Fasiglifam (TAK-875), a Free Fatty Acid Receptor 1 (FFAR1) agonist in development for the treatment of type 2 diabetes, was voluntarily terminated in phase 3 due to adverse liver effects. A mechanistic investigation described in this manuscript focused on the inhibition of bile acid (BA) transporters as a driver of the liver findings. TAK-875 was an in vitro inhibitor of multiple influx (NTCP and OATPs) and efflux (BSEP and MRPs) hepatobiliary BA transporters at micromolar concentrations. Repeat dose studies determined that TAK-875 caused a dose-dependent increase in serum total BA in rats and dogs. Additionally, there were dose-dependent increases in both unconjugated and conjugated individual BAs in both species. Rats had an increase in serum markers of liver injury without correlative microscopic signs of tissue damage. Two of 6 dogs that received the highest dose of TAK-875 developed liver injury with clinical pathology changes, and by microscopic analysis had portal granulomatous inflammation with neutrophils around a crystalline deposition. The BA composition of dog bile also significantly changed in a dose-dependent manner following TAK-875 administration. At the highest dose, levels of taurocholic acid were 50% greater than in controls with a corresponding 50% decrease in taurochenodeoxycholic acid. Transporter inhibition by TAK-875 may cause liver injury in dogs through altered bile BA composition characteristics, as evidenced by crystalline deposition, likely composed of test article, in the bile duct. In conclusion, a combination of in vitro and in vivo evidence suggests that BA transporter inhibition could contribute to TAK-875-mediated liver injury in dogs.
The poly(ethylene glycol) (PEG)-modified enzyme superoxide dismutase (PEG-SOD) was characterized using capillary electrophoresis (CE), and the results were compared to those obtained using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. A free-solution CE method was developed which separated PEG-SOD species based on the number of attached PEG chains. The identity of individual CE peaks was established by isolation and subsequent molecular weight analysis by MALDI. The accuracy of the CE method in determining the average total number of PEG chains attached to the enzyme was established by comparing it with an HPLC method that quantitates the amount of PEG after hydrolytic cleavage from the modified enzyme. In addition to determining the average number of PEG chains attached to the protein, the CE method provides information on the distribution of PEG conjugation to the SOD. The total amount and distribution of coupling of PEG to the enzyme was also directly measured using MALDI. The distribution profile of PEG modification for a typical sample of PEG-SOD as determined by CE was found to be consistent with that obtained by MALDI. A quantitative comparison of the results obtained by the two techniques on PEG-SOD samples representing a range of values of PEG modification of the enzyme demonstrated good correlation, although differences were noted in some cases. Both techniques achieved a high degree of reproducibility when properly optimized. Data were also obtained on stressed samples of PEG-SOD demonstrating the ability of the CE method to detect changes in the degree and distribution of PEG conjugation for samples on stability. These two techniques provide a convenient means of characterizing the distribution of coupling of PEG to this enzyme which may have applications to other PEG modified proteins.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTProbing conformational changes in proteins by mass spectrometrySwapan K. Chowdhury, Viswanatham Katta, and Brian T. ChaitCite this: J. Am. Chem. Soc. 1990, 112, 24, 9012–9013Publication Date (Print):November 1, 1990Publication History Published online1 May 2002Published inissue 1 November 1990https://pubs.acs.org/doi/10.1021/ja00180a074https://doi.org/10.1021/ja00180a074research-articleACS PublicationsRequest reuse permissionsArticle Views1741Altmetric-Citations558LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Quantitative assessment of hepatic clearance (CLH) of drugs is critical to accurately predict human dose and drug-drug interaction (DDI) liabilities. This is challenging for drugs that involve complex transporter-enzyme interplay. In this study, we demonstrate this interplay in the CLH and DDI effect in the presence of CYP3A4 perpetrator for pevonedistat using both the conventional clearance model (CCM) and the extended clearance model (ECM). In vitro metabolism and hepatocyte uptake data showed that pevonedistat is actively transported into the liver via multiple uptake transporters and metabolized predominantly by CYP3A4 (88%). The active uptake clearance (CLact,inf) and passive diffusion clearance (CLdiff,inf) were 21 and 8.7 ml/min/kg, respectively. The CLact,inf was underpredicted as Empirical Scaling Factor of 13 was needed to recover the in vivo plasma clearance (CLplasma). Both CCM and ECM predicted CLplasma of pevonedistat reasonably well (predicted CLplasma of 30.8 (CCM) and 32.1 (ECM) versus observed CLplasma of 32.2 ml/min/kg). However, both systemic and liver exposures in the presence of itraconazole were well predicted by ECM but not by CCM (predicted pevonedistat plasma area under the concentration-time curve ratio (AUCR) 2.73 (CCM) and 1.23 (ECM))., The ECM prediction is in accordance with the observed clinical DDI data (observed plasma AUCR of 1.14) that showed CYP3A4 inhibition did not alter pevonedistat exposure systemically, although ECM predicted liver AUCR of 2.85. Collectively, these data indicated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat and are consistent with the lack of systemic clinical DDI with itraconazole.
SIGNIFICANCE STATEMENT
In this study, we successfully demonstrated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat. Both the conventional and extended clearance models predict CLplasma of pevonedistat well however, only the ECM accurately predicted DDI effect in the presence of itraconazole, thus providing further evidence for the lack of DDI with CYP3A4 perpetrators for drugs that involve complex transporter-enzyme interplay as there are currently not many examples in the literature except prototypical OATP substrate drugs.
L. Y. Chiang, J. W. Swirczewski, C. S. Hsu, S. K. Chowdhury, S. Cameron and K. Creegan, J. Chem. Soc., Chem. Commun., 1992, 1791 DOI: 10.1039/C39920001791
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