Low-Dose Edoxaban in Very Elderly Patients with Atrial Fibrillation.

BACKGROUND: Implementation of appropriate oral anticoagulant treatment for the prevention of stroke in very elderly patients with atrial fibrillation is challenging because of concerns regarding bleeding. METHODS: We conducted a phase 3, multicenter, randomized, double-blind, placebo-controlled, event-driven trial to compare a once-daily 15-mg dose of edoxaban with placebo in elderly Japanese patients (≥80 years of age) with nonvalvular atrial fibrillation who were not considered to be appropriate candidates for oral anticoagulant therapy at doses approved for stroke prevention. The primary efficacy end point was the composite of stroke or systemic embolism, and the primary safety end point was major bleeding according to the definition of the International Society on Thrombosis and Haemostasis. RESULTS: A total of 984 patients were randomly assigned in a 1:1 ratio to receive a daily dose of 15 mg of edoxaban (492 patients) or placebo (492 patients). A total of 681 patients completed the trial, and 303 discontinued (158 withdrew, 135 died, and 10 had other reasons); the numbers of patients who discontinued the trial were similar in the two groups. The annualized rate of stroke or systemic embolism was 2.3% in the edoxaban group and 6.7% in the placebo group (hazard ratio, 0.34; 95% confidence interval [CI], 0.19 to 0.61; P<0.001), and the annualized rate of major bleeding was 3.3% in the edoxaban group and 1.8% in the placebo group (hazard ratio, 1.87; 95% CI, 0.90 to 3.89; P = 0.09). There were substantially more events of gastrointestinal bleeding in the edoxaban group than in the placebo group. There was no substantial between-group difference in death from any cause (9.9% in the edoxaban group and 10.2% in the placebo group; hazard ratio, 0.97; 95% CI, 0.69 to 1.36). CONCLUSIONS: In very elderly Japanese patients with nonvalvular atrial fibrillation who were not appropriate candidates for standard doses of oral anticoagulants, a once-daily 15-mg dose of edoxaban was superior to placebo in preventing stroke or systemic embolism and did not result in a significantly higher incidence of major bleeding than placebo. (Funded by Daiichi Sankyo; ELDERCARE-AF ClinicalTrials.gov number, NCT02801669.).

Prognosis of elderly non-valvular atrial fibrillation patients stratified by B-type natriuretic peptide: ELDERCARE-AF subanalysis.

BACKGROUND: B-type natriuretic peptide (BNP) is a risk factor for stroke and cardiac death in patients with atrial fibrillation. We hypothesized the prognostic outcomes of very elderly non-valvular atrial fibrillation patients ineligible for standard anticoagulation treatment would vary according to BNP stratification. METHODS: In this subanalysis of the ELDERCARE-AF trial, patients were stratified by BNP levels at enrollment, and clinical outcomes compared among BNP subgroups. Hazard ratios were adjusted for age, atrial fibrillation type, body mass index, creatine clearance, congestive heart failure, and D-dimer. BNP levels were measured using chemiluminescence enzyme immunoassays. RESULTS: In total, 984 patients (average age: 86.6 years) not considered eligible for oral anticoagulant therapy at approved doses for stroke prevention were included. The BNP levels at enrollment were <200 (low), 200 to <400 (moderate), and ≥400 (high) pg/mL in 428, 300, and 256 patients, respectively. The number (%) of patients with stroke or systemic embolism (SSE) was 7 (1.2%), 24 (5.9%), and 28 (8.6%) in the low, moderate, and high BNP subgroups, respectively (adjusted hazard ratio 3.82, P = .0025 for low vs moderate BNP and 4.76, P = .0007 for low vs high BNP). There was no significant difference in major bleeding incidence between the BNP subgroups. Edoxaban 15 mg was associated with a consistent reduction in SSE vs placebo in all BNP subgroups. CONCLUSIONS: Stratification by BNP level was associated with the incidence of SSE for very elderly non-valvular atrial fibrillation patients ineligible for standard anticoagulation treatment, and the effect of edoxaban 15 mg was consistent across BNP levels.

Nighttime Blood Pressure Phenotype and Cardiovascular Prognosis: Practitioner-Based Nationwide JAMP Study.

BACKGROUND: Ambulatory and home blood pressure (BP) monitoring parameters are better predictors of cardiovascular events than are office BP monitoring parameters, but there is a lack of robust data and little information on heart failure (HF) risk. The JAMP study (Japan Ambulatory Blood Pressure Monitoring Prospective) used the same ambulatory BP monitoring device, measurement schedule, and diary-based approach to data processing across all study centers and determined the association between both nocturnal hypertension and nighttime BP dipping patterns and the occurrence of cardiovascular events, including HF, in patients with hypertension. METHODS: This practitioner-based, nationwide, multicenter, prospective, observational study included patients with at least 1 cardiovascular risk factor, mostly hypertension, and free of symptomatic cardiovascular disease at baseline. All patients underwent 24-hour ambulatory BP monitoring at baseline. Patients were followed annually to determine the occurrence of primary end point cardiovascular events (atherosclerotic cardiovascular disease and HF). RESULTS: A total of 6,359 patients (68.6±11.7 years of age, 48% men) were included in the final analysis. During a mean±SD follow-up of 4.5±2.4 years, there were 306 cardiovascular events (119 stroke, 99 coronary artery disease, 88 HF). Nighttime systolic BP was significantly associated with the risk of atherosclerotic cardiovascular disease and HF (hazard ratio adjusted for demographic and clinical risk factors per 20-mm Hg increase: 1.18 [95% CI, 1.02-1.37], P=0.029; and 1.25 [95% CI, 1.00-1.55], P=0.048, respectively). Disrupted circadian BP rhythm (riser pattern, nighttime BP higher than daytime BP) was significantly associated with higher overall cardiovascular disease risk (1.48 [95% CI, 1.05-2.08]; P=0.024), and especially HF (2.45 [95% CI, 1.34-4.48]; P=0.004) compared with normal circadian rhythm. CONCLUSIONS: Nighttime BP levels and a riser pattern were independently associated with the total cardiovascular event rate, in particular for HF. These findings suggest the importance of antihypertensive strategies targeting nighttime systolic BP. Registration: URL: https://www.umin.ac.jp/ctr/; Unique identifier: UMIN000020377.

Significance of 11C-PIB PET/CT in cardiac amyloidosis compared with 99mTc-aprotinin scintigraphy: A pilot study.

BACKGROUND: This study was to investigate the significance of 11C-Pittsburgh B (PIB) PET/CT in patients with suspected cardiac amyloidosis compared with 99mTc-aprotinin scintigraphy. METHODS: Thirteen consecutive patients with suspected cardiac amyloidosis were considered for enrolment in this prospective pilot study. Participants were scheduled to undergo a series of 11C-PIB PET/CT and 99mTc-aprotinin within a 2-month period. Finally, we evaluated nine cases who underwent both imaging modalities, and compared imaging results with clinical and pathological results and prognosis. RESULTS: Six of the 9 patients who underwent both imaging modalities were diagnosed with amyloidosis, of whom 3 patients were diagnosed with cardiac amyloidosis from endomyocardial biopsy. These 3 patients with positive 11C-PIB uptake at the left ventricle wall showed worsening of cardiac function progressing in the short term or death caused by acute exacerbation of chronic heart failure. Six of 8 patients with positive uptake on 99mTc-aprotinin presented with amyloid deposition in the left ventricle wall, but symptoms remained stable if results of 11C-PIB were not positive. CONCLUSION: In a small sample of subjects, the present study showed that 11C-PIB accumulation in myocardium indicated cardiac amyloidosis with poor prognosis. Uptake of 11C-PIB may be related to progressive amyloid deposition to the heart and can predict patient prognosis.

Correction to: Performance of 99mTc-aprotinin scintigraphy for diagnosing light chain (AL) cardiac amyloidosis confirmed by endomyocardial biopsy.

This prospective study was conducted according to the principles outlined within the Declaration of Helsinki, and approved by the Ethics Review Board of National Center for Global Health and Medicine (NCGM-G-00839-01, NCGM-G-00839-02).

Performance of 99mTc-aprotinin scintigraphy for diagnosing light chain (AL) cardiac amyloidosis confirmed by endomyocardial biopsy.

BACKGROUND: Light chain (AL) cardiac amyloidosis is associated with a poor prognosis. Diagnosing at an early stage is critical for treatment and the management of cardiac complication. PURPOSE: We aimed to evaluate the diagnostic performance of 99mTc-aprotinin images in patients with AL cardiac amyloidosis. METHODS AND RESULTS: 99mTc-aprotinin scintigraphy and endomyocardial biopsy were performed in 10 patients with suspected amyloidosis. Endomyocardial biopsy showed amyloid deposits in 5 of 10 patients. 99mTc-aprotinin (planer image) was positive in 4 of 5 patients who had amyloid deposits in endomyocardial biopsy. On the other hand, all 5 patients without amyloid deposits were negative in planer image. 99mTc-aprotinin (SPECT/CT image) was positive in all 5 patients who had amyloid deposits. CONCLUSIONS: 99mTc-aprotinin scintigraphy is valuable for the non-invasive diagnosis of AL cardiac amyloidosis.

Selexipag for Chronic Thromboembolic Pulmonary Hypertension in Japanese Patients　- A Double-Blind, Randomized, Placebo-Controlled, Multicenter Phase II Study.

BACKGROUND: Selexipag is an oral prostacyclin receptor (IP receptor) agonist with a non-prostanoid structure. This study examined its efficacy and safety in Japanese patients with non-operated or persistent/recurrent chronic thromboembolic pulmonary hypertension (CTEPH).Methods and Results:This Phase II study was a randomized, double-blind, placebo-controlled parallel-group comparison. The primary endpoint was a change in pulmonary vascular resistance (PVR) from baseline to week 17. The main analysis involved a per-protocol set group of 28 subjects. The change in PVR (mean±SD) after 17 weeks of treatment in the selexipag group was -104±191 dyn·s/cm5, whereas that in the placebo group was 26±180 dyn·s/cm5. Thus, the treatment effect after 17 weeks of selexipag treatment was calculated as -130±189 dyn·s/cm5(P=0.1553). Although the primary endpoint was not met, for the group not concomitantly using a pulmonary vasodilator the PVR in the selexipag group was significantly decreased compared with placebo group (P=0.0364). The selexipag group also showed improvement in total pulmonary resistance and cardiac index. CONCLUSIONS: Selexipag treatment improved pulmonary hemodynamics in Japanese patients with CTEPH, but PVR did not show a significant difference between the selexipag and placebo groups. (Trial registration: JAPIC Clinical Trials Information [JapicCTI-111667]).

Radionuclide Imaging of Cardiac Amyloidosis and Sarcoidosis: Roles and Characteristics of Various Tracers.

Amyloidosis and sarcoidosis are systemic diseases that affect multiple organ systems. Accurate diagnosis of cardiac amyloidosis and sarcoidosis is particularly important because cardiac involvement can be fatal. Amyloidosis is characterized by the deposition of amyloid fibrils, and cardiac amyloidosis is classified into amyloid immunoglobulin light chain (AL) and amyloid transthyretin (ATTR) types. Radionuclide tracers for amyloidosis include (a) bone tracers, (b) amyloid-directed molecules, and (c) PET amyloid agents. Bone tracers are particularly sensitive in detection of ATTR type amyloidosis, whereas PET amyloid agents show a higher affinity for the AL type. In sarcoidosis, gallium 67 (67Ga) citrate scintigraphy and fluorine 18 (18F) fluorodeoxyglucose (FDG) PET are pivotal to diagnosis of cardiac sarcoidosis, and 18F-FDG PET/CT has particularly high efficacy in detection of sarcoidosis and monitoring of response to therapy. A major limitation of 18F-FDG is physiologic uptake in the myocardium, which can remain in approximately 20% of patients even after elaborate preparation (eg, prolonged fasting >12-18 hours, modification to a high-fat and low-carbohydrate diet, and injection of unfractionated heparin). This limitation has led to a search for potential new tracers. Recently introduced tracers that show promise include those used in somatostatin receptor imaging and cellular proliferation imaging, which provide detectability as high as that for 18F-FDG without requiring dietary restrictions and have potential for monitoring disease activity. ©RSNA, 2020.

Efficacy and Safety of an Orally Administered Selective Prostacyclin Receptor Agonist, Selexipag, in Japanese Patients With Pulmonary Arterial Hypertension.

BACKGROUND: Selexipag is an orally available prostacyclin receptor (IP receptor) agonist with a non-prostanoid structure. In this open-label Phase II trial, the efficacy and safety of selexipag in Japanese patients with pulmonary arterial hypertension (PAH) is examined.Methods and Results:Selexipag was administered at 200 µg twice daily and titrated up to 1,600 µg by increments of 200 µg in 37 subjects to reach the individual maximum tolerated dose. At 16 weeks, in 33 patients comprising the per-protocol set, the pulmonary vascular resistance (PVR; primary endpoint) decreased from 683.2±237.3 to 560.3±238.7 dyn·s/cm5(P<0.0001). For the secondary endpoint, the 6-min walk distance (6MWD) increased from 445.0±102.2 to 459.1±112.8 m (P=0.0324); World Health Organization functional class improved in 4 patients (12.1%), and was maintained in 29 patients (87.9%). A decrease in PVR was also shown in patients treated with selexipag, on top of a phosphodiesterase inhibitor and endothelin receptor antagonist. Most of the commonly reported adverse events were consistent with those reported for other PGI2formulations. Thirty-four patients attained the individual maximum tolerated dose (maintenance dose). CONCLUSIONS: The efficacy and tolerability of selexipag in Japanese PAH patients was confirmed by improvement in pulmonary hemodynamics, exercise capacity, symptoms. Selexipag is an efficacious treatment option for Japanese PAH patients. (Trial registration: JAPIC Clinical Trials Information [JapicCTI-111532].).

DAPagliflozin for the attenuation of albuminuria in Patients with hEaRt failure and type 2 diabetes (DAPPER study): a multicentre, randomised, open-label, parallel-group, standard treatment-controlled trial.

Background: Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the urinary albumin-to-creatinine ratio (UACR) in patients with elevated levels of albuminuria in the presence or absence of heart failure (HF) or type 2 diabetes mellitus (T2D). However, these effects have not yet been reported in the presence of both HF and T2D. This lack of evidence prompted us to conduct a clinical trial on the effects of dapagliflozin on UACR in patients with HF and T2D. Methods: DAPPER is a multicentre, randomised, open-labeled, parallel-group, standard treatment-controlled trial that enrolled patients at 18 medical facilities in Japan. Eligible participants with both HF and T2D and aged between 20 and 85 years were randomly assigned to a dapagliflozin or control (anti-diabetic drugs other than SGLT 2 inhibitors) group with a 1:1 allocation. The primary outcome was changes in UACR from baseline after a two-year observation, and secondary endpoints were cardiovascular (CV) events and parameters related to HF. This trial was registered with the UMIN-CTR registry, UMIN000025102 and the Japan Registry of Clinical Trials, jRCTs051180135. Findings: Between 12 May 2017 and 31 March 2020, 294 patients were randomly assigned to the dapagliflozin group (n = 146) or control group (n = 148). The mean age of patients was 72.1 years and 29% were female. The mean glycated hemoglobin value was 6.9%, mean NT-proBNP was 429.1 pg/mL, mean estimated GFR was 65.7 mL/min/1.73 m2, and median UACR was 25.0 (8.8-74.6) mg/g Cr in the dapagliflozin group and 25.6 (8.2-95.0) mg/g Cr in the control group. Of the 146 patients in the dapagliflozin group, 122 completed the study, and 107 (87.7%) were taking 5 mg of dapagliflozin daily at the end of the observation period. The primary outcome did not significantly differ between the dapagliflozin and control groups. Among the secondary endpoints, the mean decrease in left ventricular end-diastolic dimensions as one of the echocardiographic parameters was larger in the dapagliflozin group than in the control group. The composite endpoint, defined as CV death or hospitalisation for CV events, hospitalisation for HF events, hospitalisation for all causes, and an additional change in prescriptions for heart failure in a two-year observation, was less frequent in the dapagliflozin group than in the control group. Interpretation: Although dapagliflozin at a dose of 5 mg daily did not reduce urinary albumin excretion in patients with HF and T2D from that in the controls, our findings suggest that dapagliflozin decreased CV events and suppressed left ventricular remodeling. Funding: AstraZeneca KK, Ono Pharmaceutical Co., Ltd.

Novel comb-shaped PEG modification enhances the osteoclastic inhibitory effect and bone delivery of osteoprotegerin after intravenous administration in ovariectomized rats.

PURPOSE: Recombinant osteoprotegerin (OPG) has been proven to be useful for treating various bone disorders such as osteoporosis. To improve its in vivo pharmacological effect, OPG was conjugated to novel comb-shaped co-polymers of polyethylene glycol (PEG) allylmethylether and maleamic acid (poly(PEG), 5 kDa). Biodistribution and bioactivity were evaluated. METHODS: OPG was conjugated via lysine to poly(PEG) and to linear PEG (0.5 kDa and 5 kDa). Poly(PEG)-OPG was compared with linear PEG0.5k-OPG and PEG5k-OPG in terms of in vitro and in vivo efficacy and bone distribution. RESULTS: The in vitro receptor binding study showed that poly(PEG)-OPG could be the most bioactive among the three PEG-OPG derivatives. Pharmacokinetic studies in ovariectomized (OVX) rats showed that serum half-life and AUC of poly(PEG)-OPG were comparable with those of linear PEG-OPG derivatives. For in vivo pharmacological effect, poly(PEG)-OPG showed the strongest inhibitory effect on bone resorption activity in OVX rats. Poly(PEG)-OPG demonstrated enhanced bone marrow distribution with higher selectivity than linear PEG5k-OPG. CONCLUSION: Poly(PEG) modification could provide longer residence time in serum and higher bone-marrow specific delivery of OPG, leading to a higher in vivo pharmacological effect.

Computer simulation of clinical electrophysiological study.

BACKGROUND: Computer simulation is an important method for the basic researches of cardiac electrophysiology to prove the mechanisms and hypotheses of cardiac arrhythmias by reproducing body surface electrocardiograms. In this research, we extend the researches of heart models to the computer simulation of clinical electrophysiological study (EPS) as a clinical application of heart models. METHOD: Through setting the standard EPS pacing protocols, including extrastimuli and incremental pacing in the heart models, we simulated the corresponding excitation propagations in the heart and the intracardiac electrograms that would be measured with catheter electrodes. RESULTS: Examples of complicated cases observed during EPS are reproduced in this research, including the induction of a Wenckebach pattern, the induction and termination of supraventricular tachycardia due to the reentry loop of antidromic atrioventricular reentrant tachycardia for the Wolff-Parkinson-White (WPW) syndrome (Type A), and the localization of an accessory pathway for the WPW syndrome (Type A). CONCLUSION: This study demonstrates an application of whole-heart modeling and computer simulation to clinical EPS.

Human carboxymethylenebutenolidase as a bioactivating hydrolase of olmesartan medoxomil in liver and intestine.

Olmesartan medoxomil (OM) is a prodrug type angiotensin II type 1 receptor antagonist widely prescribed as an antihypertensive agent. Herein, we describe the identification and characterization of the OM bioactivating enzyme that hydrolyzes the prodrug and converts to its pharmacologically active metabolite olmesartan in human liver and intestine. The protein was purified from human liver cytosol by successive column chromatography and was identified by mass spectrometry to be a carboxymethylenebutenolidase (CMBL) homolog. Human CMBL, whose endogenous function has still not been reported, is a human homolog of Pseudomonas dienelactone hydrolase involved in the bacterial halocatechol degradation pathway. The ubiquitous expression of human CMBL gene transcript in various tissues was observed. The recombinant human CMBL expressed in mammalian cells was clearly shown to activate OM. By comparing the enzyme kinetics and chemical inhibition properties between the recombinant protein and human tissue preparations, CMBL was demonstrated to be the primary OM bioactivating enzyme in the liver and intestine. The recombinant CMBL also converted other prodrugs having the same ester structure as OM, faropenem medoxomil and lenampicillin, to their active metabolites. CMBL exhibited a unique sensitivity to chemical inhibitors, thus, being distinguishable from other known esterases. Site-directed mutagenesis on the putative active residue Cys(132) of the recombinant CMBL caused a drastic reduction of the OM-hydrolyzing activity. We report for the first time that CMBL serves as a key enzyme in the bioactivation of OM, hydrolyzing the ester bond of the prodrug type xenobiotics.

Distribution of KAI-9803, a novel δ-protein kinase C inhibitor, after intravenous administration to rats.

KAI-9803 is composed of a selective δ-protein kinase C (δPKC) inhibitor peptide derived from the δV1-1 portion of δPKC (termed "cargo peptide"), conjugated reversibly to the cell-penetrating peptide 11-amino acid, arginine-rich sequence of the HIV type 1 transactivator protein (TAT47₋57; termed "carrier peptide") via a disulfide bond. KAI-9803 administration at the end of ischemia has been found to reduce cardiac damage caused by ischemia-reperfusion in a rat model of acute myocardial infarction. In the study presented here, we examined the TAT47₋57-mediated distribution of KAI-9803 in rats after a single intravenous bolus administration (1 mg/kg). ¹4C-KAI-9803 was rapidly delivered to many tissues, including the heart (1.21 µg eq/g tissue), while being quickly cleared from the systemic circulation. The microautoradiography analysis showed that ¹4C-KAI-9803 was effectively delivered into various cells, including cardiac myocytes and cardiac endothelial cells within 1 min after dosing. The tissue distribution of ¹²5I-labeled KAI-9803 was compared to that of ¹²5I-labeled cargo peptide; this comparison demonstrated that the distribution of KAI-9803 to tissues such as the liver, kidney, and heart was facilitated by the reversible conjugation to TAT47₋57. In an in vitro cardiomyocyte study, the extent of ¹²5I-KAI-9803 internalization was greater at 37°C than that at 4°C, whereas the internalization of the ¹²5I-cargo peptide at 37°C was not observed, indicating that the uptake of ¹²5I-KAI-9803 into the cardiomyocytes was mediated by the TAT47₋57 carrier. Our studies demonstrated that after a single intravenous administration, KAI-9803 can be delivered into the target cells in the liver, kidney, and heart by a TAT47₋57-mediated mechanism.

Pharmacokinetics, metabolism, and disposition of rivoglitazone, a novel peroxisome proliferator-activated receptor γ agonist, in rats and monkeys.

The pharmacokinetics, metabolism, and excretion of rivoglitazone [(RS)-5-{4-[(6-methoxy-1-methyl-1H-benzimidazol-2-yl)methoxy]benzyl}-1,3-thiazolidine-2,4-dione monohydrochloride], a novel thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ selective agonist, were evaluated in male F344/DuCrlCrlj rats and cynomolgus monkeys. The total body clearance and volume of distribution of rivoglitazone were low in both animals (0.329-0.333 ml per min/kg and 0.125-0.131 l/kg for rats and 0.310-0.371 ml per min/kg and 0.138-0.166 l/kg for monkeys), and the plasma half-life was 4.55 to 4.84 h for rats and 6.21 to 6.79 h for monkeys. The oral bioavailability was high (>95% in rats and >76.1% in monkeys), and the exposure increased dose proportionally. After administration of [(14)C]rivoglitazone, radioactivity was mainly excreted in feces in rats, whereas radioactivity was excreted in urine and feces with the same ratio in monkeys. Because excreted rivoglitazone in urine and bile was low, metabolism was predicted to be the main contributor to total body clearance. The structures of 20 metabolites (M1-M20) were identified, and 5 initial metabolic pathways were proposed: O-demethylation, TZD ring opening, N-glucuronidation, N-demethylation, and TZD ring hydroxylation. O-Demethylation was the main metabolic pathway in both animals, but N-demethylation and TZD ring hydroxylation were observed only in monkeys. N-Glucuronide (M13) was nonenzymatically hydrolyzed to TZD ring-opened N-glucuronide (M9), and the amount of these metabolites in monkeys was larger than that in rats. In plasma, rivolitazone was observed as the main component in both animals, and O-demethyl-O-sulfate (M11) was observed as the major metabolite in rats but as many minor metabolites in monkeys.

In vitro metabolism of rivoglitazone, a novel peroxisome proliferator-activated receptor γ agonist, in rat, monkey, and human liver microsomes and freshly isolated hepatocytes.

The in vitro metabolism of rivoglitazone, (RS)-5-{4-[(6-methoxy-1-methyl-1H-benzimidazol-2-yl)methoxy]benzyl}-1,3-thiazolidine-2,4-dione monohydrochloride, a novel thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ selective agonist, was studied in liver microsomes and freshly isolated hepatocytes of rat, monkey, and human as well as cDNA-expressed human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes. Fourteen metabolites were detected, and these structures were elucidated by liquid chromatography-tandem mass spectrometry. Five initial metabolic pathways of rivoglitazone consisting of four oxidation pathways and one N-glucuronidation pathway were predicted in correspondence with those proposed for in vivo studies using rats and monkeys. In metabolization using liver microsomes, the TZD ring-opened mercapto amide (M22) and TZD ring-opened mercapto carboxylic acid (M23) were identified as the primary metabolite of the TZD ring-opening pathway and its sequential metabolite, which have not been detected previously from in vivo studies. Combination with S-adenosyl-L-methionine was useful to obtain the sequential S-methylated metabolites from the oxidative metabolites. N-Glucuronide and sequential TZD ring-opened metabolites were also found in liver microsomes in the presence of UDP-glucuronic acid. The O-demethyl-O-sulfate (M11), which is the major in vivo metabolite in rats and monkeys, was detected in all species of hepatocytes. In addition, a TZD ring-opened S-cysteine conjugate (M15) was detected in human hepatocytes. From these results, the in vivo metabolic pathways in humans were predicted to be the four oxidation and one N-glucuronidation pathways. The four oxidative metabolites were formed by multiple human P450 enzymes, and N-glucuronide was formed by UGT1A3 and UGT2B7.

Combination of GSH trapping and time-dependent inhibition assays as a predictive method of drugs generating highly reactive metabolites.

Covalent binding (CB) of reactive metabolites (RMs) is potentially involved in severe adverse drug reactions. Because the CB assay is of low throughput and costly, a qualitative trapping assay using agents such as [(35)S]GSH is often performed in the early stages of drug discovery. However, trapping methods alone cannot replace the CB assay. We hypothesized that the time-dependent inhibition (TDI) assay might be complementary to the [(35)S]GSH trapping assay in detecting RMs. We performed CB assays, [(35)S]GSH trapping assays, and TDI assays for 42 structurally diverse compounds. First, we showed that the [(35)S]GSH trapping assay alone does not correlate with the extent of CB. Four compounds that the [(35)S]GSH trapping assay failed to detect but that showed high extent of CB were inactivators of the enzyme in the TDI assay. There was a tendency for compounds judged as positive in the TDI assay to show a high degree of CB irrespective of the result of the [(35)S]GSH trapping assay. Finally, to combine parameters from the two assays, we introduced intrinsic clearance to describe the formation of RMs (CL(int, RMs)). The Spearman rank correlation coefficient between the extent of CB and CL(int, RMs) was 0.77 (p < 0.0001), which was better than that for the formation rates of [(35)S]GSH adducts. Therefore, we demonstrated that a combination of the [(35)S]GSH trapping and TDI assays is an effective method for detecting compounds potentially capable of generating highly reactive metabolites in the early stages of drug discovery.

Human pharmacokinetic prediction of UDP-glucuronosyltransferase substrates with an animal scale-up approach.

The aim of the current study was to evaluate the accuracy of allometric scaling methods for drugs metabolized by UDP-glucuronosyltransferases (UGTs), such as ketoprofen, imipramine, lorazepam, levofloxacin, zidovudine, diclofenac, furosemide, raloxifene, gemfibrozil, mycophenolic acid, indomethacin, and telmisartan. Human plasma clearance (CL) predictions were conducted from preclinical in vivo data by using multiple-species allometry with the rule of exponents and single-species allometric scaling (SSS) of mice, rats, monkeys, or dogs. Distribution volume at a steady state (V(ss)) was predicted by multiple-species allometry or SSS of V(ss). Oral plasma clearance (CL(po)) was calculated under the assumption that F(a) × F(g) was equivalent across species. Each of the results was compared with the observed parameter calculated from the clinical data after intravenous or oral administration. Multiple-species allometry and SSS of mice, rats, and dogs resulted in a similar accuracy of CL and CL(po) predictions. Monkeys tended to provide the most accurate predictions of human CL and CL(po). The ability to predict the half-life, which was determined from CL and V(ss) predictions, was more accurate in SSS of rats and monkeys. The in vivo fraction metabolized by glucuronidation (f(m,UGT)) in bile duct-cannulated monkeys was relatively similar to that of humans compared with other animal species, which likely contributed to the highest accuracy of SSS prediction of monkeys. On the basis of the current results, monkeys would be more reliable than other animal species in predicting human pharmacokinetics and f(m,UGT) for drugs metabolized by UGTs.