The legalization of cannabis in many parts of the United States and other countries has led to a need for a more comprehensive understanding of cannabis constituents and their potential for drug-drug interactions. Although (−)- trans -Δ 9 -tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) are the most abundant cannabinoids present in cannabis, THC metabolites are found in plasma at higher concentrations and for a longer duration than that of the parent cannabinoids. To understand the potential for drug-drug interactions, the inhibition potential of major cannabinoids and their metabolites on major hepatic cytochrome P450 (P450) enzymes was examined. In vitro assays with P450-overexpressing cell microsomes demonstrated that the major THC metabolites 11-hydroxy-Δ 9 -tetra-hydrocannabinol and 11-nor-9-carboxy-Δ 9 -THC-glucuronide competitively inhibited several major P450 enzymes, including CYP2B6, CYP2C9, and CYP2D6 (apparent K i,u values = 0.086 ± 0.066 µM and 0.90 ± 0.54 µM, 0.057 ± 0.044 µM and 2.1 ± 0.81 µM, 0.15 ± 0.067 µM and 2.3 ± 0.54 µM, respectively). 11-Nor-9-carboxy-Δ 9 - tetrahydrocannabinol exhibited no inhibitory activity against any CYP450 tested. THC competitively inhibited CYP1A2, CYP2B6, CYP2C9, and CYP2D6; CBD competitively inhibited CYP3A4, CYP2B6, CYP2C9, CYP2D6, and CYP2E1; and CBN competitively inhibited CYP2B6, CYP2C9, and CYP2E1. THC and CBD showed mixed-type inhibition for CYP2C19 and CYP1A2, respectively. These data suggest that cannabinoids and major THC metabolites are able to inhibit the activities of multiple P450 enzymes, and basic static modeling of these data suggest the possibility of pharmacokinetic interactions between these cannabinoids and xenobiotics extensively metabolized by CYP2B6, CYP2C9, and CYP2D6. SIGNIFICANCE STATEMENT Major cannabinoids and their metabolites found in the plasma of cannabis users inhibit several P450 enzymes, including CYP2B6, CYP2C9, and CYP2D6. This study is the first to show the inhibition potential of the most abundant plasma cannabinoid metabolite, THC-COO-Gluc, and suggests that circulating metabolites of cannabinoids play an essential role in CYP450 enzyme inhibition as well as drug-drug interactions.

The UDP-glucuronosyltransferase (UGT) family of enzymes play a central role in the metabolism and detoxification of a wide range of endogenous and exogenous compounds. UGTs exhibit a high degree of structural similarity and display overlapping substrate specificity, often making estimations of potential drug-drug interactions difficult to fully elucidate. One such interaction yet to be examined may be occurring between UGTs and cannabinoids, as the legalization of recreational and medicinal cannabis and subsequent co-usage of cannabis and therapeutic drugs increases in the United States and internationally. In the present study, the inhibition potential of the major cannabinoids Δ 9 -tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), as well as their major metabolites, was determined in microsomes isolated from HEK293 cells overexpressing individual recombinant UGTs and in microsomes from human liver and kidney specimens. The highest inhibition was seen by CBD against the glucuronidation activity of UGTs 1A9, 2B4, 1A6, and 2B7, with binding-corrected IC 50 values of 0.12 ± 0.020 µM, 0.22 ± 0.045 µM, 0.40 ± 0.10 µM, and 0.82 ± 0.15 µM, respectively. Strong inhibition of UGT1A9 was also demonstrated by THC and CBN, with binding-corrected IC 50 values of 0.45 ± 0.12 μM and 0.51 ± 0.063 μM, respectively. Strong inhibition of UGT2B7 was also observed for THC and CBN; no or weak inhibition was observed with cannabinoid metabolites. This inhibition of UGT activity suggests that in addition to playing an important role in drug-drug interactions, cannabinoid exposure may have important implications in patients with impaired hepatic or kidney function. SIGNIFICANCE STATEMENT Major cannabinoids found in the plasma of cannabis users inhibit several UDP-glucuronosyltransferase (UGT) enzymes, including UGT1A6, UGT1A9, UGT2B4, and UGT2B7. This study is the first to show the potential of cannabinoids and their metabolites to inhibit all the major kidney UGTs as well as the two most abundant UGTs present in liver. This study suggests that as all three major kidney UGTs are inhibited by cannabinoids, greater drug-drug interaction effects might be observed from co-use of cannabinods and therapeutics that are cleared renally.

Background: The major mode of metabolism of nicotine is by hydroxylation via cytochrome P450 (CYP) 2A6, but it can also undergo glucuronidation by UDP-glucuronosyltransferases and oxidation by flavin monooxygenases (FMO). The goal of this study was to examine the potential importance of FMOs in nicotine metabolism and assess the potential impact of missense polymorphisms in active FMOs on nicotine-N 0oxide (NOX) formation. Methods: Urine samples from 106 current Chinese smokers were analyzed for nicotine metabolites by mass spectrometry. Wild-type FMOs 1-5 and their most prevalent nonsynonymous variants were cloned and overexpressed in HEK293 cells, and were tested in oxidation reactions against nicotine. Results: A strong inverse correlation was observed between the ratio of urinary 3 0-hydroxycotinine/cotinine, a measure of CYP2A6 activity, and the urinary levels of NOX alone (r ¼ À0.383; P < 0.001) or NOX measured as a ratio of total nicotine metabolites (r ¼ À0.414; P < 0.001) in smokers. In addition to FMO1 and FMO3, the functional FMO2 427Q isoform was active against nicotine, whereas FMO4 and FMO5 exhibited low activity against nicotine (K m > 5.0 mmol/L). Significant (P < 0.05) decreases in N 0-oxidation activity (V max /K m) were observed for the FMO1 I303V , FMO3 N61S , FMO3 D132H , FMO3 V257M , and FMO3 E308G variants in vitro when compared with their respective wild-type isoforms; the truncated FMO2 Q472stop isoform exhibited no enzyme activity. Conclusions: These data indicate that increases in nicotine-N 0-oxidation occur in subjects with deficient CYP2A6 activity, and that several FMO enzymes are active in nicotine-N 0oxidation. Impact: Several common missense FMO variants are associated with altered enzyme activity against nicotine and may play an important role in nicotine metabolism in low-CYP2A6 activity subjects.

Head and neck cancers are among the most common cancers in Southeast Asia and are likely to face sharp increases in the coming years due to the widespread use of smokeless tobacco (SLT) products. Bangladesh has the second highest oral cancer mortality rate as well as the second highest rate of SLT product usage worldwide. SLT products contain high levels of the highly carcinogenic tobacco‐specific nitrosamines (TSNAs); they include N‐nitrosonornicotine (NNN), 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), N‐nitrosoanatabine (NAT), and N‐nitrosoanabasine (NAB). To date, the amount of TSNAs present in Bangladeshi SLT products has not been reported. The goal of the present study was to develop an efficient TSNA and nicotine extraction method to quantify the levels of TSNAs and nicotine present in 31 Bangladeshi SLT products. The concentration of NNN, NNK, NAT, and NAB in Bangladeshi SLT brands ranged from 1.07–59.18 μg/g SLT, 0.15–33.70 μg/g SLT, 0.79–44.64 μg/g SLT, and 0.03–12.85 μg/g SLT, respectively. Pan Masala, which is labeled as a non‐tobacco product, also contains low levels of TSNAs. Betel nut, a common flavor additive in SLT products in Bangladesh, contained no detectible TSNA levels. Bangladeshi SLT products contained significantly higher (p<0.05) levels of TSNAs as compared to SLT brands from the United States (U.S.), and were higher on average than the levels observed in brands in other regions of southern Asia. NNN and NNK levels were 16 and 19, 2 and 3.5, and 62 and 85 fold higher than U.S., Indian and Pakistani SLT brands, respectively. Additionally, the levels of NNN and NNK measured in U.S. brands in this study were 1.2‐fold higher than previously reported values from 2013. This study demonstrates that the levels of TSNAs, including the major tobacco carcinogens NNN and NNK, are far higher in Bangladeshi SLT brands than they are in other regions of the world. Exposure to such high levels of carcinogens may be the most important contributing factor to the rate of head and neck as well as other tobacco‐related cancers observed in Bangladesh. Support or Funding Information This work is supported in part by grants from NIH, National Cancer Institute (grant R01‐ES025460, to P. Lazarus) and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292, to College of Pharmacy and Pharmaceutical Sciences, Washington State University) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cannabis-based products have experienced notable increases in co-usage alongside tobacco products. Several cannabinoids exhibit inhibition of a number of cytochrome P450 (CYP) and UDP glucuronosyltransferase (UGT) enzymes, but few studies have examined their inhibition of enzymes involved in nicotine metabolism. The goal of the present study was to examine potential drug–drug interactions occurring in the nicotine metabolism pathway perpetrated by cannabidiol (CBD) and its active metabolite, 7-hydroxy-CBD (7-OH-CBD). The inhibitory effects of CBD and 7-OH-CBD were tested in microsomes from HEK293 cells overexpressing individual metabolizing enzymes and from human liver tissue. Assays with overexpressing microsomes demonstrated that CBD and 7-OH-CBD inhibited CYP-mediated nicotine metabolism. Binding-corrected IC50,u values for CBD inhibition of nicotine metabolism to cotinine and nornicotine, and cotinine metabolism to trans-3′-hydroxycotinine (3HC), were 0.27 ± 0.060, 0.23 ± 0.14, and 0.21 ± 0.14 μM, respectively, for CYP2A6; and 0.26 ± 0.17 and 0.029 ± 0.0050 μM for cotinine and nornicotine formation, respectively, for CYP2B6. 7-OH-CBD IC50,u values were 0.45 ± 0.18, 0.16 ± 0.08, and 0.78 ± 0.23 μM for cotinine, nornicotine, and 3HC formation, respectively, for CYP2A6, and 1.2 ± 0.44 and 0.11 ± 0.030 μM for cotinine and nornicotine formation, respectively, for CYP2B6. Similar IC50,u values were observed in HLM. Inhibition (IC50,u = 0.37 ± 0.06 μM) of 3HC to 3HC-glucuronide formation by UGT1A9 was demonstrated by CBD. Significant inhibition of nicotine metabolism pathways by CBD and 7-OH-CBD suggests that cannabinoids may inhibit nicotine metabolism, potentially impacting tobacco addiction and cessation.