Date of Award

2019

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Dr. Gary O. Rankin, Committee Chairperson

Second Advisor

Dr. Monica A. Valentovic

Third Advisor

Dr. Donald A. Primerano

Fourth Advisor

Dr. Travis B. Salisbury

Fifth Advisor

Dr. Richard D. Egleton

Abstract

Methadone is a synthetic, long-acting opioid prescribed as an analgesic for chronic pain. It has a single chiral center forming two enantiomers, (R)-methadone and (S)-methadone, each having specific pharmacological actions. Concentrations of (R)- and (S)-methadone above therapeutic levels have the ability to cause serious, life-threatening, and fatal side effects. Cardiotoxicity is caused by elevated (S)-methadone levels by prolonging the QT interval of the heart’s electrical cycle. In 2014, methadone accounted for only 1% of all opioids prescribed for pain, but was responsible for 3,400 of the 14,838 individuals (~23%) who died in the United States from overdoses due to prescription opioids. West Virginia (WV) and Kentucky (KY) continue to have significant increases in overdose death rates involving prescription opioids. These overdoses could be due in part to the pharmacogenetics of an individual, encompassing the ability to influence the pharmacokinetic and pharmacodynamic properties of methadone, contributing to toxicity. The principle determinant of the large interindividual variability of methadone pharmacokinetics is metabolism, causing a discordance in the relationship between dose, plasma concentrations, and side effects. Single nucleotide polymorphisms (SNPs) within the genes of drug metabolizing enzymes, cytochrome P450 (CYP), may be important contributing factors in altering CYP activity, methadone metabolism and pharmacodynamics. (S)-Methadone is stereoselectively metabolized in the liver by CYP2B6. The purpose of this study was to determine if one or more SNPs located within the CYP2B6 gene contributes to a poor metabolizer phenotype for unexpected methadone deaths. A study cohort was obtained from the WV and KY Offices of the Chief Medical Examiner consisting of 125 Caucasians who suffered methadone-only fatal overdoses. Genotypic frequency of three intronic SNPs (rs2279344, rs4803419, and rs8192719) and five exonic SNPs (rs3211371, rs3745274, rs8192709, rs12721655, and rs35979566) were investigated. The differences between the observed genotypic frequencies were compared to the regional control group of 255 deceased Caucasian individuals. The frequencies of the exonic SNP rs3745274 and intronic SNP rs8192719 were enhanced in the methadone-only group. Higher blood methadone concentrations were observed in individuals who were genotyped homozygous for SNP rs3211371. The five exonic SNPs genotyped, and two others (rs2279343 and rs35773040), were individually expressed in COS-1 cells. The effect of these SNPs on the catalytic activity of the CYP2B6 enzyme was assessed by evaluating the luminescence generated from the metabolism of luminogenic luciferin-2B6 substrate. Additionally, inhibition studies were also conducted using clopidogrel to determine that the activity observed was from the CYP2B6 enzyme. Methadone was used to evaluate competitive inhibition with luciferin-2B6 substrate at the active site. The effect on CYP2B6 activity by introducing individual SNP variants resulted in the following decreasing order: rs2279343 > rs3745274 > wild type (CYP2B6*1), rs3211371 > rs8192709 > rs35773040, rs35979566. Relative to the wild type CYP2B6, SNPs rs8192709, rs35773040, and rs35979566 yielded 57%, 81%, and 94% decreased activity, respectively. Methadone inhibited the activity of the CYP2B6 enzyme by displacing the luciferin-2B6 substrate. Therefore, our investigations suggest that the differences in catalytic activity by CYP2B6 allelic variants rs8192709, rs35773040, and rs35979566 result in poor metabolizing phenotypes which may be contributing factors in decreased metabolism of (S)-methadone. Because CYP2B6 only plays a partial role in the metabolism of (S)-methadone, additional knowledge on the combination of SNPs on CYP2B6, together with SNPs on other methadone metabolizing CYP genes, can improve the proper therapeutic dosing for methadone, patient outcome, and the development of individualized medicine.

Subject(s)

Pharmacogenetics.

Cardiology -- Research.

Methadone -- Research.

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