A conscious rat model involving bradycardia and hypotension after oral administration: a toxicokinetical study of aconitine

1.?A model of aconitine-induced bradycardia and hypotension, which is similar to aconitine poisoning in humans, was constructed in conscious rats by oral administration.

2.?Blood pressure (BP) and heart rate (HR) of Sprague-Dawley rats were measured using a volume pressure recording (VPR) system. The pharmacokinetics of toxic doses of aconitine and its metabolites were analyzed using UPLC-MS/MS.

3.?The HR was significantly decreased by 29% at 2?h after oral administration of 200?μg/kg aconitine. When the dose was increased to 400?μg/kg, systolic BP and diastolic BP were significantly decreased by 11% and 12% at 2?h after the administration, except when bradycardia occurred at 2?h and 4?h. The drug concentration-time curve showed a double-peak phenomenon in rats administered a 400?μg/kg dose. The AUC_0–12?h value in the 400?μg/kg group significantly increased 0.8-fold compared to the 200?μg/kg group. Moreover, a high plasma concentration of 16-O-demethyaconitine was found in the rats that received two toxic doses.

4.?In conclusion, bradycardia and hypotension are induced in conscious rats by a toxic dose of aconitine (400?μg/kg), and there was no significant difference in dose-normalized AUC_0–12?h values between oral administrations of 200?μg/kg and that of 400?μg/kg. However, the dose-normalized C_max and AUC_0–12?h values in 200?μg/kg and 400?μg/kg groups were significantly smaller than those in 100?μg/kg group. The metabolites of aconitine, 16-O-demethyaconitine, and benzoylaconitine may also contribute to the hypotensive response.     

Identification of the cytochrome P450 enzymes involved in the metabolism of domperidone

1.?The objective was to identify the major cytochrome P450 enzyme(s) involved in the metabolism of domperidone.

2.?Experiments were performed using human liver microsomes (HLMs), recombinant human cytochrome P450 enzymes, cytochrome P450 chemical inhibitors and monoclonal antibodies directed against cytochrome P450 enzymes.

3.?Four metabolites were identified from incubations performed with HLMs and excellent correlations were observed between the formation of domperidone hydroxylated metabolites (M1, M3 and M4), N-desalkylated domperidone metabolite (M2) and enzymatic markers of CYP3A4/5 (r²?=?0.9427, 0.951, 0.9497 and 0.8304, respectively).

4.?Ketoconazole (1?μM) decreased the formation rate of M1, M2, M3 and M4 by 83, 78, 75 and 88%, respectively, whereas the effect of other inhibitors (quinidine, furafylline and sulfaphenazole) was minimal. Important decreases in the formation rate of M1 (68%), M2 (64%) and M3 (54%) were observed with anti-CYP3A4 antibodies.

5.?Formation of M1, M2 and M3 in HLMs exhibited Michaelis–Menten kinetics (K_m: 166, 248 and 36?μM, respectively). Similar K_m values were observed for M1, M2 and M3 when incubations were performed with recombinant human CYP3A4 (K_m: 107, 273 and 34?μM, respectively).

6.?The data suggest that CYP3As are the major enzymes involved in the metabolism of domperidone.     

Preclinical metabolism and pharmacokinetics of NVS-CRF38, a potent and orally bioavailable corticotropin-releasing factor receptor 1 antagonist

Abstract

1.?The pharmacokinetic properties and metabolism of NVS-CRF38 [7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole], a novel corticotropin-releasing factor receptor 1 (CRF₁) antagonist, were determined in vitro and in animals.

2.?NVS-CRF38 undergoes near complete absorption in rats and dogs. In both species the compound has low hepatic extraction and is extensively distributed to tissues.

3.?In rat and human hepatic microsomes and cryopreserved hepatocytes from rat, dog, monkey and human, NVS-CRF38 was metabolised to form O-desmethyl NVS-CRF38 (M7) and several oxygen adducts (M1, M3, M4, M5 and M6). In hepatocytes further metabolites were observed, specifically the carboxylic acid (M2) and conjugates (sulphate and glucuronide) of M7.

4.?Formation of primary metabolites in hepatocytes was blocked by the cytochrome P450 enzyme (P450) suicide inhibitor 1-aminobenzotriazole, implicating P450 enzymes in the primary metabolism of this compound.

5.?NVS-CRF38 is weakly bound to plasma proteins from rat (fu_b?=?0.19), dog (fu_b?=?0.25), monkey (fu_b?=?0.20) and humans (fu_b?=?0.23). Blood-to-plasma partition for NVS-CRF38 approaches unity in rat and human blood.

6.?The hepatic clearance of NVS-CRF38 in humans is predicted to be low (extraction ratio?～?0.2) based on scaling from drug depletion profiles in hepatic microsomes.     

In vitro inhibition of UGT1A3, UGT1A4 by ursolic acid and oleanolic acid and drug–drug interaction risk prediction

1.?Ursolic acid (UA) and oleanolic acid (OA) may have important activity relevant to health and disease prevention. Thus, we studied the activity of UA and OA on UDP-glucuronosyltransferases (UGTs) and used trifluoperazine as a probe substrate to test UGT1A4 activity. Recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as a probe reaction for other UGT isoforms.

2.?UA and OA inhibited UGT1A3 and UGT1A4 activity but did not inhibit other tested UGT isoforms.

3.?UA-mediated inhibition of UGT1A3 catalyzed 4-MU-β-d-glucuronidation was via competitive inhibition (IC₅₀ 0.391?±?0.013?μM; K_i 0.185?±?0.015?μM). UA also competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 2.651?±?0.201?μM; K_i 1.334?±?0.146?μM).

4.?OA offered mixed inhibition of UGT1A3-mediated 4-MU-β-d-glucuronidation (IC₅₀ 0.336?±?0.013?μM; K_i 0.176?±?0.007?μM) and competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 5.468?±?0.697?μM; K_i 6.298?±?0.891?μM).

5.?Co-administering OA or UA with drugs or products that are substrates of UGT1A3 or UGT1A4 may produce drug-mediated side effects.     

Human mass balance,metabolite profile and identification of metabolic enzymes of [14C]ASP015K,a novel oral janus kinase inhibitor

Abstract

1.?The human mass balance of ¹⁴C-labelled ASP015K ([¹⁴C]ASP015K), an orally bioavailable Janus kinase (JAK) inhibitor, was characterized in six healthy male subjects after a single oral dose of [¹⁴C]ASP015K (100?mg, 3.7?MBq) in solution. [¹⁴C]ASP015K was rapidly absorbed with t_max of 1.6 and 1.8?h for ASP015K and total radioactivity in plasma, respectively. Mean recovery in urine and feces amounted to 36.8% and 56.6% of the administered dose, respectively. The main components of radioactivity in plasma and urine were ASP015K and M2 (5′-O-sulfo ASP015K). In feces, ASP015K and M4 (7-N-methyl ASP015K) were the main components.

2.?In vitro study of ASP015K metabolism showed that the major isozyme contributing to the formation of M2 was human sulfotransferase (SULT) 2A1 and of M4 was nicotinamide N-methyltransferase (NNMT).

3.?The in vitro intrinsic clearance (CL_{int_in?vitro}) of M4 formation from ASP015K in human liver cytosol (HLC) was 11-fold higher than that of M2. The competitive inhibitory effect of nicotinamide on M4 formation in the human liver was considered the reason for high CL_{int_in vitro} of M4 formation, while each metabolic pathway made a near equal contribution to the in vivo elimination of ASP015K. ASP015K was cleared by multiple mechanisms.     

Isolation and identification of phase I metabolites of butyrolactone I in rats

1.?Butyrolactone I (BL-I), one of the major secondary metabolites of fungus Aspergillus terreus, is a selective cdc2 kinase inhibitor. In the present study, the metabolism of BL-I in male Wistar rats was investigated by characterizing metabolites excreted into feces.

2.?Following an oral dose of 40?mg/kg BL-I, 10 phase I metabolites were isolated from the feces of rats, and their structures were identified on the basis of a range of spectroscopic data and ICD analysis. These metabolites were fully characterized as butyrolactone VI (M1), aspernolide E (M2), 7′′S-hydroxy-9′′-ene-butyrolactone I (M3), 7′′R-hydroxy-9′′-ene-butyrolactone I (M4), 7″S, 8″R-dihydroxy-aspernolide E (M5), 7″R, 8″S-dihydroxy-aspernolide E (M6), 7″R-acetyl-8″S-hydroxy-aspernolide E (M7), 7″S-acetyl-8″R-hydroxy-aspernolide E (M8), 7″R-methoxy-8″S-hydroxy-aspernolide E (M9), butyrolactone V (M10), respectively. It is the first time to describe the metabolites of BL-I in vivo, and metabolites M3 to M9 are new compounds.

3.?BL-I and metabolites M2 to M10 were evaluated for their antimicrobial activity and in vitro antiproliferative activities. Only M-3 and M-4 showed inhibitory effect against staphylococcus aureus both with MIC of 125?μg/ml. BL-I and metabolites M-4 and M-5 exhibited potent cancer cell growth inhibitory activities against HL-60 (human leukemia) cell lines with the IC₅₀ values of 13.2, 28.8 and 35.7?μM, respectively.

4.?On the basis of metabolites profile, a possible metabolism pathway for BL-I in rats has been proposed. This is the first systematic study on the phase I metabolites of BL-I.     

Pharmacokinetics,metabolism and excretion of [14C]-lanicemine (AZD6765), a novel low-trapping N-methyl-d-aspartic acid receptor channel blocker,in healthy subjects

Abstract

1.?(1S)-1-phenyl-2-(pyridin-2-yl)ethanamine (lanicemine; AZD6765) is a low-trapping N-methyl-d-aspartate (NMDA) channel blocker that has been studied as an adjunctive treatment in major depressive disorder. The metabolism and disposition of lanicemine was determined in six healthy male subjects after a single intravenous infusion dose of 150?mg [¹⁴C]-lanicemine.

2.?Blood, urine and feces were collected from all subjects. The ratios of C_max and AUC_(0–∞) of lanicemine to plasma total radioactivity were 84 and 66%, respectively, indicating that lanicemine was the major circulating component with T_1/2 at 16?h. The plasma clearance of lanicemine was 8.3?L/h, revealing that lanicemine is a low-clearance compound. The mean recovery of radioactivity from urine was 93.8% of radioactive dose.

3.?In urine samples, 10 metabolites of lanicemine were identified. Among which, an O-glucuronide conjugate (M1) was the most abundant metabolite (～11% of the dose in excreta). In plasma, the circulatory metabolites were identified as a para-hydroxylated metabolite (M1), an O-glucuronide (M2), an N-carbamoyl glucuronide (M3) and an N-acetylated metabolite (M6). The average amount of each of metabolite was less than 4% of total radioactivity detected in plasma or urine.

4.?In conclusion, lanicemine is a low-clearance compound. The unchanged drug and metabolites are predominantly eliminated via urinary excretion.     

Identification of the human cytochrome P450s responsible for the in vitro metabolism of a leukotriene B4 receptor antagonist,CP-195,543

1.?The major human cytochrome P450 (CYP) form(s) responsible for the metabolism of CP-195,543, a potent leukotriene B4 antagonist, were investigated.

2.?Incubation of CP-195,543 with human liver microsomes resulted in the formation of three major metabolites, M1–3. M1 and M2 were diastereoisomers and formed by oxidation on the benzylic position. M3 was formed by aromatic oxidation of the benzyl group attached to the 3-position of the benzopyran ring.

3.?The results from experiments with recombinant CYPs, correlation studies and inhibition studies with form-selective inhibitors and a CYP3A antibody strongly suggest that the CYP3A4 plays a major role in the metabolism of CP-195,543. Recombinant CYP3A5 did not metabolize CP-195,543.

4.?The apparent K_m and V_max for the formation of M1–3 in human liver microsomes were determined as 36?μM and 4.1?pmol?min^?1?pmol^?1 P450, 44?μM and 10?pmol?min^?1?pmol^?1 P450, and 34?μM and 2.0?pmol?min^?1?pmol^?1 P450, respectively. The average in vitro intrinsic clearance for M2 was the highest both in human liver microsomes and recombinant CYP3A4 compared with M1 and M3. Intrinsic clearance for M2 in human liver microsomes and recombinant CYP3A4 was 0.231 and 0.736 ml?min^?1?pmol^?1 P450, respectively. The intrinsic clearances for M1 and M3 in human liver microsomes and CYP3A4 were 0.114 and 0.060 and 0.197 and 0.088 ml?min^?1?pmol^?1 P450, respectively. This suggests that benzylic oxidation is the predominant phase I metabolic pathway of CP-195,543 in man.     

Pharmacokinetics and metabolism of [14C]-tozadenant (SYN-115), a novel A2a receptor antagonist ligand,in healthy volunteers

1.?This phase-I study (NCT02240290) was designed to investigate the human absorption, disposition and mass balance of ¹⁴C-tozadenant, a novel A2a receptor antagonist in clinical development for Parkinson s disease.

2.?Six healthy male subjects received a single oral dose of tozadenant (240?mg containing 81.47?KBq of [¹⁴C]-tozadenant). Blood, urine and feces were collected over 14 days. Radioactivity was determined by liquid scintillation counting or accelerator mass spectrometry (AMS). Tozadenant and metabolites were characterized using HPLC-MS/MS and HPLC-AMS with fraction collection.

3.?At 4?h, the C_max of tozadenant was 1.74?μg/mL and AUC_(0–t) 35.0?h?μg/mL, t_1/2 15?h, Vz/F 1.82?L/kg and CL/F 1.40?mL/min/kg. For total [¹⁴C] radioactivity, the C_max was 2.29?μg?eq/mL at 5?h post-dose and AUC_(0–t) 43.9?h?μg?eq/mL. Unchanged tozadenant amounted to 93% of the radiocarbon AUC_(0–48h). At 312?h post-dose, cumulative urinary and fecal excretion of radiocarbon reached 30.5% and 55.1% of the dose, respectively. Unchanged tozadenant reached 11% in urine and 12% of the dose in feces. Tozadenant was excreted as metabolites, including di-and mono-hydroxylated metabolites, N/O dealkylated metabolites, hydrated metabolites.

4.?The only identified species circulating in plasma was unchanged tozadenant. Tozadenant was primarily excreted in urine and feces in the form of metabolites.     

Mechanism-based inactivation of human cytochrome P450 1A2 and 3A4 isoenzymes by anti-tumor triazoloacridinone C-1305

1.?5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, is a promising anti-tumor therapeutic agent with high activity against several experimental tumors.

2.?It was determined to be a potent and selective inhibitor of liver microsomal and human recombinant cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1305 might modulate the effectiveness of other drugs used in multidrug therapy.

3.?The objective of this study was to investigate the mechanism of the observed C-1305-mediated inactivation of CYP1A2 and CYP3A4.

4.?Our findings indicated that C-1305 produced a time- and concentration-dependent decrease in 7-ethoxycoumarin O-deethylation (CYP1A2, K_I?=?10.8?±?2.14?μM) and testosterone 6β-hydroxylation (CYP3A4, K_I = 9.1?±?2.82?μM). The inactivation required the presence of NADPH, was unaffected by a nucleophilic trapping agent (glutathione) and a reactive oxygen species scavenger (catalase), attenuated by a CYP-specific substrate (7-ethoxycoumarin or testosterone), and was not reversed by potassium ferricyanide. The estimated partition ratios of 1086 and 197 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively.

5.?In conclusion, C-1305 inhibited human recombinant CYP1A2 and CYP3A4 isoenzymes by mechanism-based inactivation. The obtained knowledge about specific interactions between C-1305 and/or its metabolites, and CYP isoforms would be useful for predicting the possible drug–drug interactions in potent multidrug therapy.     

Impact of CYP2C8*3 polymorphism on in vitro metabolism of imatinib to N-desmethyl imatinib

Abstract

1.?Imatinib is metabolized to N-desmethyl imatinib by CYPs 3A4 and 2C8. The effect of CYP2C8*3 genotype on N-desmethyl imatinib formation was unknown.

2.?We examined imatinib N-demethylation in human liver microsomes (HLMs) genotyped for CYP2C8*3, in CYP2C8*3/*3 pooled HLMs and in recombinant CYP2C8 and CYP3A4 enzymes. Effects of CYP-selective inhibitors on N-demethylation were also determined.

3.?A single-enzyme Michaelis–Menten model with autoinhibition best fitted CYP2C8*1/*1 HLM (n?=?5) and recombinant CYP2C8 kinetic data (median?±?SD K_i?=?139?±?61?µM and 149?µM, respectively). Recombinant CYP3A4 showed two-site enzyme kinetics with no autoinhibition. Three of four CYP2C8*1/*3 HLMs showed single-enzyme kinetics with no autoinhibition. Binding affinity was higher in CYP2C8*1/*3 than CYP2C8*1/*1 HLM (median?±?SD K_m?=?6?±?2 versus 11?±?2?µM, P=0.04). CYP2C8*3/*3 (pooled HLM) also showed high binding affinity (K_m?=?4?µM) and single-enzyme weak autoinhibition (K_i?=?449?µM) kinetics. CYP2C8 inhibitors reduced HLM N-demethylation by 47–75%, compared to 0–30% for CYP3A4 inhibitors.

4.?In conclusion, CYP2C8*3 is a gain-of-function polymorphism for imatinib N-demethylation, which appears to be mainly mediated by CYP2C8 and not CYP3A4 in vitro in HLM.     

In vitro phase I metabolism of vinclozolin by human liver microsomes

1.?Vinclozolin (Vin) is a fungicide used in agricultural settings and is classified as an endocrine disruptor. Vin is non-enzymatically hydrolyzed to 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) metabolites. There is no information about Vin biotransformation in humans, therefore, the aim of this study was to characterize its in vitro metabolism using human liver microsomes.

2.?Vin was metabolized to the [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione] (M4) and N-(2,3,4-trihydroxy-2-methyl-1-oxo)-3,5-dichlorophenyl-1-carbamic acid (M7) metabolites, which are unstable and gradually converted to 3′,5′-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (DTMBA, formerly denoted as M5). M4 and DTMBA metabolites co-eluted in the same HPLC peak; this co-elute peak exhibited a Michaelis-Menten kinetic, whereas M7 showed a substrate inhibition kinetics. The K_{M app} for co-eluted M4/DTMBA and M7 was 24.2?±?5.6 and 116.0?±?52.6?μM, the V_{Max app} was 0.280?±?0.015 and 0.180?±?0.060 nmoles/min/mg protein, and the CL_{int app} was 11.5 and 1.5?mL/min/g protein, respectively. The K_i for M7 was 133.2?±?63.9?μM. Cytochrome P450 (CYP) chemical inhibitors furafylline (CYP1A2), ketoconazole (CYP3A4), pilocarpine (CYP2A6) and sulfaphenazole (CYP2C9) inhibited M4/DTMBA and M7 formation, suggesting that Vin is metabolized in humans by CYP.

3.?DTMBA is a stable metabolite and specific of Vin, therefore, it could be used as a biomarker of Vin exposure in humans to perform epidemiological studies.     

A microbial model of mammalian metabolism: biotransformation of 4,5-dimethoxyl-canthin-6-one using Cunninghamella blakesleeana CGMCC 3.970

1.?A filamentous fungus, Cunninghamella blakesleeana CGMCC 3.970, was applied as a microbial system to mimic mammalian metabolism of 4,5-dimethoxyl-canthin-6-one (1). Compound 1 belongs to canthin-6-one type alkaloids, which is a major bioactive constituent of a traditional Chinese medicine (the stems of Picrasma quassioides).

2.?After 72?h of incubation in potato dextrose broth, 1 was metabolized to seven metabolites as follows: 4-methoxyl-5-hydroxyl-canthin-6-one (M1), 4-hydroxyl-5-methoxyl-canthin-6-one (M2), canthin-6-one (M3), canthin-6-one N-oxide (M4), 10-hydroxyl-4,5-dimethoxyl-canthin-6-one (M5), 1-methoxycarbonl-β-carboline (M6), and 4-methoxyl-5-O-β-D-glucopyranosyl-canthin-6-one (M7).

3.?The structures of metabolites were determined using spectroscopic analyses, chemical methods, and comparison of NMR data with those of known compounds. Among them, M7 was a new compound.

4.?The metabolic pathways of 1 were proposed, and the metabolic processes involved phase I (O-demethylation, dehydroxylation, demethoxylation, N-oxidation, hydroxylation, and oxidative ring cleavage) and phase II (glycosylation) reactions.

5.?This was the first research on microbial transformation of canthin-6-one alkaloid, which could be a useful microbial model for producing the mammalian phase I and phase II metabolites of canthin-6-one alkaloids.

6.?1, M1?M5, and M7 are canthin-6-one alkaloids, whereas M6 belongs to β-carboline type alkaloids. The strain of Cunninghamella blakesleeana can supply an approach to transform canthin-6-one type alkaloids into β-carboline type alkaloids.     

Anti-HIV-1 integrase compounds from Dioscorea bulbifera and molecular docking study

Context Dioscorea bulbifera L. (Dioscoreaceae) has been used in a traditional Thai longevity medicine preparation. Isolation of inhibitors from natural products is a potential source for continuous development of new HIV-1 integrase (IN) inhibitors.

Objective The objective of this study is to isolate the compounds and evaluate their anti-HIV-1 IN activity, as well as to predict the potential interactions of the compounds with an IN.

Materials and methods The ethyl acetate and water fractions (1–100?μg/mL) of Dioscorea bulbifera bulbils were isolated and tested for their anti-HIV-1 IN activity using the multiplate integration assay (MIA). The interactions of the active compounds with IN were investigated using a molecular docking method.

Results and discussions The ethyl acetate and water fractions of Dioscorea bulbifera bulbils afforded seven compounds. Among these, allantoin (1), 2,4,3′,5′-tetrahydroxybibenzyl (2), and 5,7,4′-trihydroxy-2-styrylchromone (5) were isolated for the first time from this plant. Myricetin (4) exhibited the most potent activity with an IC₅₀ value of 3.15?μM, followed by 2,4,6,7-tetrahydroxy-9,10-dihydrophenanthrene (3, IC₅₀ value=?14.20?μM), quercetin-3-O-β-d-glucopyranoside (6, IC₅₀ value?=?19.39?μM) and quercetin-3-O-β-d-galactopyranoside (7, IC₅₀ value?=?21.80?μM). Potential interactions of the active compounds (3, 4, 6, and 7) with the IN active site were additionally investigated. Compound 4 showed the best binding affinity to IN and formed strong interactions with various amino acid residues. These compounds interacted with Asp64, Thr66, His67, Glu92, Asp116, Gln148, Glu152, Asn155, and Lys159, which are involved in both the 3′-processing and strand transfer reactions of IN. In particular, galloyl, catechol, and sugar moieties were successful inhibitors for HIV-1 IN.     

Predominant contributions of carboxylesterase 1 and 2 in hydrolysis of anordrin in humans

1.?Anordrin (2α, 17α-diethynyl-A-nor-5α-androstane-2β, 17β-diol diproprionate) is post-coital contraceptive drug that is on the market in China for more than 30 years. This study aims to elucidate enzymes involved in anordrin hydrolysis, and to evaluate the significant role of carboxylesterases in anordrin hydrolysis in humans.

2.?Human liver and intestinal microsomes, recombinant human carboxylesterase were selected as enzyme sources. In human liver microsomes, intrinsic clearance was 684?±?83?μL/min/mg protein, which was considerably higher than the value of intestine microsomes (94.6?±?13.3?μL/min/mg protein). Carboxylesterase (CES) 1 has more contribution than CES2 in human liver.

3.?Inhibition studies were performed using representative esterase inhibitors to confirm esterase isoforms involved in anordrin hydrolysis. Simvastatin strongly inhibited hydrolytic process of anordrin in liver and intestine microsomes, with IC₅₀ values of 10.9?±?0.1 and 6.94?±?0.03?μM, respectively.

4.?The present study investigated for the first time hydrolytic enzyme phenotypes of anordrin. Anordrin is predominantly catalyzed by CES1 and CES2 to generate the main active metabolite, anordiol. Moreover, anordrin and its metabolite anordiol can be altered by esterase inhibitors, such as simvastatin, upon exposure in vivo.     

Model for the drug–drug interaction responsible for CYP3A enzyme inhibition. II: establishment and evaluation of dexamethasone-pretreated female rats

1.?Cytochrome P450 (CYP) 3A catalysis of testosterone 6β-hydroxylation in female rat liver microsomes was significantly induced, then reached a plateau level after pretreatment with 80?mg?kg^?1?day^?1 dexamethasone (DEX) for 3 days.

2.?Midazolam was mainly metabolized by CYP3A in DEX-treated female rat liver microsomes from an immuno-inhibition study, and the apparent K_m was 1.8?μM, similar to that in human microsomes.

3.?Ketoconazole and erythromycin, typical CYP3A inhibitors, demonstrated extensive inhibition of midazolam metabolism in DEX-treated female rat liver microsomes, and the apparent K_i values were 0.088 and 91.2?μM, respectively. The values were similar to those in humans, suggesting that DEX-treated female rat liver microsomes have properties similar to those of humans.

4.?After oral administration of midazolam, the plasma midazolam concentration in DEX-treated female rats significantly decreased compared with control female rats. The area under the plasma concentration curve (AUC) and elimination half-life were one-11th and one-20th of those of control female rats, respectively.

5.?Using DEX-treated female rats, the effect of CYP3A inhibitors on midazolam pharmacokinetics was evaluated. The AUC and maximum concentration in plasma (C_max) increased when ketoconazole was co-administered with midazolam.

6.?It was shown that the drug–drug interaction that occurs in vitro is also observed in vivo after oral administration of midazolam. In conclusion, the DEX-treated female rat could be a useful model for evaluating drug–drug interactions based on CYP3A enzyme inhibition.     

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor

1.?The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied.

2.?The cytochrome P450s (CYPs) responsible for metabolism of IN-1130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomes?, were identified using specific CYP inhibitors. The order of disappearance of IN-1130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog.

3.?Five distinct metabolites (M1–M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomes? examined, CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19 were involved in the metabolism of IN-1130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1130 (M1–M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites.

4.?Hence, it is concluded that metabolism of IN-1130 is mediated by CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19.     

In vitro assessment of the roles of drug transporters in the disposition and drug–drug interaction potential of olaparib

1.?In vitro assessments were conducted to examine interactions between olaparib (a potent oral inhibitor of poly[ADP-ribose] polymerase) and drug transporters.

2.?Olaparib showed inhibition of the hepatic drug uptake transporters OATP1B1 (IC₅₀ values of 20.3?μM and 27.1?μM) and OCT1 (IC₅₀ 37.9?μM), but limited inhibition of OATP1B3 (25% at 100?μM); inhibition of the renal uptake transporters OCT2 (IC₅₀ 19.9?μM) and OAT3 (IC₅₀ 18.4?μM), but limited inhibition of OAT1 (13.5% at 100?μM); inhibition of the renal efflux transporters MATE1 and MATE2K (IC₅₀s 5.50?μM and 47.1?μM, respectively); inhibition of the efflux transporter MDR1 (IC₅₀ 76.0?μM), but limited inhibition of BCRP (47% at 100?μM) and no inhibition of MRP2. At clinically relevant exposures, olaparib has the potential to cause pharmacokinetic interactions via inhibition of OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K in the liver and kidney, as well as MDR1 in the liver and GI tract. Olaparib was found to be a substrate of MDR1 but not of several other transporters.

3.?Our assessments indicate that olaparib is a substrate of MDR1 and may cause clinically meaningful inhibition of MDR1, OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K.     

瑞香素抗血栓作用

瑞香素80 mg/kg iv对大鼠实验性血栓形成有明显的抑制作用,抑制率54%,在试管内当浓度为0.025,0.05及0.1 mg/ml时,可抑制ADP诱导的家兔血小板聚集。iv 80 mg/kg 5 min,对ADP诱导的家兔血小板聚集也有明显的抑制作用。iv 40及80 mg/kg 20 min,皆可使血小板粘附性降低;并能延长小鼠凝血时间。iv 80 mg/kg 5 min,对家兔纤维蛋白原含量,全血粘度及血浆比粘度均无明显影响。