Hepatocellular carcinoma (HCC) is one of the most malignant tumors in the world. Inadequate efficacy of 5-fluorouracil (5-FU) on HCC could be related to low expression of human organic anion transporter 2 (OAT2). However, the knowledge of downregulation of OAT2 in HCC remains limited. We explored the underlying mechanism focusing on protein expression regulation and attempted to design a strategy to sensitize HCC cells to 5-FU. In this study, we revealed that the 1 bp to 300 bp region of OAT2 mRNA 3' untranslated region (UTR) reduced its protein expression and uptake activity in Li-7 and PLC/PRF/5 cells. Mechanistically, it was demonstrated that staphylococcal nuclease and Tudor domain containing 1 (SND1) bound at the 1 bp to 300 bp region of OAT2 mRNA 3' UTR, leading to a decrease in OAT2 protein expression. Enrichment analysis results indicated reduction of OAT2 might be mediated by translational inhibition. Furthermore, the knockdown of SND1 upregulated OAT2 protein expression and uptake activity. Based on this, decreasing SND1 expression enhanced 5-FU-caused G1/S phase arrest in Li-7 and PLC/PRF/5 cells, resulting in suppression of cell proliferation. Additionally, the knockdown of SND1 augmented the inhibitory effect of 5-FU on PLC/PRF/5 xenograft tumor growth in vivo by increasing OAT2 protein expression and accumulation of 5-FU in the tumor. Collectively, a combination of inhibition of SND1 with 5-FU might be a potential strategy to sensitize HCC cells to 5-FU from the perspective of restoring OAT2 protein level. SIGNIFICANCE STATEMENT: We investigated the regulatory mechanism of OAT2 protein expression in HCC cells and designed a strategy to sensitize them to 5-FU (OAT2 substrate) via restoring OAT2 protein level. It found that SND1, an RNA binding protein, regulated OAT2 protein expression by interacting with OAT2 mRNA 3' UTR 1-300 bp region. Through decreasing SND1, the antitumor effect of 5-FU on HCC was enhanced in vitro and in vivo, indicating that SND1 could be a potential target for sensitizing HCC cells to 5-FU.
Two unique metabolites (M18 and M19) were detected in feces of human volunteers dosed orally with [14C]inavolisib with a molecular ion of parent plus 304 Da. They were generated in vitro by incubation with fecal homogenates and we have evidence that they are formed chemically and possibly enzymatically. Structural elucidation by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy showed that the imidazole ring of inavolisib was covalently bound to partial structures derived from stercobilin, an end-product of heme catabolism produced by the gut microbiome. The structural difference between the two metabolites was the position of methyl and ethyl groups on the pyrrolidin-2-one moieties. We propose a mechanism of M18 and M19 generation from inavolisib and stercobilin whereby nucleophilic attack from the imidazole ring of inavolisib occurs to the bridging carbon of a stercobilin molecule. The proposed mechanism was supported by computational calculations of molecular orbitals and transition geometry. SIGNIFICANCE STATEMENT: We report the characterization of two previously undescribed conjugates of the phosphoinositide 3-kinase inhibitor inavolisib, generated by reaction with stercobilin, an end-product of heme catabolism produced by the gut microbiome. These conjugates were confirmed by generating them using in vitro fecal homogenate incubation via nonenzymatic and possibly enzymatic reactions. Given the unique nature of the conjugate, it is plausible that it may have been overlooked with other small molecule drugs in prior studies.
Recently, we have proposed simple methodology to derive clearance and rate constant equations, independent of differential equations, based on Kirchhoff's Laws, a common methodology from physics used to describe rate-defining processes either in series or parallel. Our approach has been challenged in three recent publications, two published in this journal, but notably what is lacking is that none evaluate experimental pharmacokinetic data. As reviewed here, manuscripts from our laboratory have evaluated published experimental data, demonstrating that the Kirchhoff's Laws approach explains (1) why all of the experimental perfused liver clearance data appear to fit the equation that was previously believed to be the well-stirred model, (2) why linear pharmacokinetic systemic bioavailability determinations can be greater than 1, (3) why renal clearance can be a function of drug input processes, and (4) why statistically different bioavailability measures may be found for urinary excretion versus systemic concentration measurements. Our most recent paper demonstrates (5) how the universally accepted steady-state clearance approach used by the field for the past 50 years leads to unrealistic outcomes concerning the relationship between liver-to-blood Kpuu and hepatic availability FH , highlighting the potential for errors in pharmacokinetic evaluations based on differential equations. The Kirchhoff's Laws approach is applicable to all pharmacokinetic analyses of quality experimental data, those that were previously adequately explained with present pharmacokinetic theory, and those that were not. The publications that have attempted to rebut our position do not address unexplained experimental data, and we show here why their analyses are not valid. SIGNIFICANCE STATEMENT: The Kirchhoff's Laws approach to deriving clearance equations for linear systems in parallel or in series, independent of differential equations, successfully describes published pharmacokinetic data that has previously been unexplained. Three recent publications claim to refute our proposed methodology; these publications only make theoretical arguments, do not evaluate experimental data, and never demonstrate that the Kirchhoff methodology provides incorrect interpretations of experimental pharmacokinetic data, including statistically significant data not explained by present pharmacokinetic theory. We demonstrate why these analyses are invalid.
P2Y12 receptor inhibitors are commonly used in clinical antiplatelet therapy, typically alongside other medications. Vicagrel, a promising P2Y12 receptor inhibitor, has submitted a new drug marketing application to the United States Food and Drug Administration. Its primary metabolites and some metabolic pathways are identical to those of clopidogrel. The aim of this study was to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine (DCMB) on the metabolism and pharmacokinetics of vicagrel. In vitro incubation with human and rat liver microsomes revealed that DCMB significantly inhibited the methylation of vicagrel's thiol metabolite M15-1. Rats were orally administered 6 mg/kg [14C]vicagrel (100 μCi/kg) 1 hour after peritoneal injection with or without DCMB (80 mg/kg). Compared with the control group, the plasma of DCMB-pretreated rats exhibited maximum plasma concentration (C max) decrease and time to reach C max (T max) delay for all vicagrel-related substances, the methylation product of the thiol metabolite (M9-2), and the derivatization product of the active thiol metabolite (MP-M15-2). However, no significant changes in area under the curve (AUC) or half-life (t 1/2) were observed. DCMB had negligible effect on the total radiological recovery of vicagrel within 72 hours, although the rate of vicagrel excretion slowed down within 48 hours. DCMB had a negligible impact on the metabolic pathway of vicagrel. Overall, the present study found that DCMB did not significantly affect the total exposure, metabolic pathways, metabolite profiles, or total excretion rates of vicagrel-related metabolites in rats, but led to C max decrease, T max delay, and slower excretion rate within 48 hours. SIGNIFICANCE STATEMENT: This study used liquid chromatography-tandem mass spectrometry combined with radiolabeling technology to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine on the absorption, metabolism, and excretion of vicagrel in rats. This work helps to better understand the in vivo metabolism of active thiol metabolites of P2Y12 inhibitors such as clopidogrel, vicagrel, etc.
The role of the kidney as an excretory organ for exogenous and endogenous compounds is well recognized, but there is a wealth of data demonstrating that the kidney has significant metabolizing capacity for a variety of exogenous and endogenous compounds that in some cases surpass the liver. The induction of drug-metabolizing enzymes by some chemicals can cause drug-drug interactions and intraindividual variability in drug clearance. In this study, we evaluated the expression and induction of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) isoforms in 3D-cultured primary human renal proximal tubule epithelial cells (RPTEC) to elucidate their utility as models of renal drug metabolism. CYP2B6, CYP2E1, CYP3A4, CYP3A5, and all detected UGTs (UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) mRNA levels in 3D-RPTEC were significantly higher than those in 2D-RPTEC and HK-2 cells and were close to the levels in the human kidney cortex. CYP1B1 and CYP2J2 mRNA levels in 3D-RPTEC were comparable to those in 2D-RPTEC, HK-2 cells, and the human kidney cortex. Midazolam 1'-hydroxylation, trifluoperazine N-glucuronidation, serotonin O-glucuronidation, propofol O-glucuronidation, and morphine 3-glucuronidation in the 3D-RPTEC were significantly higher than the 2D-RPTEC and comparable to those in the HepaRG cells, although bupropion, ebastine, and calcitriol hydroxylations were not different between the 2D- and 3D-RPTEC. Treatment with ligands of the aryl hydrocarbon receptor and farnesoid X receptor induced CYP1A1 and UGT2B4 expression, respectively, in 3D-RPTEC compared with 2D-RPTEC. We provided information on the expression, activity, and induction abilities of P450s and UGTs in 3D-RPTEC as an in vitro human renal metabolism model. SIGNIFICANCE STATEMENT: This study demonstrated that the expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in 3D-cultured primary human renal proximal tubule epithelial cells (3D-RPTEC) was higher than those in 2D-cultured primary human renal proximal tubule epithelial cells and HK-2 cells. The results were comparable to that in the human kidney cortex. 3D-RPTEC are useful for evaluating the induction of kidney P450s, UDP-glucuronosyltransferases, and human renal drug metabolism in cellulo.
Currently, four kinds of phosphorodiamidate morpholino oligomers (PMOs), such as viltolarsen, have been approved for the treatment of Duchenne muscular dystrophy (DMD); however, it is unclear whether human efficacy can be estimated using plasma concentrations. This study summarizes the tissue distribution of viltolarsen in mice and cynomolgus monkeys and evaluates the relationship between exposure and efficacy based on exon skipping. In the tissue distribution studies, all muscles in DMD-model mice showed higher concentrations of viltolarsen than those in wild-type mice and cynomolgus monkeys, and the concentrations in skeletal muscle were correlated with the exon-skipping efficiency in mice and cynomolgus monkeys. In addition, a highly sensitive bioanalytical method using liquid chromatography with tandem mass spectrometry shows promise for determining plasma concentrations up to a later time point, and the tissue (muscle)/plasma concentration ratio (Kp) in DMD-model mice was shown to be useful for predicting changes in pharmacodynamic (PD) markers in humans. Our results suggest that pharmacokinetic (PK)/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD-model mice. This information will be useful for the efficient and effective development of PMOs as therapeutic agents. SIGNIFICANCE STATEMENT: We evaluated the relationship between the plasma or tissue concentrations and the efficiency of exon skipping for viltolarsen as an example phosphorodiamidate morpholino oligomers in the skeletal and cardiac muscle of mice and cynomolgus monkeys for pharmacokinetic/pharmacodynamic (PK/PD) analysis. The results suggest that PK/PD analysis can be conducted by using the human PK profile or tissue (muscle)/plasma concentration ratios and skipping efficiency in DMD-model mice.
4β-Hydroxycholesterol (4β-HC) in plasma has been used as a biomarker to assess CYP3A drug-drug interaction (DDI) potential during drug development. However, due to the long half-life and narrow dynamic range of 4β-HC, its use has been limited to the identification of CYP3A inducers, but not CYP3A inhibitors. The formation of 1β-hydroxydeoxycholic acid (1β-OH DCA) from deoxycholic acid (DCA) is mediated by CYP3A, thus 1β-OH DCA can potentially serve as an alternative to 4β-HC for assessment of CYP3A DDI potential. To study this feasibility, we developed a sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantitation of 1β-OH DCA and its glycine and taurine conjugates in human plasma with the lower limit of quantitation of 50 pg/ml, which enabled the quantitation of basal levels and further reduction. The method was applied to a DDI study to assess how 1β-OH DCA and its glycine and taurine conjugates would respond to CYP3A induction or inhibition. Rifampin induction resulted in an increase of 1β-OH DCA and its conjugates in plasma, with 6.8-, 7.8-, 8.3-, and 10.3-fold increases of area under the curve from the time of dosing to the last measurable concentration (AUCLST), area under the curve from the time of dosing to 24 hours (AUC24h), C max, and mean concentrations for total 1β-OH DCA (total of all three forms), respectively. Importantly, inhibition with itraconazole resulted in notable reduction of these biomarkers, with 84%, 85%, 82%, and 81% reductions of AUCLST, AUC24h, C max, and mean concentrations for total 1β-OH DCA, respectively. These preliminary data demonstrate for the first time that total 1β-OH DCA in plasma has the potential to serve as a biomarker for CYP3A DDI assessment in early clinical development and may provide key advantages over 4β-HC. SIGNIFICANCE STATEMENT: The authors have reported the use of total 1β-hydroxydeoxycholic acid (1β-OH DCA) (sum of 1β-OH DCA and its glycine and taurine conjugates) plasma exposure as a biomarker for CYP3A activity. Itraconazole inhibition led to an 81%-85% decrease of total 1β-OH DCA plasma exposures, whereas rifampin induction led to a 6.8- to 10.3-fold increase of total 1β-OH DCA plasma exposures. Using 1β-OH DCA exposures in plasma also provides the benefit of allowing pharmacokinetic and biomarker assessment using the same matrix.
Pigs are sometimes used in preclinical drug metabolism studies, with growing interest, and thus their drug-metabolizing enzymes, including the cytochromes P450 (P450 or CYP; EC 1.14.14.1), need to be examined. In the present study, novel CYP4A cDNAs were isolated and characterized, namely, pig CYP4A23 and CYP4A90; cat CYP4A37 and CYP4A106; and tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g. For comparison, the following known CYP4A cDNAs were also analyzed: pig CYP4A21 and dog CYP4A37, CYP4A38, and CYP4A39. These CYP4A cDNAs all contained open reading frames of 504-513 amino acids and had high amino acid sequence identity (74%-80%) with human CYP4As. Phylogenetic analysis of amino acid sequences revealed that these CYP4As were clustered in each species. All CYP4A genes contained 12 coding exons and formed a gene cluster in the corresponding genomic regions. A range of tissue types were analyzed, and these CYP4A mRNAs were preferentially expressed in liver and/or kidney, except for pig CYP4A90, which showed preferential expression in lung and duodenum. CYP4A enzymes, heterologously expressed in Escherichia coli, preferentially catalyzed lauric acid 12-hydroxylation and arachidonic acid 20-hydroxylation, just as human CYP4A11 does, with the same regioselectivity (i.e., at the ω-position of fatty acids). These results imply that dog, cat, pig, and tree shrew CYP4As have functional characteristics similar to those of human CYP4A11, with minor differences in lauric acid 12-hydroxylation. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450, CYP) 4As are important P450s in human biological processes because of their fatty acid-metabolizing ability. Pig CYP4A21, CYP4A23, and CYP4A90; cat CYP4A37 and CYP4A106; tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g; and dog CYP4A37, CYP4A38, and CYP4A39 cDNAs were isolated and analyzed. These CYP4A cDNAs shared relatively high sequence identities with human CYP4A11 and CYP4A22. Pig, cat, tree shrew, and dog CYP4As in the liver and kidneys are likely to catalyze the ω-hydroxylation of fatty acids.
The propensity for aldehyde oxidase (AO) substrates to be implicated in drug-drug interactions (DDIs) is not well understood due to the dearth of potent inhibitors that elicit in vivo inhibition of AO. Although there is only one reported instance of DDI that has been ascribed to the inhibition of AO to date, the supporting evidence for this clinical interaction is rather tenuous, and its veracity has been called into question. Our group recently reported that the epidermal growth factor receptor inhibitor erlotinib engendered potent time-dependent inhibition of AO with inactivation kinetic constants in the same order of magnitude as its free circulating plasma concentrations. At the same time, it was previously reported that the concomitant administration of erlotinib with the investigational drug OSI-930 culminated in a an approximately twofold increase in its systemic exposure. Although the basis underpinning this interaction remains unclear, the structure of OSI-930 contains a quinoline motif that is amenable to oxidation at the electrophilic carbon adjacent to the nitrogen atom by molybdenum-containing hydroxylases like AO. In this study, we conducted metabolite identification that revealed that OSI-930 undergoes AO metabolism to a mono-oxygenated 2-oxo metabolite and assessed its formation kinetics in human liver cytosol. Additionally, reaction phenotyping in human hepatocytes revealed that AO contributes nearly 50% to the overall metabolism of OSI-930. Finally, modeling the interaction between erlotinib and OSI-930 using a mechanistic static model projected an ∼1.85-fold increase in the systemic exposure of OSI-930, which accurately recapitulated clinical observations. SIGNIFICANCE STATEMENT: This study delineates an aldehyde oxidase (AO) metabolic pathway in the investigational drug OSI-930 for the first time and confirmed that it represented a major route of metabolism through reaction phenotyping in human hepatocytes. Our study provided compelling mechanistic and modeling evidence for the first instance of an AO-mediated clinical drug-drug interaction stemming from the in vivo inhibition of the AO-mediated quinoline 2-oxidation pathway in OSI-930 by erlotinib.
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