Proteomic characterisation of perhexiline treatment on THP-1 M1 macrophage differentiation.

Background: Dysregulated inflammation is important in the pathogenesis of many diseases including cancer, allergy, and autoimmunity. Macrophage activation and polarisation are commonly involved in the initiation, maintenance and resolution of inflammation. Perhexiline (PHX), an antianginal drug, has been suggested to modulate macrophage function, but the molecular effects of PHX on macrophages are unknown. In this study we investigated the effect of PHX treatment on macrophage activation and polarization and reveal the underlying proteomic changes induced. Methods: We used an established protocol to differentiate human THP-1 monocytes into M1 or M2 macrophages involving three distinct, sequential stages (priming, rest, and differentiation). We examined the effect of PHX treatment at each stage on the polarization into either M1 or M2 macrophages using flow cytometry, quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Quantitative changes in the proteome were investigated using data independent acquisition mass spectrometry (DIA MS). Results: PHX treatment promoted M1 macrophage polarization, including increased STAT1 and CCL2 expression and IL-1ß secretion. This effect occurred when PHX was added at the differentiation stage of the M1 cultures. Proteomic profiling of PHX treated M1 cultures identified changes in metabolic (fatty acid metabolism, cholesterol homeostasis and oxidative phosphorylation) and immune signalling (Receptor Tyrosine Kinase, Rho GTPase and interferon) pathways. Conclusion: This is the first study to report on the action of PHX on THP-1 macrophage polarization and the associated changes in the proteome of these cells.

The Antianginal Drug Perhexiline Displays Cytotoxicity against Colorectal Cancer Cells In Vitro: A Potential for Drug Repurposing.

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Perhexiline, a prophylactic anti-anginal drug, has been reported to have anti-tumour effects both in vitro and in vivo. Perhexiline as used clinically is a 50:50 racemic mixture ((R)-P) of (-) and (+) enantiomers. It is not known if the enantiomers differ in terms of their effects on cancer. In this study, we examined the cytotoxic capacity of perhexiline and its enantiomers ((-)-P and (+)-P) on CRC cell lines, grown as monolayers or spheroids, and patient-derived organoids. Treatment of CRC cell lines with (R)-P, (-)-P or (+)-P reduced cell viability, with IC50 values of ~4 µM. Treatment was associated with an increase in annexin V staining and caspase 3/7 activation, indicating apoptosis induction. Caspase 3/7 activation and loss of structural integrity were also observed in CRC cell lines grown as spheroids. Drug treatment at clinically relevant concentrations significantly reduced the viability of patient-derived CRC organoids. Given these in vitro findings, perhexiline, as a racemic mixture or its enantiomers, warrants further investigation as a repurposed drug for use in the management of CRC.

Nitrosative Stress as a Modulator of Inflammatory Change in a Model of Takotsubo Syndrome.

Previous studies have shown that patients with Takotsubo syndrome (TS) have supranormal nitric oxide signaling, and post-mortem studies of TS heart samples revealed nitrosative stress. Therefore, we first showed in a female rat model that isoproterenol induces TS-like echocardiographic changes, evidence of nitrosative stress, and consequent activation of the energy-depleting enzyme poly(ADP-ribose) polymerase-1. We subsequently showed that pre-treatment with an inhibitor of poly(ADP-ribose) polymerase-1 ameliorated contractile abnormalities. These findings thus add to previous reports of aberrant ß-adrenoceptor signaling (coupled with nitric oxide synthase activation) to elucidate mechanisms of impaired cardiac function in TS and point to potential methods of treatment.

Enantioselectivity in the tissue distribution of perhexiline contributes to different effects on hepatic histology and peripheral neural function in rats.

Perhexiline, a chiral drug, is a potent antiischemic agent whose clinical utility is limited by hepatic and neural toxicities. It inhibits mitochondrial carnitine palmitoyltransferase-1, however, excessive inhibition predisposes toward tissue steatosis. This pilot study investigated the distribution of the two enantiomers and their toxicological potential. Dark Agouti rats (n = 4 per group) were administered vehicle or 200 mg/kg daily of racemic, (+)- or (-)-perhexiline maleate orally for 8 weeks. Plasma biochemical liver function tests and Von Frey assessments of peripheral neural function were performed. Hepatic and neuronal histology, including lipid and glycogen content, was assessed using electron microscopy. Concentrations of the perhexiline enantiomers and metabolites were quantified in plasma, liver and heart. Plasma perhexiline concentrations following administration of racemate, (+)- or (-)-enantiomer were within the mid-upper clinical therapeutic range. There was extensive uptake of both enantiomers into liver and heart, with 2.5- to 4.5-fold greater net uptake of (+)- compared to (-)-perhexiline (P < .05) when administered as pure enantiomers, but not when administered as racemate. There was no biochemical or gross histological evidence of hepatotoxicity. However, livers of animals administered (+)-perhexiline had higher lipid (P < .01) and lower glycogen (P < .05) content, compared to those administered (-)-perhexiline. Animals administered racemic perhexiline had reduced peripheral neural function (P < .05) compared to controls or animals administered (-)-perhexiline. For the same plasma concentrations, differences in tissue distribution may contribute to disparities in the effects of (+)- and (-)-perhexiline on hepatic histology and neural function.

Inflammatory peroxidases promote breast cancer progression in mice via regulation of the tumour microenvironment.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, well known for their antimicrobial activity, are released in high quantities by infiltrating immune cells in breast cancer. However, the functional importance of their presence within the tumour microenvironment is unclear. We have recently described a new role for peroxidases as key regulators of fibroblast and endothelial cell functionality. In the present study, we investigate for the first time, the ability of peroxidases to promote breast cancer development and progression. Using the 4T1 syngeneic murine orthotopic breast cancer model, we examined whether increased levels of peroxidases in developing mammary tumours influences primary tumour growth and metastasis. We showed that MPO and EPO stimulation increased mammary tumour growth and enhanced lung metastases, effects that were associated with reduced tumour necrosis, increased collagen deposition and neo-vascularisation within the primary tumour. In vitro, peroxidase treatment, robustly stimulated human mammary fibroblast migration and collagen type I and type VI secretion. Mechanistically, peroxidases induced the transcription of pro-tumorigenic and metastatic MMP1, MMP3 and COX-2 genes. Taken together, these findings identify peroxidases as key contributors to cancer progression by augmenting pro-tumorigenic collagen production and angiogenesis. Importantly, this identifies inflammatory peroxidases as therapeutic targets in breast cancer therapy.

Stereoselective handling of perhexiline: implications regarding accumulation within the human myocardium.

PURPOSE: Perhexiline is a prophylactic anti-ischaemic agent with weak calcium antagonist effect which has been increasingly utilised in the management of refractory angina. The metabolic clearance of perhexiline is modulated by CYP2D6 metaboliser status and stereoselectivity. The current study sought to (1) determine whether the acute accumulation of perhexiline in the myocardium is stereoselective and (2) investigate the relationship between duration of short-term therapy and the potential stereoselective effects of perhexiline within myocardium. METHOD: Patients (n = 129) from the active arm of a randomised controlled trial of preoperative perhexiline in cardiac surgery were treated with oral perhexiline for a median of 9 days. Correlates of atrial and ventricular concentrations of enantiomers were sought via univariate followed by multivariate analyses. RESULTS: Myocardial uptake of both (+) and (-) perhexiline was greater in ventricles than in atria, and there was more rapid clearance of (-) than (+) perhexiline. The main determinants of atrial uptake of both (+) and (-) perhexiline were the plasma concentrations [(+) perhexiline: ß = -0.256, p = 0.015; (-) perhexiline: ß = -0.347, p = 0.001] and patients' age [(+) perhexiline: ß = 0.300, p = 0.004; (-) perhexiline: ß = 0.288, p = 0.005]. Atrial uptake of (+) enantiomer also varied directly with duration of therapy (ß = 0.228, p = 0.025), while atrial uptake of (-) perhexiline varied inversely with simultaneous heart rate (ß = -0.240, p = 0.015). CONCLUSION: (1) Uptake of both perhexiline enantiomers into atrium is greater with advanced age and displays evidence of both saturability and minor stereoselectivity. (2) Atrial uptake of (-) perhexiline may selectively modulate heart rate reduction.

Validation of a High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Perhexiline and Cis-Hydroxy-Perhexiline Plasma Concentrations.

BACKGROUND: The polymorphic nature of cytochrome P450 2D6 has made therapeutic drug monitoring of the anti-anginal agent perhexiline a compulsory step in reducing adverse events associated with plasma concentrations above the therapeutic range (0.15-0.60 mg/L). The aim of this study was to develop a high-performance liquid chromatography-mass spectrometry/mass spectrometry method for the determination of plasma perhexiline concentrations and its major metabolite cis-hydroxy-perhexiline to reduce sample extraction procedures and improve sample turnaround times. METHODS: The method was validated by determining the precision and accuracy of calibrators and quality control material, comparing quality assurance program samples and patient samples measured by a previously reported liquid-liquid extraction fluorescence (FL) detection high-performance liquid chromatography method and performing matrix effects investigations. RESULTS: Replicates of calibrators at concentrations of 3.00 and 0.05 mg/L demonstrated imprecision of <10.8% and inaccuracy of <8.2% for perhexiline and <10.1% and <4.5% for cis-hydroxy-perhexiline, respectively. All samples measured by the 2 methods (n = 102) demonstrated Deming regression of perhexiline = 1.20 FL + 0.00 (Sy.x = 0.08, 1/slope = 0.67); cis-hydroxy-perhexiline = 1.48 FL - 0.20 (Sy.x = 0.40, 1/slope = 0.67). CONCLUSIONS: The assay performance was deemed acceptable and integrated into the routine therapeutic drug monitoring program of the department.

Suppression of neutrophil superoxide generation by BNP is attenuated in acute heart failure: a case for 'BNP resistance'.

AIMS: The release of the B-type natriuretic peptide (BNP) is increased in heart failure (HF), a condition associated with oxidative stress. BNP is known to exert anti-inflammatory effects including suppression of neutrophil superoxide (O2(-)) release. However, BNP-based restoration of homeostasis in HF is inadequate, and the equivocal clinical benefit of a recombinant BNP, nesiritide, raises the possibility of attenuated response to BNP. We therefore tested the hypothesis that BNP-induced suppression of neutrophil O2(-) generation is impaired in patients with acute HF. METHODS AND RESULTS: We have recently characterized suppression of neutrophil O2(-) generation (PMA- or fMLP-stimulated neutrophil burst) by BNP as a measure of its physiological activity. In the present study, BNP response was compared in neutrophils of healthy subjects (n = 29) and HF patients (n = 45). Effects of BNP on fMLP-induced phosphorylation of the NAD(P)H oxidase subunit p47phox were also evaluated. In acute HF patients, the suppressing effect of BNP (1 µmol/L) on O2(-) generation was attenuated relative to that in healthy subjects (P < 0.05 for both PMA and fMLP). Analogously, BNP inhibited p47phox phosphorylation in healthy subjects but not in HF patients (P < 0.05). However, O2(-)-suppressing effects of the cell-permeable cGMP analogue (8-pCPT-cGMP) were preserved in acute HF. Conventional HF treatment for 5 weeks partially restored neutrophil BNP responsiveness (n = 25, P < 0.05), despite no significant decrease in plasma NT-proBNP levels. CONCLUSIONS: BNP inhibits neutrophil O2(-) generation by suppressing NAD(P)H oxidase assembly. This effect is impaired in acute HF patients, with partial recovery during treatment.

Reversal of hyperglycemia: effects on nitric oxide signaling.

BACKGROUND: Hyperglycemia in patients with acute coronary syndromes is associated with poor outcomes, and its rapid correction with insulin infusion has been shown to restore platelet responsiveness to nitric oxide and to suppress superoxide (O2(-)) generation. Thioredoxin-interacting protein has emerged recently as a pivotal modulator of hyperglycemia-induced inflammation, O2(-) production, and impairment of nitric oxide signaling, but it is not known whether its expression in platelets can be downregulated rapidly. METHODS: In 12 hyperglycemic patients with acute coronary syndrome, we evaluated the putative role of thioredoxin-interacting protein suppression in the platelet nitric oxide response after reversal of hyperglycemia with insulin infusion. RESULTS: Insulin infusion for 13.0 ± 0.8 (standard error of the mean) hours decreased blood glucose level from 16.6 ± 1.6 mmol/L to 8.7 ± 1.4 mmol/L (P = .002). This induced (1) sensitization of antiaggregatory response to nitric oxide (from 6.5% ± 7.7% to 39.7% ± 7.0%, P < .0001); (2) improved endothelial progenitor cell function (from a median of 45 to 180 colony-forming units, P < .05); and (3) decreases of whole blood reactive oxygen species content (P < .05). However, there was no significant suppression of platelet thioredoxin-interacting protein expression (mean decrease, 59 arbitrary units; 95% confidence interval, -193 to +74). CONCLUSIONS: Correction of hyperglycemia in patients with acute coronary syndrome rapidly reverses oxidative stress, restoring both platelet nitric oxide responsiveness and endothelial progenitor cell function, but this process is largely or entirely independent of thioredoxin-interacting protein.

Comparison of CYP2D metabolism and hepatotoxicity of the myocardial metabolic agent perhexiline in Sprague-Dawley and Dark Agouti rats.

1. Perhexiline, a chiral anti-anginal agent, may be useful to develop new cardiovascular therapies, despite its potential hepatotoxicity. 2. This study compared Dark Agouti (DA) and Sprague-Dawley (SD) rats, as models of perhexiline's metabolism and hepatotoxicity in humans. Rats (n = 4/group) received vehicle or 200 mg/kg/d of racemic perhexiline maleate for 8 weeks. Plasma and liver samples were collected to determine concentrations of perhexiline and its metabolites, hepatic function and histology. 3. Median (range) plasma and liver perhexiline concentrations in SD rats were 0.09 (0.04-0.13) mg/L and 5.42 (0.92-8.22) ng/mg, respectively. In comparison, DA rats showed higher (p < 0.05) plasma 0.50 (0.16-1.13) mg/L and liver 24.5 (9.40-54.7) ng/mg perhexiline concentrations, respectively, 2.5- and 3.7-fold higher cis-OH-perhexiline concentrations, respectively (p < 0.05), and lower plasma metabolic ratio (0.89 versus 1.55, p < 0.05). In both strains, the (+):(-) enantiomer ratio was 2:1. Perhexiline increased plasma LDH concentrations in DA rats (p < 0.05), but had no effect on plasma biochemistry in SD rats. Liver histology revealed lower glycogen content in perhexiline-treated SD rats (p < 0.05), but no effects on lipid content in either strain. 4. DA rats appeared more similar to humans with respect to plasma perhexiline concentrations, metabolic ratio, enantioselective disposition and biochemical changes suggestive of perhexiline-induced toxicity.

Relationship between plasma, atrial and ventricular perhexiline concentrations in humans: insights into factors affecting myocardial uptake.

AIM: Little is known regarding the steady-state uptake of drugs into the human myocardium. Perhexiline is a prophylactic anti-anginal drug which is increasingly also used in the treatment of heart failure and hypertrophic cardiomyopathy. We explored the relationship between plasma perhexiline concentrations and its uptake into the myocardium. METHODS: Blood, right atrium ± left ventricle biopsies were obtained from patients treated with perhexiline for a median of 8.5 days before undergoing coronary surgery in the perhexiline arm of a randomized controlled trial. Perhexiline concentrations in plasma and heart tissue were determined by HPLC. RESULTS: Atrial biopsies were obtained from 94 patients and ventricular biopsies from 28 patients. The median plasma perhexiline concentration was within the therapeutic range at 0.24 mg l⁻¹ (IQR 0.12-0.44), the median atrial concentration was 6.02 mg kg⁻¹ (IQR 2.70-9.06) and median ventricular concentration was 10.0 mg kg⁻¹ (IQR 5.76-13.1). Atrial (r² = 0.76) and ventricular (r² = 0.73) perhexiline concentrations were closely and directly correlated with plasma concentrations (both P < 0.001). The median atrial : plasma ratio was 21.5 (IQR 18.1-27.1), ventricular : plasma ratio was 34.9 (IQR 24.5-55.2) and ventricular : atrial ratio was 1.67 (IQR 1.39-2.22). Using multiple regression, the best model for predicting steady-state atrial concentration included plasma perhexiline, heart rate and age (r² = 0.83). Ventricular concentrations were directly correlated with plasma perhexiline concentration and length of therapy (r² = 0.84). CONCLUSIONS: This study demonstrates that plasma perhexiline concentrations are predictive of myocardial drug concentrations, a major determinant of drug effect. However, net myocardial perhexiline uptake is significantly modulated by patient age, potentially via alteration of myocardial:extracardiac drug uptake.