Modulation of Angiotensin II-Induced Cellular Hypertrophy by Cannflavin-C: Unveiling the Impact on Cytochrome P450 1B1 and Arachidonic Acid Metabolites.

This research aimed to clarify the impacts of cannflavin-C on angiotensin II (Ang II)-induced cardiac hypertrophy and their potential role in modulating cytochrome P450 1B1 (CYP1B1) and arachidonic acid (AA) metabolites. Currently there is no evidence to suggest that cannflavin-C; a prenylated flavonoid, has any significant effects on the heart or cardiac hypertrophy. The metabolism of arachidonic acid (AA) into midchain hydroxyeicosatetraenoic acids (HETEs), facilitated by CYP1B1 enzyme, plays a role in the development of cardiac hypertrophy which is marked by enlarged cardiac cells. Adult human ventricular cardiomyocytes cell line (AC16) were cultured and exposed to cannflavin-C in the presence and absence of Ang II. The assessment of mRNA expression pertaining to cardiac hypertrophic markers and CYPs was conducted via real-time polymerase chain reaction (PCR) while the quantification of CYPs protein levels was carried out through western blot analysis. Ang II induced hypertrophic markers myosin heavy chain (ß/α-MHC), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) and increased cell surface area, while cannflavin-C mitigated these effects. Gene and protein expression analysis revealed that cannflavin-C downregulated CYP1B1 gene expression, protein level as well as the enzyme activity assessed by 7-methoxyresorufin O-deethylase (MROD). Arachidonic acid metabolites analysis, using LC-MS/MS, demonstrated that Ang II increased midchain (R/S)-HETEs concentrations, which were attenuated by cannflavin-C. This study provides novel insights into the potential of cannflavin-C in modulating arachidonic acid metabolites and attenuating Ang II-induced cardiac hypertrophy, highlighting the importance of this compound as potential therapeutic agents for cardiac hypertrophy. Significance Statement This study demonstrates that cannflavin-C offers protection against cellular hypertrophy induced by Ang II. The significance of this research lies in its novel discovery, which elucidates a mechanistic pathway involving the inhibition of CYP 1B1 by cannflavin-C. This discovery opens up new avenues for leveraging this compound in the treatment of heart failure.

Physiological and pathophysiological roles of hepoxilins and their analogs.

The metabolism of arachidonic acid (AA) occurs via different pathways leading to the production of a great number of metabolites with a wide range of biological effects. Hepoxilins (HXs) are physiologically active AA metabolites produced through the lipoxygenase pathway. Since their discovery, several researchers have investigated their biological effects. They were proven to have pro-inflammatory, anti-apoptotic, and skin-protective effects. HXs also contribute to the processes of neutrophil activation and migration and inflammatory hyperalgesia. The major limitation to their effects is that they are highly labile and are metabolized into less active compounds which led to the synthesis of stable HXs analogs called proprietary bioactive therapeutics (PBTs). Although PBTs were synthesized to further study the effect of HXs, they showed different effects than natural HXs under some conditions. PBTs were proven to have anti-inflammatory and anti-cancer effects and were found to be potent antagonists of the thromboxane receptor. In this review article, we aimed to provide an overview of some physiological and pathophysiological effects of hepoxilins and their analogs on the skin, platelet, blood vessel, neutrophil, and cell survival.

6-Formylindolo[3,2-b]carbazole Protects Against Angiotensin II-Induced Cellular Hypertrophy through the Induction of Cytochrome P450 1A1 and Its Associated 19(S)-HETE Metabolite In Vitro.

Aryl hydrocarbon receptor (AhR) is a multifunctional receptor that regulates cytochrome P450 1A1 (CYP1A1), an arachidonic acid (AA) metabolizing enzyme producing 19-hydroxyeicosatetraenoic acid (HETE). 6-formylindolo[3,2-b]carbazole (FICZ) demonstrates great affinity toward the AhR. Recently, we have shown that 19(S)-HETE is preferentially cardioprotective. This study investigates the role of FICZ on AhR and cytochrome P450 (CYP) 1A1-mediated AA metabolism and whether it attenuates angiotensin (Ang) II-induced cardiac hypertrophy. Adult human ventricular cardiomyocytes cell line treated with FICZ in the presence and absence of Ang II 10 µM. Protein levels of AhR and CYPs were determined by Western blot analysis and the mRNA expression of cardiac hypertrophic markers and CYPs were determined by real-time polymerase chain reaction. CYP1A1 enzyme activity and proteasomal degradation were determined by 7-ethoxyresorufin O-deethylase and proteasome 20S activity assays, respectively. Liquid chromatography tandem mass spectrometry was used to measure AA metabolites. Our results show that Ang II-induced cardiac hypertrophy modulates AA metabolites in an enantioselective manner, and that FICZ activates AhR in a time-dependent manner, inhibits AhR proteasomal degradation, induces CYP1A1, increases the concentration of 19(S)-HETE, and attenuates Ang II-induced cardiac hypertrophy by inhibiting the hypertrophic markers and decreasing cell surface area through midchain-HETE-dependent mechanism. In conclusion, the results demonstrate the ability of FICZ to protect against Ang II-induced cardiac hypertrophy by increasing the concentration of 19(S)-HETE through AhR regulated enzyme induction and inhibition of midchain-HETEs metabolites. SIGNIFICANCE STATEMENT: This study shows that 6-formylindolo[3,2-b]carbazole attenuate angiotensin II-induced cellular hypertrophy. The novel findings of our investigation are in characterizing the aryl hydrocarbon receptor involvement and the enantioselective differences in arachidonic acid metabolism in cardiac hypertrophy, which opens a new pathway to tackle and eventually treat heart failure.

17-(R/S)-hydroxyeicosatetraenoic acid (HETE) induces cardiac hypertrophy through the CYP1B1 in enantioselective manners.

Cardiac cellular hypertrophy is the increase in the size of individual cardiac cells. Cytochrome P450 1B1 (CYP1B1) is an extrahepatic inducible enzyme that is associated with toxicity, including cardiotoxicity. We previously reported that 19-hydroxyeicosatetraenoic acid (19-HETE) inhibited CYP1B1 and prevented cardiac hypertrophy in enantioselective manner. Therefore, our aim is to investigate the effect of 17-HETE enantiomers on cardiac hypertrophy and CYP1B1. Human adult cardiomyocyte (AC16) cells were treated with 17-HETE enantiomers (20 µM); cellular hypertrophy was evaluated by cell surface area and cardiac hypertrophy markers. In addition, CYP1B1 gene, protein and activity were assessed. Human recombinant CYP1B1 and heart microsomes of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats were incubated with 17-HETE enantiomers (10-80 nM). Our results demonstrated that 17-HETE induced cellular hypertrophy, which is manifested by increase in cell surface area and cardiac hypertrophy markers. 17-HETE enantiomers allosterically activated CYP1B1 and selectively upregulated CYP1B1 gene and protein expression in AC16 cells at uM range. In addition, CYP1B1 was allosterically activated by 17-HETE enantiomers at nM range in recombinant CYP1B1 and heart microsomes. In conclusion, 17-HETE acts as an autocrine mediator, leading to the cardiac hypertrophy through induction of CYP1B1 activity in the heart.

Effect of inflammation on cytochrome P450-mediated arachidonic acid metabolism and the consequences on cardiac hypertrophy.

The incidence of heart failure (HF) is generally preceded by cardiac hypertrophy (CH), which is the enlargement of cardiac myocytes in response to stress. During CH, the metabolism of arachidonic acid (AA), which is present in the cell membrane phospholipids, is modulated. Metabolism of AA gives rise to hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs) via cytochrome P450 (CYP) ω-hydroxylases and CYP epoxygenases, respectively. A plethora of studies demonstrated the involvement of CYP-mediated AA metabolites in the pathogenesis of CH. Also, inflammation is known to be a characteristic hallmark of CH. In this review, our aim is to highlight the impact of inflammation on CYP-derived AA metabolites and CH. Inflammation is shown to modulate the expression of various CYP ω-hydroxylases and CYP epoxygenases and their respective metabolites in the heart. In general, HETEs such as 20-HETE and mid-chain HETEs are pro-inflammatory, while EETs are characterized by their anti-inflammatory and cardioprotective properties. Several mechanisms are implicated in inflammation-induced CH, including the modulation of NF-κB and MAPK. This review demonstrated the inflammatory modulation of cardiac CYPs and their metabolites in the context of CH and the anti-inflammatory strategies that can be employed in the treatment of CH and HF.

The enantioselective separation and quantitation of the hydroxy-metabolites of arachidonic acid by liquid chromatography - tandem mass spectrometry.

Arachidonic acid (AA) is a polyunsaturated fatty acid with a structure of 20:4(ω-6). Cytochrome P450s (CYPs) metabolize AA to several regioisomers and enantiomers of hydroxyeicosatetraenoic acids (HETEs). The hydroxy-metabolites (HETEs) exist as enantiomers in the biological system. The chiral assays developed for HETEs are so far limited to a few assays reported for midchain HETEs. The developed method is capable of quantitative analysis for midchain, subterminal HETE enantiomers, and terminal HETEs in microsomes. The peak area or height ratios were linear over concentrations ranging (0.01 -0.6 µg/ml) with r2 > 0.99. The intra-run percent error and coefficient of variation (CV) were ≤ ± 12 %. The inter-run percent error and coefficient of variation (CV)were ≤ ± 13 %, and ≤ 15 %, respectively. The matrix effect for the assay was also within the acceptable limit (≤ ± 15 %). The recovery of HETE metabolites ranged from 70 % to 115 %. The method showed a reliable and robust performance for chiral analysis of cytochrome P450-mediated HETE metabolites.

Nimodipine systemic exposure and outcomes following aneurysmal subarachnoid hemorrhage: a pilot prospective observational study (ASH-1 study).

Background: Nimodipine improves outcomes following aneurysmal subarachnoid hemorrhage (aSAH). Guidelines recommend that all patients should receive a fixed-dose nimodipine for 21 days. However, studies reported variability of nimodipine concentrations in aSAH. It is not clear if reduced systemic exposure contributes to worsening outcomes. The aim of this study was to compare nimodipine systemic exposure in those who experienced poor outcomes to those who experienced favorable outcomes. Methods: This was a pilot prospective observational study in 30 adult patients admitted to the University of Alberta Hospital with aSAH. Data were collected from the electronic health records following enrollment. Blood samples were collected around one nimodipine 60 mg dose at a steady state, and nimodipine [total, (+)-R and (-)-S enantiomers] plasma concentrations were determined. The poor outcome was defined as a modified Rankin Scale (mRS) score at 90 days of 3-6, while the favorable outcome was an mRS score of 0-2. The correlation between nimodipine concentrations and percent changes in mean arterial pressure (MAP) before and after nimodipine administration was also determined. Furthermore, covariates potentially associated with nimodipine exposure were explored. Results: In total, 20 (69%) participants had favorable outcomes and 9 (31%) had poor outcomes. Following the exclusion of those with delayed presentation (>96 h from aSAH onset), among those presented with the World Federation of Neurological Surgeons (WFNS) grade 3-5, nimodipine median (interquartile range) area under the concentration time curve (AUC0-3h) in those with favorable outcomes were 4-fold higher than in those with poor outcomes [136 (52-192) vs. 33 (23-39) ng.h/mL, respectively, value of p = 0.2]. On the other hand, among those presented with WFNS grade 1-2, nimodipine AUC0-3h in those with favorable outcomes were significantly lower than in those with poor outcomes [30 (28-36) vs. 172 (117-308) ng.h/mL, respectively, value of p = 0.03)]. (+)-R-nimodipine AUC0-3h in those who did not develop vasospasm were 4-fold significantly higher than those who had vasospasm (value of p = 0.047). (-)-S-nimodipine was significantly correlated with percentage MAP reduction. Similar results were obtained when the whole cohort was analyzed. Conclusion: The study was the first to investigate the potential association between nimodipine exposure following oral dosing and outcomes. In addition, it suggests differential effects of nimodipine enantiomers, shedding light on the potential utility of nimodipine enantiomers. Larger studies are needed.

The multifaceted role of cytochrome P450-Derived arachidonic acid metabolites in diabetes and diabetic cardiomyopathy.

Understanding lipid metabolism is a critical key to understanding the pathogenesis of Diabetes Mellitus (DM). It is known that 60-90% of DM patients are obese or used to be obese. The incidence of obesity is rising owing to the modern sedentary lifestyle that leads to insulin resistance and increased levels of free fatty acids, predisposing tissues to utilize more lipids with less glucose uptake. However, the exact mechanism is not yet fully elucidated. Diabetic cardiomyopathy seems to be associated with these alterations in lipid metabolism. Arachidonic acid (AA) is an important fatty acid that is metabolized to several bioactive compounds by cyclooxygenases, lipoxygenases, and the more recently discovered, cytochrome P450 (P450) enzymes. P450 metabolizes AA to either epoxy-AA (EETs) or hydroxy-AA (HETEs). Studies showed that EETs could have cardioprotective effects and beneficial effects in reversing abnormalities in glucose and insulin homeostasis. Conversely, HETEs, most importantly 12-HETE and 20-HETE, were found to interfere with normal glucose and insulin homeostasis and thus, might be involved in diabetic cardiomyopathy. In this review, we highlight the role of P450-derived AA metabolites in the context of DM and diabetic cardiomyopathy and their potential use as a target for developing new treatments for DM and diabetic cardiomyopathy.

Enantioselective assay of nimodipine in human plasma using liquid chromatography-tandem mass spectrometry.

Nimodipine is a dihydropyridine calcium channel blocker that exhibits higher selectivity toward cerebral blood vessels compared with other members of the same class. It has been shown to improve outcomes and prevent delayed cerebral ischemia in the setting of aneurysmal subarachnoid hemorrhage, a life-threatening brain bleed. Nimodipine is a chiral compound and it is marketed as a racemic mixture of (+)-R and (-)-S enantiomers. (-)-S-Nimodipine is approximately twice as potent a vasorelaxant as the racemic mixture and is more rapidly eliminated than the (+)-R counterpart following oral dosing. Few analytical procedures have been reported to determine nimodipine enantiomers in biological samples; however, the reported methods were time-consuming, involved multistep extraction procedures and required large sample volumes. Herein, we present an LC-MS/MS method for quantifying nimodipine enantiomers in human plasma using a small sample volume (0.3 ml) and a single liquid-liquid extraction step. The peak area ratios were linear over the tested concentration ranges (1.5-75 ng/ml) with r2 > 0.99. The intraday CV and percentage error were within ±14% while the interday values were within ±13%, making this analytical method feasible for research purposes and pharmacokinetic studies.

Nimodipine Pharmacokinetic Variability in Various Patient Populations.

Nimodipine has been shown to improve outcomes following aneurysmal subarachnoid hemorrhage. Guidelines recommend that all patients receive a fixed dose of oral nimodipine for 21 days. However, pharmacokinetic studies have suggested variability of nimodipine pharmacokinetics in subarachnoid hemorrhage and in other patient populations. The clinical relevance of such variability is unknown. Therefore, the objective of the present review is, first, to conduct a literature review and summarize nimodipine pharmacokinetic data and sources of variability in various patient groups. Second, to determine if there is any evidence reporting an association between nimodipine exposure and clinical outcomes in patients with subarachnoid hemorrhage. A systematic literature search was performed in MEDLINE and EMBASE. The following keywords were used: ("nimodipine" OR "nymalize" OR "nimotop") AND ("pharmacokinetic*", OR "PK"). The search results were limited to English language and human studies. A large interpatient variability in nimodipine pharmacokinetics has been reported. Patient-specific factors that had an influence on pharmacokinetic parameters are age, comorbidities, variabilities in metabolism due to genetic polymorphism and co-administered medications, as well as nimodipine administration technique. The association between nimodipine exposure and clinical outcomes remains unclear and data available are too scarce to reach a firm conclusion. Here, we present a narrative review with a systematic literature search discussing nimodipine pharmacokinetic variability in various patient populations. It is not clear if minimal or lack of systemic exposure to nimodipine denies its benefit and contributes to worsening outcomes in patients with subarachnoid hemorrhage. Further studies are needed to determine if such an association exists.

The Impact of Nimodipine Administration through Feeding Tube on Outcomes in Patients with Aneurysmal Subarachnoid Hemorrhage.

PURPOSE: Delayed cerebral ischemia (DCI) and vasospasm are the main challenges contributing to unfavorable outcomes following aneurysmal subarachnoid hemorrhage. Nimodipine has been shown to decrease the incidence of delayed cerebral ischemia and improve outcomes. In patients who are unable to swallow, nimodipine tablets are crushed and administered through enteral feeding tubes. However, it is not clear whether this may result in reduced clinical effectiveness. The aims of the study were to investigate the impact of nimodipine administration through enteral feeding tubes, in the first 7 days and over the 21-days period on patient outcomes. METHODS: A retrospective chart review of subarachnoid hemorrhage patients admitted at the University of Alberta Hospital, Edmonton, Alberta, Canada was carried out. Logistic regression modelling was utilized to identify predictors of vasospasm and delayed cerebral ischemia. Main outcome measures were angiographic evidence of moderate to severe vasospasm, development of delayed cerebral ischemia and hospital mortality. RESULTS: 85 patients were included. Following adjustment for disease severity, nimodipine administration technique was associated with vasospasm in the first 7 days of patient admission where patients receiving nimodipine via enteral feeding tubes had increased odds of vasospasm compared to those administered it as whole tablets (OR 8.9, 95% CI 1.1-73.1, p value 0.042). When analyzed over the 21-day period, nimodipine administration by feeding tube was associated with increased odds of DCI compared to whole tablets (OR 38.1, 95% CI 1.4-1067.9, p value 0.032). CONCLUSIONS: Our findings suggest that nimodipine administration via enteral feeding tubes may be associated with vasospasm and DCI in subarachnoid hemorrhage patients secondary to reduced exposure. Prospective studies are needed to confirm such association and alternate methods of administration should be explored to ensure patients are getting the benefits of nimodipine.