David Fairman

The Antianginal Agent Trimetazidine Does Not Exert Its Functional Benefit via Inhibition of Mitochondrial Long-Chain 3-Ketoacyl Coenzyme A Thiolase

Abstract— Trimetazidine acts as an effective antianginal clinical agent by modulating cardiac energy metabolism. Recent published data support the hypothesis that trimetazidine selectively inhibits long-chain 3-ketoacyl CoA thiolase (LC 3-KAT), thereby reducing fatty acid oxidation resulting in clinical benefit. The aim of this study was to assess whether trimetazidine and ranolazine, which may also act as a metabolic modulator, are specific inhibitors of LC 3-KAT. We have demonstrated that trimetazidine and ranolazine do not inhibit crude and purified rat heart or recombinant human LC 3-KAT by methods that both assess the ability of LC 3-KAT to turnover specific substrate, and LC 3-KAT activity as a functional component of intact cellular &bgr;-oxidation. Furthermore, we have demonstrated that trimetazidine does not inhibit any component of &bgr;-oxidation in an isolated human cardiomyocyte cell line. Ranolazine, however, did demonstrate a partial inhibition of &bgr;-oxidation in a dose-dependent manner (12% at 100 &mgr;mol/L and 30% at 300 &mgr;mol/L). Both trimetazidine (10 &mgr;mol/L) and ranolazine (20 &mgr;mol/L) improved the recovery of cardiac function after a period of no flow ischemia in the isolated working rat heart perfused with a buffer containing a relatively high concentration (1.2 mmol/L) of free fatty acid. In summary, both trimetazidine and ranolazine were able to improve ischemic cardiac function but inhibition of LC 3-KAT is not part of their mechanism of action. The full text of this article is available online at http://www.circresaha.org.

Progress Curve Analysis of CYP1A2 Inhibition: A More Informative Approach to the Assessment of Mechanism-Based Inactivation?

Mechanism-based cytochrome P450 inactivation is defined as a time- and NADPH-dependent inactivation that is not reversible upon extensive dialysis. Current methodologies use dilution approaches to estimate the rate of inactivation and offer limited mechanistic insight and are significantly influenced by experimental conditions. We investigated the potential of progress curve analysis because this experimental design allows investigation of both the reversible (Kiapp) and irreversible (Ki, Kinact) components of the reaction mechanism. The human liver microsomal CYP1A2 inactivation kinetics of resveratrol, oltipraz, furafylline, and dihydralazine (Fig. 2) were evaluated. The inactivation results for furafylline (Ki, 0.8 μM; Kinact, 0.16 min–1) are within 2-fold to pub-lished data (Ki, 1.6 μM; Kinact, 0.19 min–1). Resveratrol and dihydralazine results are within a 4.3-fold range of published data, which compares well with ranges of estimates of these parameters across publications (e.g., furafylline has estimates ranging of Ki from 1.6 to 22.3 μM and Kinact from 0.19 to 0.87 min–1). This range of estimates highlights the potential caveats surrounding the existing methodologies that have been previously discussed in depth. In addition to these inactivation parameters, we have been able to demonstrate a variation in balance of reversible versus irreversible inhibition within these inactivators. Oltipraz and resveratrol have Kiapp values similar to their Ki, indicating a significant early onset reversible inhibition, whereas furafylline and dihydralazine are dominated by irreversible inactivation. This approach allows a more mechanistic investigation of an inactivator and in the future may improve the prediction of clinical drug-drug interactions.

Effects of multikinase inhibitors on pressure overload-induced right ventricular remodeling

BACKGROUND Little is known about the effects of current PAH therapies and receptor tyrosine kinase inhibitors on heart remodeling. We sought to investigate the effects of the multikinase inhibitors sunitinib (PDGFR-, VEGFR- and KIT-inhibitor) and sorafenib (raf1/b-, VEGFR-, PDGFR-inhibitor) on pressure overload induced right ventricular (RV) remodeling. METHODS We investigated the effects of the kinase inhibitors on hemodynamics and remodeling in rats subjected either to monocrotaline (MCT)-induced PH or to surgical pulmonary artery banding (PAB). MCT rats were treated from days 21 to 35 with either vehicle, sunitinib (1mg/kg, 5mg/kg and 10mg/kg/day) or sorafenib (10mg/kg/day). PAB rats were treated with vehicle, sunitinib (10mg/kg/day) or sorafenib (10mg/kg/day) from days 7 to 21. RV function and remodeling were determined using echocardiography, invasive hemodynamic measurement and histomorphometry. RESULTS Treatment with both sorafenib and sunitinib decreased right ventricular systolic pressure, pulmonary vascular remodeling, RV hypertrophy and fibrosis in MCT rats. This was associated with an improvement of RV function. Importantly, after PAB, both compounds reversed RV chamber and cellular hypertrophy, reduced RV interstitial and perivascular fibrosis, and improved RV function. CONCLUSION We demonstrated that sunitinib and sorafenib reversed RV remodeling and significantly improved RV function measured via a range of invasive and non-invasive cardiopulmonary endpoints in experimental models of RV hypertrophy.

Inhalation by Design: Novel Tertiary Amine Muscarinic M3 Receptor Antagonists with Slow Off-Rate Binding Kinetics for Inhaled Once-Daily Treatment of Chronic Obstructive Pulmonary Disease

A novel tertiary amine series of potent muscarinic M(3) receptor antagonists are described that exhibit potential as inhaled long-acting bronchodilators for the treatment of chronic obstructive pulmonary disease. Geminal dimethyl functionality present in this series of compounds confers very long dissociative half-life (slow off-rate) from the M(3) receptor that mediates very long-lasting smooth muscle relaxation in guinea pig tracheal strips. Optimization of pharmacokinetic properties was achieved by combining rapid oxidative clearance with targeted introduction of a phenolic moiety to secure rapid glucuronidation. Together, these attributes minimize systemic exposure following inhalation, mitigate potential drug-drug interactions, and reduce systemically mediated adverse events. Compound 47 (PF-3635659) is identified as a Phase II clinical candidate from this series with in vivo duration of action studies confirming its potential for once-daily use in humans.

Acidic triazoles as soluble guanylate cyclase stimulators

A series of acidic triazoles with activity as soluble guanylate cyclase stimulators is described. Incorporation of the CF(3) triazole improved the overall physicochemical and drug-like properties of the molecule and is exemplified by compound 25.

Small, non-peptide C5a receptor antagonists: Part 1

Starting from 2, several highly potent C5a receptor antagonists were synthesised through alpha-amide substitution. Attempts to increase the polarity of these compounds through the introduction of basic centres or incorporation into weakly basic heterocycles is described.

Optimized glucuronidation of dual pharmacology β-2 agonists/M3 antagonists for the treatment of COPD

‘Inhalation by design’ concepts were developed to create novel dual pharmacology β-2 agonists-M3 antagonists, for the treatment of chronic obstructive pulmonary disorder. A key feature of this work is the combination of balanced potency and pharmacological duration with optimised glucuronidation through the incorporation of metabolically vulnerable phenols.

Regulation of leukotriene and 5oxoETE synthesis and the effect of 5-lipoxygenase inhibitors: a mathematical modeling approach

Background5-lipoxygenase (5-LO) is a key enzyme in the synthesis of leukotrienes and 5-Oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (oxoETE). These inflammatory signaling molecules play a role in the pathology of asthma and so 5-LO inhibition is a promising target for asthma therapy. The 5-LO redox inhibitor zileuton (Zyflo IR/CR®) is currently marketed for the treatment of asthma in adults and children, but widespread use of zileuton is limited by its efficacy/safety profile, potentially related to its redox characteristics. Thus, a quantitative, mechanistic description of its functioning may be useful for development of improved anti-inflammatory targeting this mechanism.ResultsA mathematical model describing the operation of 5-LO, phospholipase A2, glutathione peroxidase and 5-hydroxyeicosanoid dehydrogenase was developed. The catalytic cycles of the enzymes were reconstructed and kinetic parameters estimated on the basis of available experimental data. The final model describes each stage of cys-leukotriene biosynthesis and the reactions involved in oxoETE production. Regulation of these processes by substrates (phospholipid concentration) and intracellular redox state (concentrations of reduced glutathione, glutathione (GSH), and lipid peroxide) were taken into account. The model enabled us to reveal differences between redox and non-redox 5-LO inhibitors under conditions of oxidative stress. Despite both redox and non-redox inhibitors suppressing leukotriene A4 (LTA4) synthesis, redox inhibitors are predicted to increase oxoETE production, thus compromising efficacy. This phenomena can be explained in terms of the pseudo-peroxidase activity of 5-LO and the ability of lipid peroxides to transform 5-LO into its active form even in the presence of redox inhibitors.ConclusionsThe mathematical model developed described quantitatively different mechanisms of 5-LO inhibition and simulations revealed differences between the potential therapeutic outcomes for these mechanisms.

Effects of Multikinase inhibitors on pressure overload-induced right ventricular remodelling

Little is known about the effects of current PAH therapies and receptor tyrosine kinase inhibitors on heart remodelling. We sought to investigate the effects of the multikinase inhibitors sunitinib (PDGFR-, VEGFR- and KIT-inhibitor) and sorafenib (raf1/b-, VEGFR-, PDGFR-inhibitor) on pressure overload induced right ventricular (RV) remodelling. We investigated the effects of the kinase inhibitors on hemodynamics and remodelling in rats subjected either to monocrotaline (MCT)-induced PH or to surgical pulmonary artery banding (PAB). MCT rats were treated from day 21 to 35 with either vehicle, sunitinib (1 mg/kg, 5 mg/kg and 10 mg/kg/day) or sorafenib (10 mg/kg/day). PAB rats were treated with vehicle, sunitinib (10 mg/kg/day) or sorafenib (10mg/kg/day) from day 7 to 21. RV function and remodelling were determined using echocardiography, invasive hemodynamic measurement and histomorphometry. Treatment with both sorafenib and sunitinib decreased right ventricular systolic pressure, pulmonary vascular remodelling, RV hypertrophy and fibrosis in MCT rats. This was associated with an improvement of RV function. Importantly, after PAB, both compounds reversed RV chamber and cellular hypertrophy, reduced RV interstitial and perivascular fibrosis, and improved RV function. We demonstrated that sunitinib and sorafenib reversed RV remodelling and significantly improved RV function measured via a range of invasive and non-invasive cardiopulmonary endpoints in experimental models of RV hypertrophy.