The Intoxication Equivalency of 11-Hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) Relative to Δ9-tetrahydrocannabinol (THC).

Δ9-tetrahydrocannabinol (THC) is a psychoactive phytocannabinoid found in the Cannabis sativa plant. THC is primarily metabolized into 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (COOH-THC), that may themselves be psychoactive. There is very little research-based evidence concerning the pharmacokinetics and pharmacodynamics of 11-OH-THC as an individual compound. Male C57BL/6 mice were treated with THC or 11-OH-THC via i.p injection, tail vein i.v injection, or oral gavage (p.o), and whole blood compound levels were measured to determine pharmacokinetic parameters (Cmax, Tmax, t1/2, AUC, Vd, ClS, k and F) while also monitoring changes in catalepsy, body temperature, and nociception. 11-OH-THC achieved Tmax at 30 min for all routes of administration. The maximum concentration at 30 min was not different between i.v. and i.p. routes, but the p.o. Cmax was significantly lower. THC had a 10 min time to the maximum concentration - which was the first blood collection time point - for i.v. and i.p., and 60 min for p.o. with a lower Cmax for i.p. and p o. compared to i.v When accounting for circulating compound levels and ED50 responses, these data suggest that 11-OH-THC was 153% as active as THC in the tail-flick test of nociception, and 78% as active as THC for catalepsy. Therefore, 11-OH-THC displayed equal or greater activity than the parent compound THC, even when accounting for PK differences. Thus, the THC metabolite 11-OH-THC likely plays a critical role in the bioactivity of cannabis; understanding its activity when administered directly will aid in the interpretation of future animal and human studies. Significance Statement In this study we establish that the primary metabolite of THC - 11-OH-THC - displays equal or greater activity than THC in a mouse model of cannabinoid activity when directly administered and even when accounting for route of administration, sex, pharmacokinetic, and pharmacodynamic differences. These data provide critical insight into the bioactivity of THC metabolites that will inform the interpretation of future cannabinoid research and represent a model for how THC consumption and metabolism may affect cannabis use in humans.

Central Nervous System Distributional Kinetics of Selected Histone Deacetylase Inhibitors.

Histone deacetylase expression and activity are often dysregulated in central nervous system (CNS) tumors, providing a rationale for investigating histone deacetylase inhibitors (HDACIs) in selected brain tumor patients. Although many HDACIs have shown potential in in vitro studies, they have had modest efficacy in vivo This lack of activity could be due to insufficient CNS exposure to the unbound drug. In this study, we investigated the systemic pharmacokinetics and subsequent CNS distribution of two potent HDACIs, vorinostat and quisinostat, in the murine model. Both compounds undergo in vitro degradation in mouse plasma, requiring precautions during sample processing. They also have short half-lives in vivo, in both plasma and CNS, which may lead to diminished efficacy. Transgenic transporter-deficient mouse models show that the CNS delivery of vorinostat was not limited by the two major blood-brain barrier efflux transporters, p-glycoprotein and breast-cancer-resistance protein. Vorinostat had an unbound CNS tissue-to-plasma partition coefficient of 0.06 {plus minus} 0.02. Conversely, the exposure of unbound quisinostat in the brain was only 0.02 {plus minus} 0.001 of that in the plasma, and the CNS distribution of quisinostat was limited by the activity of p-glycoprotein. To gain further context for these findings, the CNS distributional kinetics for vorinostat and quisinostat were compared to another hydroxamic acid HDACI, panobinostat. A comprehensive understanding of the CNS target exposure to unbound HDACI, along with known potencies from in vitro testing, can inform the prediction of a therapeutic window for HDACIs that have limited CNS exposure to unbound drug and guide targeted dosing strategies. Significance Statement This study indicates that quisinostat and vorinostat are susceptible to enzymatic degradation in the plasma, and to a lesser degree, in the target CNS tissues. Employing techniques that minimize the post-sampling degradation in plasma, brain and spinal cord, accurate CNS distributional kinetic parameters for these potentially useful compounds were determined. A knowledge of CNS exposure (Kp,uu), time to peak, and duration can inform dosing strategies in preclinical and clinical trials in selected CNS tumors.

Absorption, Distribution, Metabolism, and Excretion of Icenticaftor (QBW251) in Healthy Male Volunteers at Steady State and In Vitro Phenotyping of Major Metabolites.

Icenticaftor (QBW251) is a potentiator of the CFTR protein and is currently in clinical development for the treatment of chronic obstructive pulmonary disease and chronic bronchitis. An absorption, distribution, metabolism, and excretion (ADME) study was performed at steady state to determine the pharmacokinetics, mass balance, and metabolite profiles of icenticaftor in humans. In this open-label study, six healthy men were treated with unlabeled oral icenticaftor (400 mg b.i.d.) for 4 days. A single oral dose of [14C]icenticaftor was administered on Day 5, and unlabeled icenticaftor was administered b.i.d. from the evening of Day 5 to Day 12. Unchanged icenticaftor accounted for 18.5% of plasma radioactivity. Moderate to rapid absorption of icenticaftor was observed (median Tmax: 4 hours), with 93.4% of the dose absorbed. It exhibited moderate distribution (Vz/F: 335 L) and was extensively metabolized, principally through N-glucuronidation, O-glucuronidation, and/or O-demethylation. The metabolites M8 and M9, formed by N-glucuronidation and O-glucuronidation of icenticaftor, respectively, represented the main entities detected in plasma (35.3% and 14.5%, respectively) in addition to unchanged icenticaftor (18.5%). The apparent mean T1/2 of icenticaftor was 15.4 hours in blood and 20.6 hours in plasma. Icenticaftor was eliminated from the body mainly through metabolism followed by renal excretion, and excretion of radioactivity was complete after 9 days. In vitro phenotyping of icenticaftor showed that cytochrome P450 and uridine diphosphate glucuronosyltransferase were responsible for 31% and 69% of the total icenticaftor metabolism in human liver microsomes, respectively. This study provided invaluable insights into the disposition of icenticaftor. Significance Statement The ADME of a single radioactive oral dose of icenticaftor was evaluated at steady state to investigate the nonlinear pharmacokinetics observed previously with icenticaftor. [14C]Icenticaftor demonstrated good systemic availability after oral administration and was extensively metabolized and moderately distributed to peripheral tissues. The most abundant metabolites, M8 and M9, were formed by N-glucuronidation and O-glucuronidation of icenticaftor, respectively. Phenotyping demonstrated that [14C]icenticaftor was metabolized predominantly by UGT1A9 with a remarkably low Km value.

Investigations into the concentrations and metabolite profiles of doping agents and antidepressants in human seminal fluid using liquid chromatography-mass spectrometry.

Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called Adverse Analytical Finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offences and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from non-athletes were analysed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e. samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the non-approved PPARδ agonist GW1516) in a total of 81 samples, with 91 % of these suspected cases being verified by the confirmation method. Besides the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. Significance Statement This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing.

Involvement of proton-coupled SLC49A4-mediated transport in the export of lysosomally trapped pyrilamine.

Our recent study revealed that SLC49A4, known as disrupted in renal carcinoma 2, is a H+-coupled lysosomal exporter for pyridoxine (vitamin B6), a cationic compound, and involved in the regulation of its lysosomal and cellular levels. We here examined a possibility that this transporter might also transport cationic amphiphilic drugs (CADs) that are known to undergo lysosomal trapping, using pyrilamine, an H1-antagonist, as a model CAD and the COS-7 cell line as a model cell system for transient introduction of human SLC49A4 and a recombinant SLC49A4 protein (SLC49A4-AA), in which the N-terminal dileucine motif involved in lysosomal localization was removed by replacing with dialanine for redirected localization to the plasma membrane. The introduction of SLC49A4 into COS-7 cells induced a significant decrease in the accumulation of pyrilamine in the intracellular compartments in the cells treated with digitonin for permeabilization of plasma membranes, suggesting its operation for lysosomal pyrilamine export. Accordingly, functional analysis using the SLC49A4-AA mutant, which operates for cellular uptake at the plasma membrane, in transiently transfected COS-7 cells demonstrated its H+-coupled operation for pyrilamine transport, which was saturable with a Michaelis constant of 132 µM at pH 5.5. In addition, many CADs that may potentially undergo lysosomal trapping, which include imipramine, propranolol, verapamil, and some others, were found to inhibit SLC49A4-AA-mediated pyrilamine transport, suggesting their affinity for SLC49A4. These results suggest that SLC49A4 is involved in the lysosomal trapping of pyrilamine, operating for its exit. The CADs that inhibited SLC49A4-AA-mediated pyrilamine transport could also be SLC49A4 substrate candidates. Significance Statement SLC49A4 mediates the transport of pyrilamine in a H+-coupled manner at the lysosomal membrane. This could be a newly identified mechanism for lysosomal export involved in its lysosomal trapping.

Hepatocellular Disposition and Transporter Interactions with Tolvaptan and Metabolites in Sandwich-Cultured Human Hepatocytes.

Tolvaptan is a selective V2-receptor antagonist primarily metabolized by CYP3A. The present study investigated the hepatocellular disposition of tolvaptan and the generated tolvaptan metabolites, DM-4103 and DM-4107, as well as the potential for drug-drug interaction (DDIs) with metabolic and transport proteins in sandwich-cultured human hepatocytes (SCHH). Tolvaptan was incubated with SCHH and quantified by LC-MS/MS. Pioglitazone, verapamil, MK-571 and elacridar were used as inhibitors to investigate mechanisms of transport and metabolism of tolvaptan and metabolites. Taurocholate (TCA), pravastatin, digoxin, and metformin were used as transporter probes to investigate which transport proteins were inhibited by tolvaptan and metabolites. Cellular accumulation of tolvaptan (0.15-50 µM), DM-4103 and DM-4107 in SCHH was concentration dependent. Tolvaptan accumulation (15 µM) in SCHH was not altered markedly by 50 µM pioglitazone, verapamil or MK-571, or 10 µM elacridar. Co-incubation of tolvaptan with pioglitazone, verapamil, MK-571 and elacridar reduced DM-4107 accumulation by 45.6, 79.8, 94.5 and 23.0%, respectively, relative to control. Co-incubation with increasing tolvaptan concentrations (0.15-50 µM) decreased TCA (2.5 µM) cell+bile accumulation and the TCA biliary excretion index (BEI; from 76% to 51%), consistent with inhibition of the bile salt export pump (BSEP). Tolvaptan (15 µM) had no effect on the cellular accumulation of 2.5 µM pravastatin or metformin. Digoxin cellular accumulation increased and the BEI of digoxin decreased from 23.9% to 8.1% in the presence of 15 µM tolvaptan, consistent with inhibition of P-glycoprotein (P-gp). In summary, SCHH studies revealed potential metabolic- and transporter-mediated DDIs involving tolvaptan and metabolites.

Phosphocholine conjugation: an unexpected in vivo conjugation pathway associated with hepatitis c ns5b inhibitors featuring a bicyclo[1.1.1]pentane.

During a medicinal chemistry campaign to identify inhibitors of the hepatitis C virus nonstructural protein 5B (RNA-dependent RNA polymerase), a bicyclo[1.1.1]pentane was introduced into the chemical scaffold to improve metabolic stability. The inhibitors bearing this feature, 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)-4-fluorophenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (1) and 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (2), exhibited low turnover in incubations with liver S9 or hepatocytes (rat, human), with hydroxylation of the bicyclic moiety being the only metabolic pathway observed. In subsequent disposition studies using bile-duct-cannulated rats, the metabolite profiles of bile samples revealed, in addition to multiple products of bicyclopentane-oxidation, unexpected metabolites characterized by molecular masses that were 181 Da greater than those of 1 or 2. Further LC/MSn and NMR analysis of the isolated metabolite of 1 demonstrated the presence of a phosphocholine (POPC) moiety bound to the methine carbon of the bicyclic moiety through an ester bond. The POPC conjugate of the NS5B inhibitors was assumed to result from two sequential reactions: hydroxylation of the bicyclic methine to a tertiary alcohol and addition of POPC by CDP-choline: 1,2-diacylglycerol cholinephosphotransferase, an enzyme responsible for the final step in the biosynthesis of phosphatidylcholine. However, this pathway could not be recapitulated using CDP-choline-supplemented liver S9 or hepatocytes due to inadequate formation of the hydroxylation product in vitro. The observation of this unexpected pathway prompted concerns about the possibility that 1 and 2 might interfere with routine phospholipid synthesis. These results demonstrate the participation in xenobiotic metabolism of a process whose function is ordinarily limited to the synthesis of endogenous compounds.