.

Inhibitory effects of propafenone on the pharmacokinetics of caffeine in humans.

CYP1A2 is involved in the metabolism of both caffeine and propafenone, a class Ic antiarrhythmic agent. Despite the widespread consumption of caffeine, drug-drug interactions with this agent are often overlooked. This study investigated effects of propafenone on the pharmacokinetics of caffeine. Eight healthy volunteers were included in our study. A total of 300 mg of caffeine was given on 2 occasions, once alone and once during the coadministration of 300 mg propafenone. Serial blood samples were collected and pharmacokinetic parameters were estimated using a population pharmacokinetic approach. A one-compartment PK model with first-order absorption and elimination described plasma concentration profiles. Concomitant administration of propafenone decreased caffeine oral clearance from 8.3 ± 0.9 L/h to 5.4 ± 0.7 L/h (P < 0.05). Elimination half-life of caffeine was also increased 54% by propafenone. One of our volunteers was a poor metabolizer of CYP2D6. Concomitant administration of propafenone to this volunteer caused the greatest increase in caffeine plasma concentrations. These results support the concept of competitive inhibition between propafenone and caffeine. Our results suggest that propafenone causes significant inhibition of CYP1A2 activity leading to a decrease in the clearance of caffeine. Caffeine has intrinsic proarrhythmic effects; thus, its coadministration with an antiarrhythmic agent such as propafenone should be used with caution, especially in patients with poor CYP2D6 activity.

Highly sensitive LC–MS/MS methods for the determination of seven human CYP450 activities using small oral doses of probe-drugs in human

Cocktails composed of several Cytochrome P450 (CYP450)-selective probe drugs have been shown of value to characterize in vivo drug-metabolism activities. Our objective was to develop and validate highly sensitive and selective LC-MS/MS assays allowing the determination of seven major human CYP450 isoenzyme activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. The seven-drug cocktail was composed of caffeine, bupropion, tolbutamide, omeprazole, dextromethorphan, midazolam (all administered concomitantly) and chlorzoxazone (administered separately) to phenotype for CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1, respectively. Serial plasma and urine samples were collected over an 8h period. The probe-drugs and their respective metabolites were measured in both human plasma and urine, except for omeprazole (plasma only) and chlorzoxazone (urine only). Samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS) using a Phenomenex Luna PFP (2) analytical column (3μm PFP(2) 150×3mm) for chromatographic separation. Optimal detection was achieved based on 3 different analytical methods; (1) isocratic elution with a mobile phase consisting of acetonitrile and water both fortified with 0.01% formic acid for the analysis of bupropion, tolbutamide, chlorzoxazone and their respective metabolites; (2) isocratic elution with a mobile phase composed of acetonitrile and ammonium formate (pH 3; 10mM) for omeprazole, dextromethorphan, midazolam and their metabolites; (3) for caffeine and paraxanthine, gradient elution using acetonitrile and 0.01% formic acid in water was used. All calibration functions were linear for all probe drugs and metabolites in both matrices over wide analytical ranges. The main advantages of our methods are the use of specific probe drugs available in most countries, the administration of small doses of probe drugs, small volume of plasma required for the analyses and simple and rapid extraction procedures. The methods met all requirements of specificity, sensitivity, linearity, precision and accuracy and stability generally accepted in bioanalytical chemistry. Determination of CYP450 phenotype in patients will permit characterization of their capacities to metabolize drugs through CYP450 under specific conditions at a definite time. This tool will be highly clinically relevant since wide intersubject variability observed in drug response is largely explained by variation in drug metabolism; it will be particularly useful in polymedicated patients with multiple comorbidities. So far, our CYP450 cocktail assays have been successfully applied to phenotype CYP450 activities in patients.