Youssef Daali

Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety

Background and purpose:  The major drug‐metabolizing enzymes for the oxidation of oxycodone are CYP2D6 and CYP3A. A high interindividual variability in the activity of these enzymes because of genetic polymorphisms and/or drug–drug interactions is well established. The possible role of an active metabolite in the pharmacodynamics of oxycodone has been questioned and the importance of CYP3A‐mediated effects on the pharmacokinetics and pharmacodynamics of oxycodone has been poorly explored.

The AmpliChip CYP450 test: cytochrome P450 2D6 genotype assessment and phenotype prediction

Polymorphisms of the cytochrome P450 2D6 (CYP2D6) gene affecting enzyme activity are involved in interindividual variability in drug efficiency/toxicity. Four phenotypic groups are found in the general population: ultra rapid (UM), extensive (EM), intermediate (IM) and poor (PM) metabolizers. The AmpliChip CYP450 test is the first genotyping array allowing simultaneous analysis of 33 CYP2D6 alleles. The main aim of this study was to evaluate the performance of this test in CYP2D6 phenotype prediction. We first verified the AmpliChip CYP450 test genotyping accuracy for five CYP2D6 alleles routinely analysed in our laboratory (alleles 3,4,5,6, × N; n=100). Results confirmed those obtained by real-time PCR. Major improvements using the array are the detection of CYP2D6 intermediate alleles and identification of the duplicated alleles. CYP2D6 phenotype was determined by assessing urinary elimination of dextromethorphan and its metabolite dextrorphan and compared to the array prediction (n=165). Although a low sensitivity of UM prediction by genotyping was observed, phenotype prediction was optimal for PM and satisfying for EM and IM.

The effects of CYP2D6 and CYP3A activities on the pharmacokinetics of immediate release oxycodone

Background and purpose:  There is high interindividual variability in the activity of drug‐metabolizing enzymes catalysing the oxidation of oxycodone [cytochrome P450 (CYP) 2D6 and 3A], due to genetic polymorphisms and/or drug–drug interactions. The effects of CYP2D6 and/or CYP3A activity modulation on the pharmacokinetics of oxycodone remains poorly explored.

On-line desorption of dried blood spots coupled to hydrophilic interaction/reversed-phase LC/MS/MS system for the simultaneous analysis of drugs and their polar metabolites

An assay for the simultaneous analysis of pharmaceutical compounds and their metabolites from micro-whole blood samples (i.e. 5 microL) was developed using an on-line dried blood spot (on-line DBS) device coupled with hydrophilic interaction/reversed-phase (HILIC/RP) LC/MS/MS. Filter paper is directly integrated to the LC device using a homemade inox desorption cell. Without any sample pretreatment, analytes are desorbed from the paper towards an automated system of valves linking a zwitterionic-HILIC column to an RP C18 column. In the same run, the polar fraction is separated by the zwitterionic-HILIC column while the non-polar fraction is eluted on the RP C18. Both fractions are detected by IT-MS operating in full scan mode for the survey scan and in product ion mode for the dependant scan using an ESI source. The procedure was evaluated by the simultaneous qualitative analysis of four probes and their relative phase I and II metabolites spiked in whole blood. In addition, the method was successfully applied to the in vivo monitoring of buprenorphine metabolism after the administration of an intraperitoneal injection of 30 mg/kg on adult female Wistar rat.

Analgesics in Patients with Hepatic Impairment

The physiological changes that accompany hepatic impairment alter drug disposition. Porto-systemic shunting might decrease the first-pass metabolism of a drug and lead to increased oral bioavailability of highly extracted drugs. Distribution can also be altered as a result of impaired production of drug-binding proteins or changes in body composition. Furthermore, the activity and capacity of hepatic drug metabolizing enzymes might be affected to various degrees in patients with chronic liver disease. These changes would result in increased concentrations and reduced plasma clearance of drugs, which is often difficult to predict.The pharmacology of analgesics is also altered in liver disease. Pain management in hepatically impaired patients is challenging owing to a lack of evidence-based guidelines for the use of analgesics in this population. Complications such as bleeding due to antiplatelet activity, gastrointestinal irritation, and renal failure are more likely to occur with nonsteroidal anti-inflammatory drugs in patients with severe hepatic impairment. Thus, this analgesic class should be avoided in this population.The pharmacokinetic parameters of paracetamol (acetaminophen) are altered in patients with severe liver disease, but the short-term use of this drug at reduced doses (2 grams daily) appears to be safe in patients with nonalcoholic liver disease.The disposition of a large number of opioid drugs is affected in the presence of hepatic impairment. Certain opioids such as codeine or tramadol, for instance, rely on hepatic biotransformation to active metabolites. A possible reduction of their analgesic effect would be the expected pharmacodynamic consequence of hepatic impairment. Some opioids, such as pethidine (meperidine), have toxic metabolites. The slower elimination of these metabolites can result in an increased risk of toxicity in patients with liver disease, and these drugs should be avoided in this population.The drug clearance of a number of opioids, such as morphine, oxycodone, tramadol and alfentanil, might be decreased in moderate or severe hepatic impairment. For the highly excreted morphine, hydromorphone and oxycodone, an important increase in bioavailability occurs after oral administration in patients with hepatic impairment. Lower doses and/or longer administration intervals should be used when these opioids are administered to patients with liver disease to avoid the risk of accumulation and the potential increase of adverse effects. Finally, the pharmacokinetics of phenylpiperidine opioids such as fentanyl, sufentanil and remifentanil appear to be unaffected in hepatic disease. All opioid drugs can precipitate or aggravate hepatic encephalopathy in patients with severe liver disease, thus requiring cautious use and careful monitoring.

Automated system for on-line desorption of dried blood spots applied to LC/MS/MS pharmacokinetic study of flurbiprofen and its metabolite

This paper illustrates the development of an automated system for the on-line bioanalysis of dried blood spots (on-line DBS). In this way, a prototype was designed for integration into a conventional LC/MS/MS, allowing the successive extraction of 30 DBS toward the analytical system without any sample pretreatment. The developed method was assessed for the DBS analysis of flurbiprofen (FLB) and its metabolite 4-hydroxyflurbiprofen (OH-FLB) in human whole blood (i.e. 5 μL). The automated procedure was fully validated based on international criteria and showed good precision, trueness, and linearity over the expected concentration range (from 10 to 1000 ng/mL and 100 to 10,000 ng/mL for OH-FLB and FLB respectively). Furthermore, the prototype showed good results in terms of recovery and carry-over. Stability of both analytes on filter paper was also investigated and the results suggested that DBS could be stored at ambient temperature for over 1 month. The on-line DBS automated system was then successfully applied to a pharmacokinetic study performed on healthy male volunteers after oral administration of a single 50-mg dose of FLB. Additionally, a comparison between finger capillary DBS and classic venous plasma concentrations was investigated. A good correlation was observed, demonstrating the complementarity of both sampling forms. The automated system described in this article represents an efficient tool for the LC/MS/MS analysis of DBS samples in many bioanalytical applications.

NADPH Oxidase NOX2 Defines a New Antagonistic Role for Reactive Oxygen Species and cAMP/PKA in the Regulation of Insulin Secretion

In insulin-secreting cells, expression of NADPH oxidase (NOX), a potent source of ROS, has been reported, along with controversial findings regarding its function. Here, the role of NOXs was investigated: first by expression and cellular localization in mouse and human pancreatic islets, and then by functional studies in islets isolated from Nox isoform–specific knockout mice. Both human and mouse β-cells express NOX, in particular NOX2. With use of Nox isoform–specific knockout mice, functional analysis revealed Nox2 as the predominant isoform. In human islets, NOX2 colocalized with both insulin granules and endosome/lysosome membranes. Nox2-deficient islets stimulated with 22.8 mmol/L glucose exhibited potentiation of insulin release compared with controls, an effect confirmed with in vitro knockdown of Nox2. The enhanced secretory function in Nox2-deficient islets was associated with both lower superoxide levels and elevated cAMP concentrations. In control islets, GLP-1 and other cAMP inducers suppressed glucose-induced ROS production similarly to Nox2 deficiency. Inhibiting cAMP-dependent protein kinase reduced the secretory response in Nox2-null islets, although not in control islets. This study ascribes a new role for NOX2 in pancreatic β-cells as negative modulator of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation. Results also show reciprocal inhibition between the cAMP/PKA pathway and ROS.

Influence of the paraoxonase-1 Q192R genetic variant on clopidogrel responsiveness and recurrent cardiovascular events: a systematic review and meta-analysis

Summary.  Background:  A poor biological response to clopidogrel is associated with an increased risk of major cardiovascular ischemic events (MACE). Paraoxonase 1 (PON1) enzyme activity is modulated by the PON1‐Q192R variant (rs662) and was recently suggested to be strongly involved in clopidogrel bioactivation, but the influence of the PON1‐Q192R variant on the risk of MACE in clopidogrel‐treated patients is controversial.

HZ166, a novel GABAA receptor subtype-selective benzodiazepine site ligand, is antihyperalgesic in mouse models of inflammatory and neuropathic pain.

Diminished GABAergic and glycinergic inhibition in the spinal dorsal horn contributes significantly to chronic pain of different origins. Accordingly, pharmacological facilitation of GABAergic inhibition by spinal benzodiazepines (BDZs) has been shown to reverse pathological pain in animals as well as in human patients. Previous studies in GABA(A) receptor point-mutated mice have demonstrated that the spinal anti-hyperalgesic effect of classical BDZs is mainly mediated by GABA(A) receptors containing the α2 subunit (α2-GABA(A) receptors), while α1-GABA(A) receptors, which mediate the sedative effects, do not contribute. Here, we investigated the potential analgesic profile of HZ166, a new partial BDZ-site agonist with preferential activity at α2- and α3-GABA(A) receptors. HZ166 showed a dose-dependent anti-hyperalgesic effect in mouse models of neuropathic and inflammatory pain, triggered by chronic constriction injury (CCI) of the sciatic nerve and by subcutaneous injection of the yeast extract zymosan A, respectively. This antihyperalgesic activity was antagonized by flumazenil and hence mediated via the BDZ-binding site of GABA(A) receptors. A central site of action of HZ166 was consistent with its pharmacokinetics in the CNS. When non-sedative doses of HZ166 and gabapentin, a drug widely used in the clinical management of neuropathic pain, were compared, the efficacies of both drugs against CCI-induced pain were similar. At doses producing already maximal antihyperalgesia, HZ166 was devoid of sedation and motor impairment, and showed no loss of analgesic activity during a 9-day chronic treatment period (i.e. no tolerance development). These findings provide further evidence that compounds selective for α2- and α3-GABA(A) receptors might constitute a novel class of analgesics suitable for the treatment of chronic pain.

A cocktail approach for assessing the in vitro activity of human cytochrome P450s: An overview of current methodologies

An assessment of cytochrome P450 (CYP) enzyme activity is essential for characterizing the phase I metabolism of biological systems or to evaluate the inhibition/induction properties of xenobiotics. CYPs have generally been investigated individually by single probes, and metabolite formation has been monitored by liquid chromatography-mass spectrometry (LC-MS). To increase the throughput, many probes have been applied to assess multiple CYP activities simultaneously within a single experiment. This strategy is called the cocktail approach, and it has already been reviewed for in vivo applications, but never for in vitro ones. This review focuses for the first time on an in vitro cocktail approach, and it references the most notable articles on this topic. The advantages and limitations of applying cocktails for the in vitro activity assessment of major human CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and subfamily CYP3A, are discussed. This article considers the probe reaction selections for each CYP according to regulatory recommendations, probe metabolic properties (i.e., specificity and turnover), probe concentrations and analytical sensitivity, but it also highlights a challenge specific to cocktail design, which is probe-probe interaction. The last part of the review reports some methodologies for incubating these cocktails and discusses some important issues regarding the incubation time, enzyme concentrations and sample preparation.