High-resolution melting curve analysis (HRM) of polymerase chain reaction (PCR) amplicons has been described as a fast, cheap, and reliable closed-tube method of genotyping with no need for labeled primers or labeled probes. We adapted this melting analysis assay for the detection of the most common nonfunctional alleles of cytochrome P-450 (CYP) 2D6 in the Caucasian population that affect the metabolism of many commonly used drugs. We used this method to genotype 91 patients under paroxetine therapy. The presence and the constitution of the most common single-nucleotide polymorphisms (1846G>A, 2988G>A, 100C>T, 2549delA, 2615_2617delAAG, and 1707delT) in poor and intermediate metabolizers from the Caucasian population were detected in short amplicons (≤148 bp). After fluorescence normalization, the wild-type, homozygous, and heterozygous samples were easily distinguishable from each other by their specific melting curve shape. A total of 92.6% of the 1846G>A heterozygotes, 96% of the 100C>T heterozygotes, and 100% of the 2988G>A, 2549delA, 2615_2617delAAG, and 1707delT heterozygotes have been correctly distinguished from the wild types. One hundred percent of all the homozygotes in this group of patients have been detected without any error. HRM of short amplicons is a simple tool for effective, rapid, and reliable CYP2D6 genotyping that does not require real-time PCR, labeled probes, processing or any separations after PCR. The reaction is performed in a closed-tube system and is highly specific and sensitive. We proved that this technique is highly reliable for use in routine diagnostics.
- MeSH
- alely MeSH
- běloši genetika MeSH
- cytochrom P-450 CYP2D6 genetika metabolismus MeSH
- DNA primery genetika MeSH
- frekvence genu MeSH
- genotypizační techniky metody MeSH
- heterozygot MeSH
- homozygot MeSH
- jednonukleotidový polymorfismus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
WHAT IS KNOWN AND OBJECTIVE: Paroxetine is both a substrate and an inhibitor of CYP2D6. The objective of the presented study was to determine the persistence of CYP2D6 inhibition after short term (6 weeks) and long term (18·7 ± 10·6 weeks) paroxetine treatment. METHODS: Two the studies consisted of 30 depressive/anxiety patients each. In the first study, patients were subdivided into three groups treated with paroxetine (A1), alprazolam (A2) and paroxetine + alprazolam (A3). After 6 weeks, all the patients (A1+A2+A3) were switched to alprazolam treatment; metabolic activity was evaluated at the beginning, after 6 weeks of paroxetine/alprazolam/alprazolam + paroxetine treatment (A1/A2/A3) and 4 weeks after the switch to alprazolam treatment (Week 0, 6, 10). In the second study patients on previous long term paroxetine treatment were subdivided into two groups treated with mirtazapine (B1) or paroxetine (B2); metabolic activity of CYP2D6 was evaluated at the beginning and after 6 weeks of therapy. RESULTS AND DISCUSSION: Metabolic ratio of dextromethorphan to dextrorphan has normalized in all subjects after 4 weeks of paroxetine wash out in the first study. In the second study, 6 weeks after paroxetine discontinuation, restoration of metabolic activity of CYP2D6 was observed in only five of eight originally poor metabolizers. WHAT IS NEW AND CONCLUSION: We conclude that a wash-out period of 4 weeks seems to be sufficient for CYP2D6 disinhibition after short-term paroxetine treatment (6 weeks). On the other hand, treatment with a CYP2D6 substrate less than 6 weeks after long-term paroxetine treatment (18·7 weeks on average) could result in elevated drug plasma levels and occasionally also in drug toxicity.
- MeSH
- alprazolam farmakologie MeSH
- cytochrom P-450 CYP2D6 metabolismus MeSH
- deprese farmakoterapie enzymologie metabolismus MeSH
- dextromethorfan farmakologie MeSH
- dextrorfan farmakologie MeSH
- dospělí MeSH
- dvojitá slepá metoda MeSH
- inhibitory cytochromu P450 CYP2D6 MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- paroxetin farmakologie MeSH
- senioři MeSH
- úzkost farmakoterapie enzymologie metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- randomizované kontrolované studie MeSH
WHAT IS KNOWN AND OBJECTIVE: Accurate prediction of actual CYP2D6 metabolic activity may prevent some adverse drug reactions and improve therapeutic response in patients receiving CYP2D6 substrates. Dextromethorphan-to-dextrorphan metabolic ratio (MR(DEM/DOR)) is well established as a marker of CYP2D6 metabolizer status. The relationship between urine and plasma or serum MR(DEM/DOR) is not well established nor is there evidence of antimode for separation of intermediate and especially poor metabolizers (PM) from extensive metabolizers (EM). This study addressed whether CYP2D6 phenotyping using molar metabolic ratio of dextromethorphan to dextrorphan (MR(DEM/DOR)) in serum is usable and reliable in clinical practice as urinary MR(DEM/DOR). METHODS: We measured MR(DEM/DOR) in serum and CYP2D6 genotype in 51 drug-naive patients and 30 volunteers. Receiver-operator characteristic (ROC) analysis was used for the evaluation of optimum cut-off value for discriminating between extensive, intermediate and PM. In addition, we studied the correlation of serum MR(DEM/DOR) with urine MR(DEM/DOR) in the 30 healthy volunteers. RESULTS AND DISCUSSION: A trimodal distribution of log MR(DEM/DOR) in serum was observed, with substantial overlap between extensive and intermediate metabolizer groups. We obtained an acceptable cut-off serum MR(DEM/DOR) value to discriminate between PM and either extensive or extensive + intermediate metabolizers. Using serum MR(DEM/DOR), it seems to be unreliable to discriminate EM from intermediate metabolizers (IM). A strong correlation between serum MR(DEM/DOR) and urine MR(DEM/DOR) was found. WHAT IS NEW AND CONCLUSION: Serum MR(DEM/DOR) (3 h) correlated with MR(DEM/DOR) in urine (0-8 h). Serum MR(DEM/DOR) discriminated between extensive and PM and between extensive + intermediate and PM. Our CYP2D6 phenotyping using serum dextromethorphan/dextrorphan molar ratio appears reliable but requires independent validation.
- MeSH
- cytochrom P-450 CYP2D6 genetika MeSH
- dextromethorfan aplikace a dávkování farmakokinetika MeSH
- dextrorfan farmakokinetika MeSH
- dospělí MeSH
- fenotyp MeSH
- genotyp MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- ROC křivka MeSH
- senioři MeSH
- studie případů a kontrol MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- klinické zkoušky MeSH
- práce podpořená grantem MeSH
