Efficiency in Drug Discovery: Liver S9 Fraction Assay As a Screen for Metabolic Stability

Richardson, Samantha; Bai, April; Kulkarni, Ashutosh A.; Moghaddam, Mehran F.

doi:10.2174/1872312810666160223121836

Cited by 105 publications

(88 citation statements)

References 9 publications

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“…The livers were collected from wild-type and Bmal1-KO mice euthanized at ZT2 and ZT14. Liver S9 fraction were prepared by centrifugation at 9000 g for 10 minutes (Richardson et al, 2016). Liver sulfation activity was determined using an incubation method as previously described .…”

Section: Methodsmentioning

confidence: 99%

The Clock Protein Bmal1 Regulates Circadian Expression and Activity of Sulfotransferase 1a1 in Mice

Guo

Zhang

et al. 2018

Drug Metab Dispos

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Sulfotransferase 1a1 (Sult1a1) is a phase II enzyme that contributes extensively to metabolism and detoxification of various drugs and chemicals. Here we aimed to investigate a potential role of the clock protein Bmal1 (brain and muscle Arnt-like protein-1) in circadian regulation of Sult1a1 in mice. The regulatory effects of Bmal1 on Sult1a1 were assessed both in vivo (using Bmal1- deficient mice) and in vitro (using both normal and serum-shocked Hepa-1c1c7 cells). The relative mRNA and protein levels of Sult1a1 in the cells or mouse livers were measured by RT-qPCR and Western blotting, respectively. Sulfation activities of two Sult1a1 substrates (i.e., -nitrophenol and galangin) were determined using mouse liver S9 fractions. Transcriptional regulation of Sult1a1 by Bmal1 was investigated using luciferase reporter, electrophoretic mobility shift (EMSA), and chromatin immunoprecipitation (ChIP) assays. We first showed that hepatic Sult1a1 was rhythmically expressed at both mRNA and protein levels (higher expressions during the night than the daytime). Consistently, the liver sulfation activities toward two Sult1a1 substrates were circadian time dependent with a higher activity at ZT14 than at ZT2. Furthermore, deletion of in mice blunted the circadian rhythmicity of hepatic Sult1a1 (with reduced expression levels). Likewise, Bmal1 positively regulated Sult1a1 expression in conventionally cultured Hepa-1c1c7 cells, and knockdown blunted expression rhythmicity of Sult1a1 in serum-shocked Hepa-1c1c7 cells. A combination of promoter analysis, EMSA and ChIP assays revealed that Bmal1 stimulated Sult1a1 transcription through its specific binding to the-571- to -554-bp region (an E-box element) in the promoter. In conclusion, Bmal1 activated the transcription of Sult1a1 and controlled circadian expression and activity of the enzyme.

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Section: Methodsmentioning

confidence: 99%

The Clock Protein Bmal1 Regulates Circadian Expression and Activity of Sulfotransferase 1a1 in Mice

Guo

Zhang

et al. 2018

Drug Metab Dispos

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“…at 10 mg/kg (n = 5/dose group) to male SD rats (8 weeks old; (255-275) g; Koatech Inc., Gyeonggi, Republic of Korea) housed individually in metabolic cages. Urine samples were collected for ((0-4), (4)(5)(6)(7)(8) and (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)) h. Pooled urine samples were mixed with equal volume of acetonitrile containing HSDH (1 µg/mL) as IS, followed by filtration with a syringeless filter device (Mini-UniPrep™, PTFE filter media with polypropylene housing, 0.45 µm). The filtrate was analyzed by Agilent 6530 Q-TOF LC-MS/MS system equipped with dual AJS ESI ion source and Agilent 1200 series HPLC system, as follows.…”

Section: In Vivo Metabolite Identificationmentioning

confidence: 99%

“…Circulating and urinary metabolites were tentatively identified using a high-resolution LC-MS/MS system in the animals and the corresponding metabolic pathways were proposed. The metabolic stability of SP-8356 was examined using liver S9 fractions of the animals and humans, well-established in vitro system being widely used for drug metabolism studies [11], in the presence of relevant cofactors such as β-nicotinamide adenine dinucleotide 2 -phosphate (NADPH), uridine 5 -diphosphoglucuronic acid (UDPGA), 3'-phosphoadenosyl-5'-phosphosulfate (PAPS), S-(5 -adenosyl)-l-methionine (SAM) and glutathione (GSH). The possibility of bioactivation and the role of competing metabolic pathways in negating the bioactivation were explored as well.…”

Section: Introductionmentioning

confidence: 99%

Metabolism and Pharmacokinetics of SP-8356, a Novel (1S)-(−)-Verbenone Derivative, in Rats and Dogs and Its Implications in Humans

Zhou

Kim

et al. 2020

Molecules

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(1S,5R)-4-((E)-3,4-dihydroxy-5-methoxystryryl)-6,6-dimethylbicylco[3.1.1]hept-3-en-2-one (SP-8356) is a novel (1S)-(−)-verbenone derivative that is currently in preclinical development for the treatment of ischemic stroke and atherosclerosis. This report aimed at characterization of the metabolism and pharmacokinetic properties of SP-8356. Following intravenous dose in rats and dogs, plasma concentrations of SP-8356 declined rapidly with high clearance (CL) and short half-life; after oral administration in both species, its plasma levels were below the quantitation limit. Fourteen circulating metabolites, formed by mono-oxygenation, demethylation, glucuronidation, catechol O-methylation, sulfation and oxidation (bioactivation) followed by glutathione (GSH) conjugation, were tentatively identified in both species. Urinary excretion of SP-8356 appeared to be minimal in rats, compared to its metabolites. GSH conjugate of SP-8356 was also formed during incubation with rat liver S9 fraction consistent with oxidative bioactivation; this bioactivation was almost completely inhibited by the cofactors for glucuronidation, sulfation and methylation, indicating that it may be abolished by competing metabolic reactions in the body. The human pharmacokinetics of SP-8356 was predicted to be similar to that of the animals based on the current in vitro metabolic stability results. In summary, rapid phase II metabolism appears to be mainly responsible for its suboptimal pharmacokinetics, such as high CL and low oral absorption. Because of competing metabolic reactions, potential safety risks related to SP-8356 bioactivation may be low.

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“…The in vitro half‐life ( t 1/2 ) and the intrinsic clearance (CL int ) were calculated as follows:

t_{italic1 / italic2} = - 0.693 / normalK

where K is the slope of the linear regression

{CL}_{int .} = ((), 0.693 / italicin vitro t_{italic1 / italic2}) ((), incubation volume / mg of microsomal protein) ((), 45 mg microsomal protein / gm of liver) ((), 45 normalg liver / kg body weight)

…”

Section: Resultsmentioning

confidence: 99%

Roflumilast analogs with improved metabolic stability, plasma protein binding, and pharmacokinetic profile

Moussa

El‐Zaher

El-Ashrey

et al. 2019

Drug Testing and Analysis

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With the aim of studying their in vitro and in vivo pharmacokinetics, new chromatographic methods were developed for the determination of three new roflumilast synthetic analogs (I−III) as PDE‐4B inhibitors in rat liver S9 fraction, phosphate buffered saline, pH 7.4, and human and rat plasma. The developed high performance liquid chromatography−ultra violet (HPLC−UV) methods were performed on a Zorbax Eclipse C8 column and UV detection was carried out at 215 nm. The three compounds were tested for their metabolic stability and were found to be metabolically more stable than roflumilast especially the 2‐mercaptobenzothiazol‐6‐yl analog (III) which displayed an in vitro half‐life time (247.55 minutes) higher than that of roflumilast (12.29 minutes) and a low in vitro clearance of 5.67 mL/min./kg. Possible phase I metabolites were investigated using ultra‐performance liquid chromatography−tandem mass spectrometry (UPLC–MS/MS) showing hydroxylation of the unsubstituted benzothiazol‐2‐yl (I) and benzothiazole‐6‐yl (II) analogs and a cleaved benzothiazole metabolite of the 2‐mercaptobenzothiazol‐6‐yl analog (III). Plasma protein binding affinity was tested using equilibrium membrane dialysis method showing a very high percentage (more than 95%) of plasma protein binding of compounds I and II where compound III exhibited lower percentage (53.71%) demonstrating its accessibility for tissue distribution. Also, a UPLC–MS/MS method was developed using an Acquity UPLC BEH shield RP C18 column to be applied to an in vivo pharmacokinetic study in rats following a subcutaneous dose (1 mg/kg). Compounds I−III showed improved in vivo pharmacokinetic parameters especially compound III which displayed a half‐life 3‐fold greater than roflumilast (21 hours) and a Cmax value of 113.958 ng/mL. Accordingly, this new chemical entity should be subjected to further investigation as it can be a good drug candidate for treating chronic obstructive pulmonary disease.

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Efficiency in Drug Discovery: Liver S9 Fraction Assay As a Screen for Metabolic Stability

Cited by 105 publications

References 9 publications

The Clock Protein Bmal1 Regulates Circadian Expression and Activity of Sulfotransferase 1a1 in Mice

The Clock Protein Bmal1 Regulates Circadian Expression and Activity of Sulfotransferase 1a1 in Mice

Metabolism and Pharmacokinetics of SP-8356, a Novel (1S)-(−)-Verbenone Derivative, in Rats and Dogs and Its Implications in Humans

Roflumilast analogs with improved metabolic stability, plasma protein binding, and pharmacokinetic profile

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