Metoprolol elicits neurobehavioral insufficiency and oxidative damage in nontarget Nauphoeta cinerea nymphs.

Metoprolol, a drug for hypertension and cardiovascular diseases, has become a contaminant of emerging concern because of its frequent detection in various environmental matrices globally. The dwindling in the biodiversity of useful insects owing to increasing presence of environmental chemicals is currently a great interest to the scientific community. In the current research, the toxicological impact of ecologically relevant concentrations of metoprolol at 0, 0.05, 0.1, 0.25, and 0.5 µg/L on Nauphoeta cinerea nymphs following exposure for 42 consecutive days was evaluated. The insects' behavior was analyzed with automated video-tracking software (ANY-maze, Stoelting Co, USA) while biochemical assays were done using the midgut, head and fat body. Metoprolol-exposed nymphs exhibited significant diminutions in the path efficiency, mobility time, distance traveled, body rotation, maximum speed and turn angle cum more episodes, and time of freezing. In addition, the heat maps and track plots confirmed the metoprolol-mediated wane in the exploratory and locomotor fitness of the insects. Compared with control, metoprolol exposure decreased acetylcholinesterase activity in insects head. Antioxidant enzymes activities and glutathione level were markedly decreased whereas indices of inflammation and oxidative injury to proteins and lipids were significantly increased in head, midgut and fat body of metoprolol-exposed insects. Taken together, metoprolol exposure induces neurobehavioral insufficiency and oxido-inflammatory injury in N. cinerea nymphs. These findings suggest the potential health effects of environmental contamination with metoprolol on ecologically and economically important nontarget insects.

The Activity of Members of the UDP-Glucuronosyltransferase Subfamilies UGT1A and UGT2B is Impaired in Patients with Liver Cirrhosis.

BACKGROUND AND OBJECTIVE: The impact of liver cirrhosis on the activity of UDP-glucuronosyltransferases (UGTs) is currently not well characterized. We investigated the glucuronidation capacity and glucuronide accumulation in patients with liver cirrhosis. METHODS: We administered the Basel phenotyping cocktail (caffeine, efavirenz, flurbiprofen, omeprazole, metoprolol, midazolam) to patients with liver cirrhosis (n = 16 Child A, n = 15 Child B, n = 5 Child C) and n = 12 control subjects and obtained pharmacokinetic profiles of substrates and primary metabolites and their glucuronides. RESULTS: Caffeine and its metabolite paraxanthine were only slightly glucuronidated. The metabolic ratio (AUCglucuronide/AUCparent, MR) was not affected for caffeine but decreased by 60% for paraxanthine glucuronide formation in Child C patients. Efavirenz was not glucuronidated whereas 8-hydroxyefavirenz was efficiently glucuronidated. The MR of 8-hydroxyefavirenz-glucuronide formation increased three-fold in Child C patients and was negatively correlated with the glomerular filtration rate. Flurbiprofen and omeprazole were not glucuronidated. 4-Hydroxyflurbiprofen and 5-hydroxyomeprazole were both glucuronidated but the corresponding MRs for glucuronide formation were not affected by liver cirrhosis. Metoprolol, but not α-hydroxymetoprolol, was glucuronidated, and the MR for metoprolol-glucuronide formation dropped by 60% in Child C patients. Both midazolam and its metabolite 1'-hydroxymidazolam underwent glucuronidation, and the corresponding MRs for glucuronide formation dropped by approximately 80% in Child C patients. No relevant glucuronide accumulation occurred in patients with liver cirrhosis. CONCLUSIONS: Detailed analysis revealed that liver cirrhosis may affect the activity of UGTs of the UGT1A and UGT2B subfamilies according to liver function. Clinically significant glucuronide accumulation did not occur in the population investigated. CLINICAL TRIAL REGISTRATION: NCT03337945.

Sympathetic Activation Promotes Cardiomyocyte Apoptosis in a Rabbit Susceptibility Model of Hyperthyroidism-Induced Atrial Fibrillation via the p38 MAPK Signaling Pathway.

Excess thyroid hormone secretion can cause endocrine metabolic disorders, which can lead to cardiovascular diseases, including heart enlargement, atrial fibrillation (AF), and heart failure. The present study investigated the molecular mechanisms of hyperthyroidism-induced AF. A rabbit susceptibility model of hyperthyroidism-induced AF was constructed, and metoprolol treatment was administered. Norepinephrine levels were determined using enzyme-linked immunosorbent assay; quantitative reverse transcription polymerase chain reaction and immunohistochemistry were used to detect the expression of markers for sympathetic remodeling (growth associated protein 43 and tyrosine hydroxylase in atrial myocardial tissues and stellate ganglia). Primary rabbit cardiomyocytes were cultured and identified by immunofluorescence staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to measure cardiomyocyte apoptosis; western blot was used to detect the expression of apoptosis-related proteins, including Bax, Bcl-2, and cleaved caspase-3, as well as to measure the phosphorylation states of p38 mitogen-activated protein kinase (MAPK) pathway proteins. Metoprolol inhibited sympathetic activation and cardiomyocyte apoptosis in the rabbit model by inhibiting the p38 MAPK signaling pathway. Immunofluorescence staining results revealed that the rabbit cardiomyocytes were isolated successfully. Inhibition of p38 MAPK signaling alleviated norepinephrine-induced apoptosis in cardiomyocytes. Sympathetic activation promotes apoptosis in cardiomyocytes with hyperthyroidism-induced AF via the p38 MAPK signaling pathway. The results of the present study provide a novel theoretical basis for the potential clinical treatment of patients with hyperthyroidism and AF.

Enantiospecific Uptake and Depuration Kinetics of Chiral Metoprolol and Venlafaxine in Marine Medaka (Oryzias melastigma): Tissue Distribution and Metabolite Formation.

The increasing use of chiral pharmaceuticals has led to their widespread presence in the environment. However, their toxicokinetics have rarely been reported. Therefore, the tissue-specific uptake and depuration kinetics of two pairs of pharmaceutical enantiomers, S-(-)-metoprolol versus R-(+)-metoprolol and S-(+)-venlafaxine versus R-(-)-venlafaxine, were studied in marine medaka (Oryzias melastigma) during a 28-day exposure and 14-day clearance period. The toxicokinetics of the studied pharmaceuticals, including uptake and depuration rate constants, depuration half-life (t1/2), and bioconcentration factor (BCF), were reported for the first time. The whole-fish results demonstrated a higher S- than R-venlafaxine bioaccumulation potential, whereas no significant difference was observed between S- and R-metoprolol. O-desmethyl-metoprolol (ODM) and α-hydroxy-metoprolol (AHM) were the main metoprolol metabolites identified by suspect screening, and the ratios of ODM to AHM were 3.08 and 1.35 for S- and R-metoprolol, respectively. N,O-Didesmethyl-venlafaxine (NODDV) and N-desmethyl-venlafaxine (NDV) were the main venlafaxine metabolites, and the ratios of NODDV to NDV were 1.55 and 0.73 for S- and R-venlafaxine, respectively. The highest tissue-specific BCFs of the four enantiomers were all found in the eyes, meriting in-depth investigation.

Neutrophil ß1 adrenoceptor blockade blunts stroke-associated neuroinflammation.

BACKGROUND AND PURPOSE: Reperfusion therapy is the standard of care for ischaemic stroke; however, there is a need to identify new therapeutic targets able to ameliorate cerebral damage. Neutrophil ß1 adrenoceptors (ß1AR) have been linked to neutrophil migration during exacerbated inflammation. Given the central role of neutrophils in cerebral damage during stroke, we hypothesize that ß1AR blockade will improve stroke outcomes. EXPERIMENTAL APPROACH: Rats were subjected to middle cerebral artery occlusion-reperfusion to evaluate the effect on stroke of the selective ß1AR blocker metoprolol (12.5 mg·kg-1 ) when injected i.v. 10 min before reperfusion. KEY RESULTS: Magnetic resonance imaging and histopathology analysis showed that pre-reperfusion i.v. metoprolol reduced infarct size. This effect was accompanied by reduced cytotoxic oedema at 24 h and vasogenic oedema at 7 days. Metoprolol-treated rats showed reduced brain neutrophil infiltration and those which infiltrated displayed a high proportion of anti-inflammatory phenotype (N2, YM1+ ). Additional inflammatory models demonstrated that metoprolol specifically blocked neutrophil migration via ß1AR and excluded a significant effect on the glia compartment. Consistently, metoprolol did not protect the brain in neutrophil-depleted rats upon stroke. In patients suffering an ischaemic stroke, ß1AR blockade by metoprolol reduced circulating neutrophil-platelet co-aggregates. CONCLUSIONS AND IMPLICATIONS: Our findings describe that ß1AR blockade ameliorates cerebral damage by targeting neutrophils, identifying a novel therapeutic target to improve outcomes in patients with stroke. This therapeutic strategy is in the earliest stages of the translational pathway and should be further explored.

Inhibition of ß1-AR/Gαs signaling promotes cardiomyocyte proliferation in juvenile mice through activation of RhoA-YAP axis.

The regeneration potential of the mammalian heart is incredibly limited, as cardiomyocyte proliferation ceases shortly after birth. ß-adrenergic receptor (ß-AR) blockade has been shown to improve heart functions in response to injury; however, the underlying mechanisms remain poorly understood. Here, we inhibited ß-AR signaling in the heart using metoprolol, a cardio-selective ß blocker for ß1-adrenergic receptor (ß1-AR) to examine its role in heart maturation and regeneration in postnatal mice. We found that metoprolol enhanced cardiomyocyte proliferation and promoted cardiac regeneration post myocardial infarction, resulting in reduced scar formation and improved cardiac function. Moreover, the increased cardiomyocyte proliferation was also induced by the genetic deletion of Gnas, the gene encoding G protein alpha subunit (Gαs), a downstream effector of ß-AR. Genome wide transcriptome analysis revealed that the Hippo-effector YAP, which is associated with immature cardiomyocyte proliferation, was upregulated in the cardiomyocytes of ß-blocker treated and Gnas cKO hearts. Moreover, the increased YAP activity is modulated by RhoA signaling. Our pharmacological and genetic studies reveal that ß1-AR-Gαs-YAP signaling axis is involved in regulating postnatal cardiomyocyte proliferation. These results suggest that inhibiting ß-AR-Gαs signaling promotes the regenerative capacity and extends the cardiac regenerative window in juvenile mice by activating YAP-mediated transcriptional programs.

Carvedilol Selectively Stimulates ßArrestin2-Dependent SERCA2a Activity in Cardiomyocytes to Augment Contractility.

Heart failure (HF) carries the highest mortality in the western world and ß-blockers [ß-adrenergic receptor (AR) antagonists] are part of the cornerstone pharmacotherapy for post-myocardial infarction (MI) chronic HF. Cardiac ß1AR-activated ßarrestin2, a G protein-coupled receptor (GPCR) adapter protein, promotes Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a SUMO (small ubiquitin-like modifier)-ylation and activity, thereby directly increasing cardiac contractility. Given that certain ß-blockers, such as carvedilol and metoprolol, can activate ßarrestins and/or SERCA2a in the heart, we investigated the effects of these two agents on cardiac ßarrestin2-dependent SERCA2a SUMOylation and activity. We found that carvedilol, but not metoprolol, acutely induces ßarrestin2 interaction with SERCA2a in H9c2 cardiomyocytes and in neonatal rat ventricular myocytes (NRVMs), resulting in enhanced SERCA2a SUMOylation. However, this translates into enhanced SERCA2a activity only in the presence of the ß2AR-selective inverse agonist ICI 118,551 (ICI), indicating an opposing effect of carvedilol-occupied ß2AR subtype on carvedilol-occupied ß1AR-stimulated, ßarrestin2-dependent SERCA2a activation. In addition, the amplitude of fractional shortening of NRVMs, transfected to overexpress ßarrestin2, is acutely enhanced by carvedilol, again in the presence of ICI only. In contrast, metoprolol was without effect on NRVMs' shortening amplitude irrespective of ICI co-treatment. Importantly, the pro-contractile effect of carvedilol was also observed in human induced pluripotent stem cell (hIPSC)-derived cardiac myocytes (CMs) overexpressing ßarrestin2, and, in fact, it was present even without concomitant ICI treatment of human CMs. Metoprolol with or without concomitant ICI did not affect contractility of human CMs, either. In conclusion, carvedilol, but not metoprolol, stimulates ßarrestin2-mediated SERCA2a SUMOylation and activity through the ß1AR in cardiac myocytes, translating into direct positive inotropy. However, this unique ßarrestin2-dependent pro-contractile effect of carvedilol may be opposed or masked by carvedilol-bound ß2AR subtype signaling.

Metoprolol Inhibits Developmental Brain Sterol Biosynthesis in Mice.

De novo sterol synthesis is a critical homeostatic mechanism in the brain that begins during early embryonic development and continues throughout life. Multiple medications have sterol-biosynthesis-inhibiting side effects, with potentially detrimental effects on brain health. Using LC-MS/MS, we investigated the effects of six commonly used beta-blockers on brain sterol biosynthesis in vitro using cell lines. Two beta-blockers, metoprolol (MTP) and nebivolol, showed extreme elevations of the highly oxidizable cholesterol precursor 7-dehydrocholesterol (7-DHC) in vitro across multiple cell lines. We followed up on the MTP findings using a maternal exposure model in mice. We found that 7-DHC was significantly elevated in all maternal brain regions analyzed as well as in the heart, liver and brain of the maternally exposed offspring. Since DHCR7-inhibiting/7-DHC elevating compounds can be considered teratogens, these findings suggest that MTP utilization during pregnancy might be detrimental for the development of offspring, and alternative beta-blockers should be considered.

Identification of Metoprolol Tartrate-Derived Reactive Metabolites Possibly Correlated with Its Cytotoxicity.

As a selective ß1-receptor antagonist, metoprolol tartrate (MTA) is commonly used to treat cardiovascular diseases such as hypertension and angina pectoris. There have been cases of liver injury induced by MTA, but the mechanism of hepatotoxicity induced by MTA is not clear. The purposes of this study were to identify the reactive metabolites of MTA, to determine the pathway for the metabolic activation of MTA, and to define a possible correlation between the metabolic activation and cytotoxicity of MTA. Three oxidative metabolites (M1-M3), a glutathione (GSH) conjugate (M4), and an N-acetyl cysteine (NAC) conjugate (M5) were detected in rat liver microsomal incubations containing MTA and GSH or NAC. M4 was also detected in cultured rat primary hepatocytes and bile of rats given MTA, and M5 was detected in the urine of MTA-treated rats. A quinone methide intermediate may be produced from the metabolic activation process in vitro and in vivo. The metabolite was reactive to glutathione and N-acetyl cysteine. MTA induced marked cytotoxicity in cultured rat primary hepatocytes. Pretreatment of aminobenzotriazole, a nonselective P450 enzyme inhibitor, attenuated the susceptibility of hepatocytes to MTA cytotoxicity.

ß-Blocker Use and Risk of Mortality in Heart Failure Patients Initiating Maintenance Dialysis.

RATIONAL & OBJECTIVE: Beta-blockers are recommended for patients with heart failure (HF) but their benefit in the dialysis population is uncertain. Beta-blockers are heterogeneous, including with respect to their removal by hemodialysis. We sought to evaluate whether ß-blocker use and their dialyzability characteristics were associated with early mortality among patients with chronic kidney disease with HF who transitioned to dialysis. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: Adults patients with chronic kidney disease (aged≥18 years) and HF who initiated either hemodialysis or peritoneal dialysis during January 1, 2007, to June 30, 2016, within an integrated health system were included. EXPOSURES: Patients were considered treated with ß-blockers if they had a quantity of drug dispensed covering the dialysis transition date. OUTCOMES: All-cause mortality within 6 months and 1 year or hospitalization within 6 months after transition to maintenance dialysis. ANALYTICAL APPROACH: Inverse probability of treatment weights using propensity scores was used to balance covariates between treatment groups. Cox proportional hazard analysis and logistic regression were used to investigate the association between ß-blocker use and study outcomes. RESULTS: 3,503 patients were included in the study. There were 2,115 (60.4%) patients using ß-blockers at transition. Compared with nonusers, the HR for all-cause mortality within 6 months was 0.79 (95% CI, 0.65-0.94) among users of any ß-blocker and 0.68 (95% CI, 0.53-0.88) among users of metoprolol at transition. There were no observed differences in all-cause or cardiovascular-related hospitalization. LIMITATIONS: The observational nature of our study could not fully account for residual confounding. CONCLUSIONS: Beta-blockers were associated with a lower rate of mortality among incident hemodialysis patients with HF. Similar associations were not observed for hospitalizations within the first 6 months following transition to dialysis.

In Vitro Metabolic Transformation of Pharmaceuticals by Hepatic S9 Fractions from Common Carp (Cyprinus carpio).

Water from wastewater treatment plants contains concentrations of pharmaceutically active compounds as high as micrograms per liter, which can adversely affect fish health and behavior, and contaminate the food chain. Here, we tested the ability of the common carp hepatic S9 fraction to produce the main metabolites from citalopram, metoprolol, sertraline, and venlafaxine. Metabolism in fish S9 fractions was compared to that in sheep. The metabolism of citalopram was further studied in fish. Our results suggest a large difference in the rate of metabolites formation between fish and sheep. Fish hepatic S9 fractions do not show an ability to form metabolites from venlafaxine, which was also the case for sheep. Citalopram, metoprolol, and sertraline were metabolized by both fish and sheep S9. Citalopram showed concentration-dependent N-desmethylcitalopram formation with Vmax = 1781 pmol/min/mg and Km = 29.7 µM. The presence of ellipticine, a specific CYP1A inhibitor, in the incubations reduced the formation of N-desmethylcitalopram by 30-100% depending on the applied concentration. These findings suggest that CYP1A is the major enzyme contributing to the formation of N-desmethylcitalopram. In summary, the results from the present in vitro study suggest that common carp can form the major metabolites of citalopram, metoprolol, and sertraline.

Charged Tags for the Identification of Oxidative Drug Metabolites Based on Electrochemistry and Mass Spectrometry.

Most of the active pharmaceutical ingredients like Metoprolol are oxidatively metabolized by liver enzymes, such as Cytochrome P450 monooxygenases into oxygenates and therefore hydrophilic products. It is of utmost importance to identify the metabolites and to gain knowledge on their toxic impacts. By using electrochemistry, it is possible to mimic enzymatic transformations and to identify metabolic hot spots. By introducing charged-tags into the intermediate, it is possible to detect and isolate metabolic products. The identification and synthesis of initially oxidized metabolites are important to understand possible toxic activities. The gained knowledge about the metabolism will simplify interpretation and predictions of metabolitic pathways. The oxidized products were analyzed with high performance liquid chromatography-mass spectrometry using electrospray ionization (HPLC-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. For proof-of-principle, we present a synthesis of one pyridinated main oxidation product of Metoprolol.

Calycosin Influences the Metabolism of Five Probe Drugs in Rats.

BACKGROUND: Calycosin (CAL), a type of O-methylated isoflavone extracted from the herb Astralagusmembranaceus (AM), is a bioactive chemical with antioxidative, antiphlogistic and antineoplastic activities commonly used in traditional alternative Chinese medicine. AM has been shown to confer health benefits as an adjuvant in the treatment of a variety of diseases. AIM: The main objective of this study was to determine whether CAL influences the cytochrome P450 (CYP450) system involved in drug metabolism. METHODS: Midazolam, tolbutamide, omeprazole, metoprolol and phenacetin were selected as probe drugs. Rats were randomly divided into three groups, specifically, 5% Carboxymethyl cellulose (CMC) for 8 days (Control), 5% CMC for 7 days + CAL for 1 day (single CAL) and CAL for 8 days (conc CAL), and metabolism of the five probe drugs evaluated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: No significant differences were observed for omeprazole and midazolam, compared to the control group. T max and t1/2 values of only one probe drug, phenacetin, in the conc CAL group were significantly different from those of the control group (T max h: 0.50±0.00 vs 0.23±0.15; control vs conc CAL). C max of tolbutamide was decreased about two-fold in the conc CAL treatment group (conc vs control: 219.48 vs 429.56, P<0.001). CONCLUSION: Calycosin inhibits the catalytic activities of CYP1A2, CYP2D6 and CYP2C9. Accordingly, we recommend caution, particularly when combining CAL as a modality therapy with drugs metabolized by CYP1A2, CYP2D6 and CYP2C9, to reduce the potential risks of drug accumulation or ineffective treatment.

Interrogation of CYP2D6 Structural Variant Alleles Improves the Correlation Between CYP2D6 Genotype and CYP2D6-Mediated Metabolic Activity.

The cytochrome P450 2D6 (CYP2D6) gene locus is challenging to accurately genotype due to numerous single nucleotide variants and complex structural variation. Our goal was to determine whether the CYP2D6 genotype-phenotype correlation is improved when diplotype assignments incorporate structural variation, identified by the bioinformatics tool Stargazer, with next-generation sequencing data. Using CYP2D6 activity measured with substrates dextromethorphan and metoprolol, activity score explained 40% and 34% of variability in metabolite formation rates, respectively, when diplotype calls incorporated structural variation, increasing from 36% and 31%, respectively, when diplotypes did not incorporate structural variation. We also investigated whether the revised Clinical Pharmacogenetics Implementation Consortium (CPIC) recommendations for translating genotype to phenotype improve CYP2D6 activity predictions over the current system. Although the revised recommendations do not improve the correlation between activity score and CYP2D6 activity, perhaps because of low frequency of the CYP2D6*10 allele, the correlation with metabolizer phenotype group was significantly improved for both substrates. We also measured the function of seven rare coding variants: one (A449D) exhibited decreased (44%) and another (R474Q) increased (127%) activity compared with reference CYP2D6.1 protein. Allele-specific analysis found that A449D is part of a novel CYP2D6*4 suballele, CYP2D6*4.028. The novel haplotype containing R474Q was designated CYP2D6*138 by PharmVar; another novel haplotype containing R365H was designated CYP2D6*139. Accuracy of CYP2D6 phenotype prediction is improved when the CYP2D6 gene locus is interrogated using next-generation sequencing coupled with structural variation analysis. Additionally, revised CPIC genotype to phenotype translation recommendations provides an improvement in assigning CYP2D6 activity.

False positive amphetamines and 3,4-methylenedioxymethamphetamine immunoassays in the presence of metoprolol-two cases reported in clinical toxicology.

Amphetamines, frequently used recreational drugs with high risk of toxicity, are commonly included in urine drug screens. This screening is based on enzyme immunoassay, which is a quick and easy-to-perform technique, but may lack specificity resulting from cross-reactivity with other compounds, causing false positive results. We present two cases of presumed false positive MULTIGENT® amphetamine/methamphetamine and MULTIGENT® ecstasy (Abbott®) immunoassays with the beta-blocker metoprolol. Both metoprolol-poisoned patients presented positive urine screening despite no history of drug abuse. No confirmation for amphetamine molecular structures was found with gas chromatography-mass spectrometry. The cross-reactivity was further investigated by doping urine samples with metoprolol and its two major phase-I metabolites. Metoprolol showed positive results for both amphetamine and MDMA tests at low concentrations (200 and 150 µg/mL, respectively). Metoprolol metabolites cross-reacted with the amphetamines immunoassay only, but at higher concentrations (i.e., 2000 µg/mL for α-hydroxymetoprolol and 750 µg/mL for O-demethylmetoprolol). In conclusion, false positive results in amphetamines and MDMA immunoassays are possible in the presence of metoprolol. Toxicologists should be aware of frequent analytical interferences with immunoassays and a detailed medication history should be taken into consideration for interpretation. In vitro investigation of suspected cross-reactivity should include not only the parent drug but also its related metabolites.

Evaluation of OptiFlow™-MS/MS for bioanalysis of pharmaceutical drugs and metabolites.

Aim: Microflow tandem mass spectrometry-based methods have been proposed as options to improve sensitivity and selectivity while improving sample utility and solvent consumption. Here, we evaluate a newly introduced microflow source, OptiFlow™, for quantitative performance. Results/methodology: We performed a comparison of the OptiFlow and IonDrive™ sources, respectively, on the same triple quadrupole mass spectrometer. The comparison used a neat cocktail of commercially available drugs and extracted plasma samples monitoring midazolam and alprazolam metabolites. Microflow produced a 2-4× signal increase for the neat drug cocktail and a 5-10× increase for extracted plasma samples. Conclusion: The OptiFlow method consistently gave increased signal response relative to the IonDrive method and enabled a better lower limit of quantitation for defining phamacokinetics.

UHPLC-MS/MS assay for simultaneous determination of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin with its active metabolites in human plasma, for population-scale drug-drug interactions studies in people living with HIV.

Thanks to highly active antiretroviral treatments, HIV infection is now considered as a chronic condition. Consequently, people living with HIV (PLWH) live longer and encounter more age-related chronic co-morbidities, notably cardiovascular diseases, leading to polypharmacy. As the management of drug-drug interactions (DDIs) constitutes a key aspect of the care of PLWH, the magnitude of pharmacokinetic DDIs between cardiovascular and anti-HIV drugs needs to be more thoroughly characterized. To that endeavour, an UHPLC-MS/MS bioanalytical method has been developed for the simultaneous determination in human plasma of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin and its active metabolites. Plasma samples were subjected to protein precipitation with methanol, followed by evaporation at room temperature under nitrogen of the supernatant, allowing to attain measurable plasma concentrations down to sub-nanogram per milliliter levels. Stable isotope-labelled analytes were used as internal standards. The five drugs and two metabolites were analyzed using a 6-min liquid chromatographic run coupled to electrospray triple quadrupole mass spectrometry detection. The method was validated over the clinically relevant concentrations ranging from 0.3 to 480 ng/mL for amlodipine, atorvastatin and p-OH-atorvastatin, and 0.4 to 480 ng/mL for pravastatin, 0.5 to 480 ng/mL for rosuvastatin and o-OH-atorvastatin, and 3 to 4800 ng/mL for metoprolol. Validation performances such as trueness (95.4-110.8%), repeatability (1.5-13.4%) and intermediate precision (3.6-14.5%) were in agreement with current international recommendations. Accuracy profiles (total error approach) were lying within the limits of ±30% accepted in bioanalysis. This rapid and robust UHPLC-MS/MS assay allows the simultaneous quantification in plasma of the major currently used cardiovascular drugs and offers an efficient analytical tool for clinical pharmacokinetics as well as DDIs studies.

Comparative research on the metabolism of metoprolol by four CYP2D6 allelic variants in vitro with LC-MS/MS.

Specific study about the effect of cytochrome P450 2D6 (CYP2D6) polymorphisms on the metabolism of clinic drugs is of great significance for drug safety investigation. Here, the interaction between CYP2D6 variants (*1, *2, *10, *39) and metoprolol (MET) was intensively researched in vitro from the aspect of drug-enzyme kinetic study. To obtain quantitative data, α-hydroxymetoprolol (main metabolite of MET) was selected as an ideal analyte and an LC-MS/MS method was adopted for sample determination. Firstly, by selecting suitable internal standard and optimizing separation condition, the LC-MS/MS method was established and validated. Then, the drug-enzyme incubation system was optimized by two parameters: incubation time and amount of enzyme. Lastly, the interaction between CYP2D6 allelic variants and MET was characterized by Km, Vmax and CLint. As a result, four CYP2D6 enzymes displayed diverse Km or Vmax towards MET and the values of CLint showed a wide range from 8.91 to 100%. Relative to CYP2D6*1 (CLint*1 = 100%), CYP2D6*2 demonstrated the second high catalytic activity (CLint*2/*1 = 74.87%) while CYP2D6*39 (CLint*39/*1 = 29.65%) and CYP2D6*10 (CLint*10/*1 = 8.91%) showed minimal catalytic activity. This comprehensive in vitro data suggested the prominent influence of CYP2D6 polymorphisms on the metabolism of MET, which could offer valuable information for personalized administration of MET in clinic.

Comparing PyroMark Q24 Pyrosequencing and MALDI-TOF MS for Identification of CYP2D6*10.

BACKGROUND: CYP2D6*10 is mainly responsible for the large pharmacokinetic variability of routinely administered metoprolol in middle-aged and elderly Asian patients. Utilizing an efficient method for identifying the CYP2D6*10 genotypes is clinically important for evaluating the pharmacokinetic effect of administration of metoprolol. This study attempted to evaluate the effectiveness of the two methods used to detect the rs1065852 and rs1135840 SNPs of the CYP2D6*10 gene. METHODS: Blood samples were processed for the collection of genomic DNA from 198 subjects across Chinese population, and detection of CYP2D6*10 (rs1065852 and rs1135840) was performed using the PyroMark Q24 pyrose-quencing and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS). The discordant results were further validated with Sanger sequencing. We eventually attempted to assess some features of these two methods including reliability, rapidness, being appropriate, and cost-effectiveness. RESULTS: Genotyping of rs1065852 and rs1135840 detected by MALDI-TOF MS were concordant with those identified by PyroMark Q24 pyrosequencing in all 198 (100%) individuals. The hands-on-time and the turnaround time were shorter in the PyroMark Q24 pyrosequencing method than in the MALDI-TOF MS method for SNP of CYP2D6*10. In terms of being cost-effective and high-throughput, the MALDI-TOF MS method outperformed the PyroMark Q24 pyrosequencing method. CONCLUSIONS: CYP2D6*10 genotypes detected by PyroMark Q24 pyrosequencing and MALDI-TOF-MS showed that both methods were reliable, rapid, appropriate, and cost-effective methods. These methods are valuable for clinical applications.

A UHPLC-MS/MS method coupled with liquid-liquid extraction for the quantitation of phenacetin, omeprazole, metoprolol, midazolam and their metabolites in rat plasma and its application to the study of four CYP450 activities.

Drug-drug interactions (DDIs) are thought to be associated with the inhibition of cytochrome P450 activities. The cocktail method with analysis of the metabolism of two or more probe drugs is used to determine CYP450 activities. In this study, we established a UHPLC-MS/MS method for simultaneous quantitation of four CYP450 probe drugs (phenacetin, omeprazole, metoprolol and midazolam) and their metabolites (acetaminophen, 5'-hydroxy omeprazole, α-hydroxy metoprolol and 1'-hydroxy midazolam) in rat plasma. Sample preparation by plasma protein precipitation was combined with a liquid-liquid extraction method. The separation was carried out on a ZORBAX Eclipse Plus C18 Rapid Resolution High Definition column with a gradient elution, using water containing 0.1% formic acid (A) and acetonitrile (B) in a run time of only 3.0 min. Detection was conducted with a 6420 series triple-quadrupole tandem mass spectrometer, using ESI in positive ion mode with multiple reaction monitoring (MRM). The calibration curves were linear over the concentration range 10-5000 ng/mL for phenacetin, omeprazole, metoprolol and midazolam, and 1-500 ng/mL for their metabolites. Intra- and inter-day precisions were within 15%, and the accuracies were in the range of 87-112%. The method was successfully applied to the pharmacokinetic study of probe drugs/metabolites and DDIs with 3-n-butylphthalide (NBP) after administration of a single oral dose of phenacetin, omeprazole, metoprolol and midazolam in rats.