(+)-9-Trifluoroethoxy-α-Dihydrotetrabenazine as a Highly Potent Vesicular Monoamine Transporter 2 Inhibitor for Tardive Dyskinesia

Valbenazine and deutetrabenazine are the only two therapeutic drugs approved for tardive dyskinesia based on blocking the action of vesicular monoamine transporter 2 (VMAT2). But there exist demethylated inactive metabolism at the nine position for both them resulting in low availability, and CYP2D6 plays a major role in this metabolism resulting in the genetic polymorphism issue. 9-trifluoroethoxy-dihydrotetrabenazine (13e) was identified as a promising lead compound for treating tardive dyskinesia. In this study, we separated 13e via chiral chromatography and acquired R,R,R-13e [(+)-13e] and S,S,S-13e [(−)-13e], and we investigated their VMAT2-inhibitory activity and examined the related pharmacodynamics and pharmacokinetics properties using in vitro and in vivo models (+)-13e displayed high affinity for VMAT2 (Ki = 1.48 nM) and strongly inhibited [3H]DA uptake (IC50 = 6.11 nM) in striatal synaptosomes. Conversely, its enantiomer was inactive. In vivo, (+)-13e decreased locomotion in rats in a dose-dependent manner. The treatment had faster, stronger, and longer-lasting effects than valbenazine at an equivalent dose. Mono-oxidation was the main metabolic pathway in the liver microsomes and in dog plasma after oral administration, and glucuronide conjugation of mono-oxidized and/or demethylated products and direct glucuronide conjugation were also major metabolic pathways in dog plasma. O-detrifluoroethylation of (+)-13e did not occur. Furthermore, CYP3A4 was identified as the primary isoenzyme responsible for mono-oxidation and demethylation metabolism, and CYP2C8 was a secondary isoenzyme (+)-13e displayed high permeability across the Caco-2 cell monolayer, and it was not a P-glycoprotein substrate as demonstrated by its high oral absolute bioavailability (75.9%) in dogs. Thus, our study findings highlighted the potential efficacy and safety of (+)-13e in the treatment of tardive dyskinesia. These results should promote its clinical development.

Contributions of UDP-Glucuronosyltransferases to Human Hepatic and Intestinal Metabolism of Ticagrelor and Inhibition of UGTs and Cytochrome P450 Enzymes by Ticagrelor and its Glucuronidated Metabolite

Ticagrelor is the first reversibly binding, direct-acting, oral P2Y12 receptor inhibitor. The contribution of UDP-glucuronosyltransferases (UGTs) enzymes to the metabolism of ticagrelor to its glucuronide conjugation, ticagrelor-O-glucuronide, in human liver microsomes (HLM) and human intestinal microsomes (HIM), was well characterized in the current study. The inhibition potential of human major UGTs by ticagrelor and ticagrelor-O-glucuronide was explored. The inhibitory effects of ticagrelor-O-glucuronide on cytochrome P450s (CYPs) enzymes were investigated as well. Ticagrelor glucuronidation exhibits substrate inhibition kinetics in both HLM and HIM with apparent Km values of 5.65 and 2.52 μM, Vmax values of 8.03 and 0.90 pmol min−1·mg protein−1, Ksi values of 1,343.0 and 292.9 respectively. The in vitro intrinsic clearances (Vmax/Km) for ticagrelor glucuronidation by HLM and HIM were 1.42 and 0.36 μl min−1·mg protein−1, respectively. Study with recombinant human UGTs suggested that multiple UGT isoforms including UGT1A9, UGT1A7, UGT1A3, UGT1A4, UGT1A1, UGT2B7 and UGT1A8 are involved in the conversion of ticagrelor to ticagrelor-O-glucuronide with UGT1A9 showing highest catalytic activity. The results were further supported by the inhibition studies on ticagrelor glucuronidation with typical UGT inhibitors in pooled HLM and HIM. Little or no inhibition of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7 by ticagrelor and ticagrelor-O-glucuronide was noted. Ticagrelor-O-glucuronide also exhibited limited inhibitory effects toward CYP2C8, CYP2D6 and CYP3A4. In contrast, ticagrelor-O-glucuronide weakly inhibited CYP2B6, CYP2C9 and CYP2C19 activity with apparent IC50 values of 45.0, 20.0 and 18.8 μM, respectively. The potential of ticagrelor-O-glucuronide to cause drug-drug interactions warrant further study.

Health Status Is Affected, and Phase I/II Biotransformation Activity Altered in Young Women Using Oral Contraceptives Containing Drospirenone/Ethinyl Estradiol

Combined oral contraceptive (COC) use has been associated with various adverse effects. Formulations containing drospirenone (DRSP) and ethinyl estradiol (EE) are generally regarded as milder COCs. Whether long term use of these pills indeed has a low health risk remains questionable. COC use may affect the biotransformation balance by increasing the toxic load or by interfering with the pharmacokinetics of other drugs. This may negatively impact overall health via the production of toxic biotransformation metabolites and induction of oxidative stress. Although individual enzymes involved in biotransformation are known to be regulated by COCs, the effect of COC use on the overall liver biotransformation efficiency has not been reported. Here, we evaluated the general subjective health status and overall liver biotransformation efficiency of healthy young women who were either long term chronic users of COCs containing DRSP/EE, or who were not using any hormonal products. COC users suffered from moderate to severe fatigue and reported more health-related symptoms. Furthermore, phase I (CYP1A2) activity was reduced whereas phase II conjugation reactions (glucuronide conjugation and glycine conjugation) were increased in COC users. Finally, serum peroxide levels were markedly elevated and antioxidant capacity of plasma was reduced in COC users. COCs containing DRSP/EE may, therefore, adversely affect health status and disturb the balance between phase I and II biotransformation reactions. These effects may be mediated by oxidative stress.

Determination of metabolites of phloretin in rats using UHPLC-LTQ-Orbitrap mass spectrometry

Purpose: To study the metabolites of phloretin in vivo using ultra-high performance liquid chromatography linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap). Methods: After administration of phloretin (50 mg/kg; oral route) to six rats, blood samples were taken from each animal. Each sample was then subjected to solid-phase extraction to prepare it for chromatographic/spectroscopic analysis. Finally, each sample was analyzed using UHPLC-LTQOrbitrap with a negative-mode electrospray ionization source. Results: Based on mass measurements, chromatographic retention times, and MS2 fragmentation ions, we detected and identified phloretin and 16 metabolites of the drug in vivo in rats. Metabolic reactions of phloretin included glucosylation and glucuronide conjugation, diglucuronide conjugation, glucosylation and sulfate conjugation, sulfate conjugation, glucuronide conjugation, and glucosylation and hydroxylation. Conclusion: The findings provide a better understanding of phloretin metabolism and metabolites, and new information about their effective forms, pharmacological actions, metabolic fate, and toxic actions in vivo.

Rupatadine – a panacea for allergies? A literature review

Allergic diseases are one of the most common chronic diseases. The incidence of allergies is constantly increasing, and it is currently estimated that around 40% of the population suffers from at least one allergy. Current guidelines for the treatment of allergic rhinoconjunctivitis and urticaria recommend modern second-generation antihistamines. Rupatadine is the first hybrid molecule with high affinity for the H1 receptor resulting from remarkably strong binding to this receptor. It exhibits significant H1 receptor binding selectivity, greater in the lungs than in the cerebellum. It has antihistamine effect that is about 25 and 75 times stronger than that of cetirizine and loratadine, respectively. The second mechanism of its action is a strong antagonism of the platelet-activating factor (PAF) receptor. Rupatadine is rapidly absorbed, which correlates with the onset of anti-H1 and anti-PAF activity. Rupatadine is approximately 99% bound to plasma proteins, but at the same time it is very well distributed to other tissues. It undergoes metabolism in the liver through oxidative processes and glucuronide conjugation. Many studies indicate that rupatadine (10 mg) was at least as effective as desloratadine, cetirizine, loratadine and ebastine in reducing allergic symptoms in allergic rhinitis and urticaria. It has also been confirmed to be effective in relieving the symptoms of insect bite response. Rupatadine has a positive effect on the daily functioning of patients with allergic rhinitis and urticaria, and improves the quality of life of these people. At the same time, many cross-sectional studies have shown that rupatadine has a good safety and tolerability profile in adults and children and has not shown adverse cardiovascular effects.

UPLC–ESI-Q-TOF/MS based metabolic profiling of protosappanin B in rat plasma, bile, feces, urine and intestinal bacteria samples

Background: Caesalpinia sappan L. is a traditional medicinal plant that is used to promote blood circulation and treat stroke in China. Protosappanin B (PTB) is a unique homoisoflavone compound isolated from Sappan Lignum (the heartwood of Caesalpinia sappan L). In a previous study, the metabolic fate of PTB remained unknown. Objective: To explore whether PTB is extensively metabolized, the metabolites of PTB in bile, plasma, urine, feces, and intestinal bacteria samples in rats were investigated. Method: The biosamples were investigated by ultraperformance liquid chromatography combined with time-of-flight mass spectrometry (UPLC-TOF-MS/MS) with MetabolitePilot software. Result: 28 metabolites were identified in the biosamples: 18 metabolites in rat bile, 8 in plasma, 20 in feces, 7 in urine and 2 in intestinal bacteria samples. Both phase I and phase II metabolites were observed. Metabolite conversion occurred via 9 proposed pathways: sulfate conjugation, glucuronide conjugation, bis-glucuronide conjugation, glucose conjugation, dehydration, oxidation, hydrolysis, methylation and hydroxymethylene loss. The metabolic pathways differed among biosamples and exhibited different distributions. Among these pathways, the most important were sulfate and glucuronide conjugation. Conclusion: The results showed that the small intestinal and biliary routes play an important role in the clearance and excretion of PTB. The main sites of metabolism in the PTB chemical structure were the phenolic hydroxyl and the side-chains on the eight-element ring.

Pharmacokinetics and Metabolism of Liposome-Encapsulated 2,4,6-Trihydroxygeranylacetophenone in Rats Using High-Resolution Orbitrap Liquid Chromatography Mass Spectrometry

2,4,6-trihydroxy-3-geranylacetophenone (tHGA) is a bioactive compound that shows excellent anti-inflammatory properties. However, its pharmacokinetics and metabolism have yet to be evaluated. In this study, a sensitive LC-HRMS method was developed and validated to quantify tHGA in rat plasma. The method showed good linearity (0.5–80 ng/mL). The accuracy and precision were within 10%. Pharmacokinetic investigations were performed on three groups of six rats. The first two groups were given oral administrations of unformulated and liposome-encapsulated tHGA, respectively, while the third group received intraperitoneal administration of liposome-encapsulated tHGA. The maximum concentration (Cmax), the time required to reach Cmax (tmax), elimination half-life (t1/2) and area under curve (AUC0–24) values for intraperitoneal administration were 54.6 ng/mL, 1.5 h, 6.7 h, and 193.9 ng/mL·h, respectively. For the oral administration of unformulated and formulated tHGA, Cmax values were 5.4 and 14.5 ng/mL, tmax values were 0.25 h for both, t1/2 values were 6.9 and 6.6 h, and AUC0–24 values were 17.6 and 40.7 ng/mL·h, respectively. The liposomal formulation improved the relative oral bioavailability of tHGA from 9.1% to 21.0% which was a 2.3-fold increment. Further, a total of 12 metabolites were detected and structurally characterized. The metabolites were mainly products of oxidation and glucuronide conjugation.

A Complete Study of Farrerol Metabolites Produced in Vivo and in Vitro

Although farrerol, a characteristically bioactive constituent of Rhododendron dauricum L., exhibits extensive biological and pharmacological activities (e.g., anti-oxidant, anti-immunogenic, and anti-angiogenic) as well as a high drug development potential, its metabolism remains underexplored. Herein, we employed ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry coupled with multiple data post-processing techniques to rapidly identify farrerol metabolites produced in vivo (in rat blood, bile, urine and feces) and in vitro (in rat liver microsomes). As a result, 42 in vivo metabolites and 15 in vitro metabolites were detected, and farrerol shown to mainly undergo oxidation, reduction, (de)methylation, glucose conjugation, glucuronide conjugation, sulfate conjugation, N-acetylation and N-acetylcysteine conjugation. Thus, this work elaborates the metabolic pathways of farrerol and reveals the potential pharmacodynamics forms of farrerol.

Absorption and metabolism of olive oil secoiridoids in the small intestine

The secoiridoids 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4-DHPEA-EDA) account for approximately 55 % of the phenolic content of olive oil and may be partly responsible for its reported human health benefits. We have investigated the absorption and metabolism of these secoiridoids in the upper gastrointestinal tract. Both 3,4-DHPEA-EDA and 3,4-DHPEA-EA were relatively stable under gastric conditions, only undergoing limited hydrolysis. Both secoiridoids were transferred across a human cellular model of the small intestine (Caco-2 cells). However, no glucuronide conjugation was observed for either secoiridoid during transfer, although some hydroxytyrosol and homovanillic alcohol were formed. As Caco-2 cells are known to express only limited metabolic activity, we also investigated the absorption and metabolism of secoiridoids in isolated, perfused segments of the jejunum and ileum. Here, both secoiridoids underwent extensive metabolism, most notably a two-electron reduction and glucuronidation during the transfer across both the ileum and jejunum. Unlike Caco-2 cells, the intact small-intestinal segments contain NADPH-dependent aldo-keto reductases, which reduce the aldehyde carbonyl group of 3,4-DHPEA-EA and one of the two aldeydic carbonyl groups present on 3,4-DHPEA-EDA. These reduced forms are then glucuronidated and represent the major in vivo small-intestinal metabolites of the secoiridoids. In agreement with the cell studies, perfusion of the jejunum and ileum also yielded hydroxytyrosol and homovanillic alcohol and their respective glucuronides. We suggest that the reduced and glucuronidated forms represent novel physiological metabolites of the secoiridoids that should be pursued in vivo and investigated for their biological activity.