Exploring the role of mu opioid receptor (OPRM1) and CYP2B6 gene variations for methadone pharmacogenomics. Can these variations be used to advance toxicological interpretation post-mortem?.

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Bunten, Hannah;
  • Subject: aeo | med

Methadone is increasingly involved in drug overdose cases and the molecular actions of the drug in vivo are largely unknown requiring elucidation. This study set out to examine the relationship between methadone toxicity and CYP2B6 and mu (μ) opioid receptor (OPRM1) sin... View more
  • References (16)
    16 references, page 1 of 2

    1.1 Background……………………………………………………………………... 1.1.1 Study Aim and Objectives…………………………………………...

    1.2 Methadone pharmacology and mechanisms of action…………………………...

    1.3 Methadone safety profile (adverse drug reactions)……………………………

    1.4 Methadone-related deaths……………………………………………………...... 1.4.1 Cardio-toxicity………………………………………………………….. 1.4.2 Respiratory depression …………………………………………………. 1.4.3 Influence of drug-drug interactions…………………………………….. 1.4.4 Drug Tolerance…………………………………………………………..

    1.5 Toxicological interpretation of methadone cases……………………………….. 1.5.1 Post-mortem redistribution……………………………………………… 1.5.2 Individual differences in drug metabolism………………………………

    1.6 Pharmacogenomics ……………………………………………………………... 1.6.1 Toxicological interpretation and pharmacogenomics…………………… 1.6.2 Pharmacogenomic influences on methadone metabolism………………. 1.6.3 Pharmacogenomic influences on methadone-receptor binding and response………………………………………………………………….

    1.7 Summary………………………………………………………………………...

    2.2 Control Sample Examination………………………………………………….. 2.2.1 DNA Extraction from buccal swabs………………………………… 2.2.2 Buccal DNA Quantification………………………………………… 2.2.3 A118G Genotyping………………………………………………….. 2.3.3.1 Software set-up…………………………………………………… 2.3.3.2 Sample preparation……………………………………………….. 2.2.4 T750C Genotyping………………………………………………….. 2.3.4.1 Software set-up…………………………………………………… 2.3.4.2 Sample preparation……………………………………………….. 2.2.5 G516T Genotyping………………………………………………….. 2.3.5.1 Software set-up…………………………………………………… 2.3.5.2 Sample preparation……………………………………………….. 2.2.6 A785G Genotyping………………………………………………….. 2.3.6.1 Software set-up…………………………………………………….. 2.3.6.2 Sample preparation…………………………………………………

    2.3 CYP2B6 Cloning………………………………………………………………... 2.3.1 Primary Cloning strategy……………………………………………. 2.3.2 Plasmid DNA preparation……………………………………………

    3.1 Method Optimisation………………………………………………………….... 3.1.1 DNA Extraction……………………………………………………... 3.1.1.1 DNA extraction from buccal swabs……………………………….. 3.1.1.2 DNA extraction from whole blood………………………………... 3.1.2 PCR Amplification of SNP regions of the human OPRM1 gene …... 3.1.2.1 MgCl2……………………………………………………………… 3.1.2.2 Temperature………………………………………………………. 3.1.2.2.1 Gradient PCR reaction………………………………………. 3.1.2.2.2 Temperatures selected from the Gradient Reaction…………. 3.1.2.2.3 Temperature optimisation to increase specificity…………… 3.1.2.2.4 SNPs revealed by DNA sequencing results…………………. 3.1.2.3 A118G identification………………………………………………. 3.1.2.3.1 A118G sequencing results…………………………………... 3.1.3 PCR Amplification of SNP regions of the human CYP2B6 gene…. 3.1.3.1 Primer validation…………………………………………………. 3.1.3.2 T750C PCR validation……………………………………………. 3.1.3.2.1 Temperature…………………………………………………. 3.1.3.2.2 MgCl2………………………………………………………... 3.1.3.3 T750C Identification……………………………………………… 3.1.3.3.1 T750C sequencing results…………………………………… 3.1.3.4 G516T PCR validation…………………………………………… 3.1.3.4.1 Temperature…………………………………………………. 3.1.3.4.2 MgCl2………………………………………………………... 3.1.3.5 G516T Identification…………………………………………….. 3.1.3.5.1 G516T sequencing results……………………………………. 3.1.3.6 A785G validation…………………………………………………. 3.1.3.6.1 Temperature………………………………………………….. 3.1.3.7 A785G Identification…………………………………………….. 3.1.3.7.1 A785G sequencing results…………………………………….

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