作者: Kulkarni, Priyanka ; Murray, Bernard P ; Prasad, Bhagwat ; Subash, Sandhya ; Smith, Bill J ; Ahire, Deepak S ; Singh, Dilip K ; Jones, Robert S ; Cronin, Ciarán N ; Zientek, Michael A ; Heyward, Scott ; Khojasteh, S Cyrus

We investigated the effect of variability and instability in aldehyde oxidase (AO) content and activity on the scaling of in vitro metabolism data. AO content and activity in human liver cytosol (HLC) and five recombinant human AO preparations (rAO) were determined using targeted proteomics and carbazeran oxidation assay, respectively. AO content was highly variable as indicated by the relative expression factor (REF; i.e., HLC to rAO content) ranging from 0.001 to 1.7 across different in vitro systems. The activity of AO in HLC degrades at a 10-fold higher rate in the presence of the substrate as compared with the activity performed after preincubation without substrate. To scale the metabolic activity from rAO to HLC, a protein-normalized activity factor (pnAF) was proposed wherein the activity was corrected by AO content, which revealed up to sixfold higher AO activity in HLC versus rAO systems. A similar value of pnAF was observed for another substrate, ripasudil. Physiologically based pharmacokinetic (PBPK) modeling revealed a significant additional clearance (CL; 66%), which allowed for the successful prediction of in vivo CL of four other substrates, i.e., O-benzyl guanine, BIBX1382, zaleplon, and zoniporide. For carbazeran, the metabolite identification study showed that the direct glucuronidation may be contributing to around 12% elimination. Taken together, this study identified differential protein content, instability of in vitro activity, role of additional AO clearance, and unaccounted metabolic pathways as plausible reasons for the underprediction of AO-mediated drug metabolism. Consideration of these factors and integration of REF and pnAF in PBPK models will allow better prediction of AO metabolism. SIGNIFICANCE STATEMENT: This study elucidated the plausible reasons for the underprediction of aldehyde oxidase (AO)-mediated drug metabolism and provided recommendations to address them. It demonstrated that integrating protein content and activity differences and accounting for the loss of AO activity, as well as consideration of extrahepatic clearance and additional pathways, would improve the in vitro to in vivo extrapolation of AO-mediated drug metabolism using physiologically based pharmacokinetic modeling.

2023-10-01·NMR in biomedicine

Real-time influence of intracellular acidification and Na⁺ /H⁺ exchanger inhibition on in-cell pyruvate metabolism in the perfused mouse heart: A ³¹ P-NMR and hyperpolarized ¹³ C-NMR study.

Article

作者: Gomori, J Moshe ; Sosna, Jacob ; Shaul, David ; Sapir, Gal ; Katz-Brull, Rachel ; Joskowicz, Leo ; Lev-Cohain, Naama

Disruption of acid-base balance is linked to various diseases and conditions. In the heart, intracellular acidification is associated with heart failure, maladaptive cardiac hypertrophy, and myocardial ischemia. Previously, we have reported that the ratio of the in-cell lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) activities is correlated with cardiac pH. To further characterize the basis for this correlation, these in-cell activities were investigated under induced intracellular acidification without and with Na⁺ /H⁺ exchanger (NHE1) inhibition by zoniporide. Male mouse hearts (n = 30) were isolated and perfused retrogradely. Intracellular acidification was performed in two ways: (1) with the NH₄ Cl prepulse methodology; and (2) by combining the NH₄ Cl prepulse with zoniporide. ³¹ P NMR spectroscopy was used to determine the intracellular cardiac pH and to quantify the adenosine triphosphate and phosphocreatine content. Hyperpolarized [1-¹³ C]pyruvate was obtained using dissolution dynamic nuclear polarization. ¹³ C NMR spectroscopy was used to monitor hyperpolarized [1-¹³ C]pyruvate metabolism and determine enzyme activities in real time at a temporal resolution of a few seconds using the product-selective saturating excitation approach. The intracellular acidification induced by the NH₄ Cl prepulse led to reduced LDH and PDH activities (-16% and -39%, respectively). This finding is in line with previous evidence of reduced myocardial contraction and therefore reduced metabolic activity upon intracellular acidification. Concomitantly, the LDH/PDH activity ratio increased with the reduction in pH, as previously reported. Combining the NH₄ Cl prepulse with zoniporide led to a greater reduction in LDH activity (-29%) and to increased PDH activity (+40%). These changes resulted in a surprising decrease in the LDH/PDH ratio, as opposed to previous predictions. Zoniporide alone (without intracellular acidification) did not change these enzyme activities. A possible explanation for the enzymatic changes observed during the combination of the NH₄ Cl prepulse and NHE1 inhibition may be related to mitochondrial NHE1 inhibition, which likely negates the mitochondrial matrix acidification. This effect, combined with the increased acidity in the cytosol, would result in an enhanced H⁺ gradient across the mitochondrial membrane and a temporarily higher pyruvate transport into the mitochondria, thereby increasing the PDH activity at the expense of the cytosolic LDH activity. These findings demonstrate the complexity of in-cell cardiac metabolism and its dependence on intracellular acidification. This study demonstrates the capabilities and limitations of hyperpolarized [1-¹³ C]pyruvate in the characterization of intracellular acidification as regards cardiac pathologies.

2021-09-21·Circulation1区 · 医学

Therapeutic Inhibition of Acid-Sensing Ion Channel 1a Recovers Heart Function After Ischemia–Reperfusion Injury

1区 · 医学

Article

作者: Cuellar-Partida, Gabriel ; Quaife-Ryan, Gregory A. ; Petrou, Steven ; Chow, Chun Yuen ; Cordwell, Stuart J. ; Fraser, John F. ; Reichelt, Melissa E. ; Scheuer, Sarah E. ; Thomas, Walter G. ; Hudson, James E. ; Joshi, Yashutosh ; White, Melanie Y. ; Chen, Xiaoli ; Redd, Meredith A. ; Macdonald, Peter S. ; Alzubaidi, Mubarak A.H. ; Xia, Di ; Yoshikawa, Yusuke ; Hicks, Mark ; Maljevic, Snezana ; Porrello, Enzo R. ; Sun, Yuliangzi ; See Hoe, Louise E. ; Saez, Natalie J. ; Suen, Jacky Y. ; Rollo, Ben ; Peart, Jason N. ; Villanueva, Jeanette E. ; Hatch, Robert J. ; Chiu, Han Sheng ; Gao, Ling ; Palpant, Nathan J. ; King, Glenn F.

Background:Ischemia–reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function.Methods:We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell–derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents.Results: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell–derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. Conclusions:Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

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适应症	最高研发状态	国家/地区	公司	日期
心血管疾病	临床3期	-	Pfizer Inc.	-

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研究	分期	人群特征	评价人数	分组	结果	评价	发布日期


ASN2010	N/A	高血压 Na-K ATPase \| NHE-1	-	Ouabain	(夢獵積願齋襯鹽簾窪淵) = 選糧簾窪襯築選鑰廠鬱廠衊遞網夢願壓構製襯 (積艱獵憲艱憲鹽蓋憲淵, 3.26)	积极	2010-11-16
				Vehicle	(夢獵積願齋襯鹽簾窪淵) = 選範網積鑰鬱鑰繭膚簾廠衊遞網夢願壓構製襯 (積艱獵憲艱憲鹽蓋憲淵, 5.32)