Major Facilitator Superfamily Domain Containing 5 Inhibition Reduces Lipoprotein(a) Uptake and Calcification in Valvular Heart Disease.

BACKGROUND: High circulating levels of Lp(a) (lipoprotein[a]) increase the risk of atherosclerosis and calcific aortic valve disease, affecting millions of patients worldwide. Although atherosclerosis is commonly treated with low-density lipoprotein-targeting therapies, these do not reduce Lp(a) or risk of calcific aortic valve disease, which has no available drug therapies. Targeting Lp(a) production and catabolism may provide therapeutic benefit, but little is known about Lp(a) cellular uptake. METHODS: Here, unbiased ligand-receptor capture mass spectrometry was used to identify MFSD5 (major facilitator superfamily domain containing 5) as a novel receptor/cofactor involved in Lp(a) uptake. RESULTS: Reducing MFSD5 expression by a computationally identified small molecule or small interfering RNA suppressed Lp(a) uptake and calcification in primary human valvular endothelial and interstitial cells. MFSD5 variants were associated with aortic stenosis (P=0.027 after multiple hypothesis testing) with evidence suggestive of an interaction with plasma Lp(a) levels. CONCLUSIONS: MFSD5 knockdown suppressing human valvular cell Lp(a) uptake and calcification, along with meta-analysis of MFSD5 variants associating with aortic stenosis, supports further preclinical assessment of MFSD5 in cardiovascular diseases, the leading cause of death worldwide.

A Combined Gas-Phase Separation Strategy for ADP-ribosylated Peptides.

ADP-ribosylation (ADPr) is a post-translational modification that is best studied using mass spectrometry. Method developments that are permissive with low inputs or baseline levels of protein ribosylation represent the next frontier in the field. High-field asymmetric waveform ion mobility spectrometry (FAIMS) reduces peptide complexity in the gas phase, providing a means to achieve maximal ADPr peptide sequencing depth. We therefore investigated the extent to which FAIMS with or without traditional gas-phase fractionation-separation (GPS) can increase the number of ADPr peptides. We examined ADPr peptides enriched from mouse spleens. We gleaned additional insight by also reporting findings from the corresponding non-ADPr peptide contaminants and the peptide inputs for ADPr peptide enrichment. At increasingly higher negative compensation voltages, ADPr peptides were more stable, whereas the non-ADPr peptides were filtered out. A combination of 3 GPS survey scans, each with 8 compensation voltages, resulted in 790 high-confidence ADPr peptides, compared to 90 with GPS alone. A simplified acquisition strategy requiring only two injections corresponding to two MS1 scan ranges coupled to optimized compensation voltage settings provided 402 ADPr peptides corresponding to 234 ADPr proteins. We conclude that our combined GPS strategy is a valuable addition to any ADP-ribosylome workflow. The data are available via ProteomeXchange with identifier PXD040898.

NLRP3 Inflammasome Activation in Peripheral Arterial Disease.

Background Peripheral arterial disease (PAD) is estimated to affect 7% of the adult population in the United States; however, there is currently little understanding of the key cellular and molecular pathways at play. With PAD characterized by vascular inflammation and associated calcification, the current study set out to elucidate the role of NLRP3 (nucleotide oligomerization domain-like receptor family, pyrin domain containing 3) inflammasome activation in the current cohort. Methods and Results Global proteomics of human vessels with and without PAD from a total of 14 donors revealed an increase of proinflammatory associated ontologies, specifically acute phase and innate immunity. Targeted mass spectrometry showed a significant increase in NLRP3, confirmed by NLRP3 ELISA. Histological analysis from the same patients demonstrated expression of NLRP3, colocalizing in immunoreactive CD68 (cluster of differentiation 68) and CD209 (cluster of differentiation 209) macrophages. Moreover, transmission electron microscopy showed the locality of macrophage-like cells in the presence of calcification, with confocal microscopy further validating the localization of CD68, NLRP3, and calcification via near-infrared calcium tracer. Systemic inflammation and the presence of the NLRP3 inflammasome was assessed via flow cytometry and ELISA, respectively. Compared with patients without PAD, NLRP3 expression was significantly increased in serum. In addition, proinflammatory cytokine presence was significantly increased in disease versus control, with IL (interleukin)-1ß, TNF-α (tumor necrosis factor α), and IL-33 demonstrating the greatest disparity, correlating with NLRP3 activation. Conclusions The current findings demonstrate a link between NLRP3, macrophage accumulation, and calcification in arteries of patients with PAD, suggesting an association or possible driver of PAD in these patients.

Sortilin enhances fibrosis and calcification in aortic valve disease by inducing interstitial cell heterogeneity.

AIMS: Calcific aortic valve disease (CAVD) is the most common valve disease, which consists of a chronic interplay of inflammation, fibrosis, and calcification. In this study, sortilin (SORT1) was identified as a novel key player in the pathophysiology of CAVD, and its role in the transformation of valvular interstitial cells (VICs) into pathological phenotypes is explored. METHODS AND RESULTS: An aortic valve (AV) wire injury (AVWI) mouse model with sortilin deficiency was used to determine the effects of sortilin on AV stenosis, fibrosis, and calcification. In vitro experiments employed human primary VICs cultured in osteogenic conditions for 7, 14, and 21 days; and processed for imaging, proteomics, and transcriptomics including single-cell RNA-sequencing (scRNA-seq). The AVWI mouse model showed reduced AV fibrosis, calcification, and stenosis in sortilin-deficient mice vs. littermate controls. Protein studies identified the transition of human VICs into a myofibroblast-like phenotype mediated by sortilin. Sortilin loss-of-function decreased in vitro VIC calcification. ScRNA-seq identified 12 differentially expressed cell clusters in human VIC samples, where a novel combined inflammatory myofibroblastic-osteogenic VIC (IMO-VIC) phenotype was detected with increased expression of SORT1, COL1A1, WNT5A, IL-6, and serum amyloid A1. VICs sequenced with sortilin deficiency showed decreased IMO-VIC phenotype. CONCLUSION: Sortilin promotes CAVD by mediating valvular fibrosis and calcification, and a newly identified phenotype (IMO-VIC). This is the first study to examine the role of sortilin in valvular calcification and it may render it a therapeutic target to inhibit IMO-VIC emergence by simultaneously reducing inflammation, fibrosis, and calcification, the three key pathological processes underlying CAVD.

Induced pluripotent stem cell-derived smooth muscle cells to study cardiovascular calcification.

Cardiovascular calcification is the lead predictor of cardiovascular events and the top cause of morbidity and mortality worldwide. To date, only invasive surgical options are available to treat cardiovascular calcification despite the growing understanding of underlying pathological mechanisms. Key players in vascular calcification are vascular smooth muscle cells (SMCs), which transform into calcifying SMCs and secrete mineralizing extracellular vesicles that form microcalcifications, subsequently increasing plaque instability and consequential plaque rupture. There is an increasing, practical need for a large scale and inexhaustible source of functional SMCs. Here we describe an induced pluripotent stem cell (iPSC)-derived model of SMCs by differentiating iPSCs toward SMCs to study the pathogenesis of vascular calcification. Specifically, we characterize the proteome during iPSC differentiation to better understand the cellular dynamics during this process. First, we differentiated human iPSCs toward an induced-SMC (iSMC) phenotype in a 10-day protocol. The success of iSMC differentiation was demonstrated through morphological analysis, immunofluorescent staining, flow cytometry, and proteomics characterization. Proteomics was performed throughout the entire differentiation time course to provide a robust, well-defined starting and ending cell population. Proteomics data verified iPSC differentiation to iSMCs, and functional enrichment of proteins on different days showed the key pathways changing during iSMC development. Proteomics comparison with primary human SMCs showed a high correlation with iSMCs. After iSMC differentiation, we initiated calcification in the iSMCs by culturing the cells in osteogenic media for 17 days. Calcification was verified using Alizarin Red S staining and proteomics data analysis. This study presents an inexhaustible source of functional vascular SMCs and calcifying vascular SMCs to create an in vitro model of vascular calcification in osteogenic conditions, with high potential for future applications in cardiovascular calcification research.

The RiboMaP Spectral Annotation Method Applied to Various ADP-Ribosylome Studies Including INF-γ-Stimulated Human Cells and Mouse Tissues.

ADP-ribosylation is a post-translational modification that is catalyzed by the ADP-ribosyltransferase enzyme family. Major emphasis to date has been ADP-ribosylation's role in cancer; however, there is growing interest in its role in inflammation and cardiovascular disease. Despite a recent boom in ADP-ribosylation mass spectrometry-based proteomics, there are limited computational resources to evaluate the quality of reported ADP-ribosylated (ADPr) proteins. We recently developed a novel mass spectral annotation strategy (RiboMaP) that facilitates identification and reporting of ADPr peptides and proteins. This strategy can monitor the fragmentation properties of ADPr peptide-unique fragment ions, termed m-ions and p-ions, that in turn provide spectral quality scores for candidate ADP-ribosyl peptides. In this study, we leveraged the availability of publicly available ADP-ribosylome data, acquired on various mass spectrometers, to evaluate the broader applicability of RiboMaP. We observed that fragmentation spectra of ADPr peptides vary considerably across datasets; nonetheless, RiboMaP improves ADPr peptide spectral annotation across all studies. We then reanalyzed our own previously published in vitro ADP-ribosylome data to determine common responses to the pro-inflammatory cytokine, IFN-γ. We conclude that despite these recent advances in the field of ADPr proteomics, studies in the context of inflammation and cardiovascular disease still require further bench-to-informatics workflow development in order to capture ADPr signaling events related to inflammatory pathways.

Lipoprotein(a) Induces Vesicular Cardiovascular Calcification Revealed With Single-Extracellular Vesicle Analysis.

Lipoprotein(a) (Lp[a]) blood levels >50 mg/dL is a major cardiovascular disease risk factor in humans. Lp(a) associates with increased cardiovascular calcification, a critical pathology with no clinically available drug therapies. The mechanisms through which Lp(a) increases cardiovascular calcification risk remain undefined. We hypothesized that Lp(a) promotes the release of calcifying extracellular vesicles (EVs) that contribute to formation of microcalcification in cardiovascular tissues. Here, we show Lp(a) increased calcification in both primary human smooth muscle cells (SMCs) and valvular interstitial cells (VICs), potentially through inflammation-related mechanisms that were suppressed with E06 antibody that neutralizes pro-inflammatory oxidized phospholipids. Incubating human SMCs and VICs with Lp(a) altered the composition of EVs, increasing CD29+/tetraspanin- microvesicle release, demonstrated with a tailored single-EV microarray assay that can distinguish multivesicular body-derived exosomes and plasma membrane budded microvesicles at a single-vesicle level. Lp(a) stimulation led to release of SMC and VIC EVs that readily calcified in acellular 3D-collagen hydrogels mimicking formation of ectopic microcalcification occurring in extracellular matrix of human atherosclerotic arteries and stenotic aortic valves. Our study mechanistically demonstrates that Lp(a) partially mediates cardiovascular calcification formation via inducing the release of calcifying EVs. Additionally, we provide a customized method to assess calcifying EVs at a single-vesicle level that can be more broadly applied to assist in quantitatively differentiating exosome and microvesicle EV subpopulations.

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ.

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.

ApoC-III is a novel inducer of calcification in human aortic valves.

Calcific aortic valve disease (CAVD) occurs when subpopulations of valve cells undergo specific differentiation pathways, promoting tissue fibrosis and calcification. Lipoprotein particles carry oxidized lipids that promote valvular disease, but low-density lipoprotein-lowering therapies have failed in clinical trials, and there are currently no pharmacological interventions available for this disease. Apolipoproteins are known promoters of atherosclerosis, but whether they possess pathogenic properties in CAVD is less clear. To search for a possible link, we assessed 12 apolipoproteins in nonfibrotic/noncalcific and fibrotic/calcific aortic valve tissues by proteomics and immunohistochemistry to understand if they were enriched in calcified areas. Eight apolipoproteins (apoA-I, apoA-II, apoA-IV, apoB, apoC-III, apoD, apoL-I, and apoM) were enriched in the calcific versus nonfibrotic/noncalcific tissues. Apo(a), apoB, apoC-III, apoE, and apoJ localized within the disease-prone fibrosa and colocalized with calcific regions as detected by immunohistochemistry. Circulating apoC-III on lipoprotein(a) is a potential biomarker of aortic stenosis incidence and progression, but whether apoC-III also induces aortic valve calcification is unknown. We found that apoC-III was increased in fibrotic and calcific tissues and observed within the calcification-prone fibrosa layer as well as around calcification. In addition, we showed that apoC-III induced calcification in primary human valvular cell cultures via a mitochondrial dysfunction/inflammation-mediated pathway. This study provides a first assessment of a broad array of apolipoproteins in CAVD tissues, demonstrates that specific apolipoproteins associate with valvular calcification, and implicates apoC-III as an active and modifiable driver of CAVD beyond its potential role as a biomarker.

CROT (Carnitine O-Octanoyltransferase) Is a Novel Contributing Factor in Vascular Calcification via Promoting Fatty Acid Metabolism and Mitochondrial Dysfunction.

OBJECTIVE: Vascular calcification is a critical pathology associated with increased cardiovascular event risk, but there are no Food and Drug Administration-approved anticalcific therapies. We hypothesized and validated that an unbiased screening approach would identify novel mediators of human vascular calcification. Approach and Results: We performed an unbiased quantitative proteomics and pathway network analysis that identified increased CROT (carnitine O-octanoyltransferase) in calcifying primary human coronary artery smooth muscle cells (SMCs). Additionally, human carotid artery atherosclerotic plaques contained increased immunoreactive CROT near calcified regions. CROT siRNA reduced fibrocalcific response in calcifying SMCs. In agreement, histidine 327 to alanine point mutation inactivated human CROT fatty acid metabolism enzymatic activity and suppressed SMC calcification. CROT siRNA suppressed type 1 collagen secretion, and restored mitochondrial proteome alterations, and suppressed mitochondrial fragmentation in calcifying SMCs. Lipidomics analysis of SMCs incubated with CROT siRNA revealed increased eicosapentaenoic acid, a vascular calcification inhibitor. CRISPR/Cas9-mediated Crot deficiency in LDL (low-density lipoprotein) receptor-deficient mice reduced aortic and carotid artery calcification without altering bone density or liver and plasma cholesterol and triglyceride concentrations. CONCLUSIONS: CROT is a novel contributing factor in vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction, as such CROT inhibition has strong potential as an antifibrocalcific therapy.

Computational Screening Strategy for Drug Repurposing Identified Niclosamide as Inhibitor of Vascular Calcification.

Vascular calcification is a cardiovascular disorder with no therapeutic options. We recently reported that o-octanoyltransferase (CROT) suppression can inhibit vascular calcification in vivo and in vitro through amelioration of mitochondrial function and fatty acid metabolism. Inhibiting calcification with a small molecule compound targeting CROT-associated mechanisms will be a promising non-invasive treatment of vascular calcification. Here we used a computational approach to search for existing drugs that can inhibit vascular calcification through the CROT pathway. For screening of the compounds that reduce CROT expression, we utilized the Connectivity Map encompassing the L1000 computational platform that contains transcription profiles of various cell lines and perturbagens including small molecules. Small molecules (n = 13) were identified and tested in human primary smooth muscle cells cultured in osteogenic media to induce calcification. Niclosamide, an FDA-improved anthelmintic drug, markedly inhibited calcification along with reduced alkaline phosphatase activity and CROT mRNA expression. To validate this compound in vivo, LDL receptor (Ldlr)-deficient mice fed a high fat diet were given oral doses of niclosamide (0 or 750 ppm admixed with diet) for 10 weeks. Niclosamide treatment decreased aortic and carotid artery calcification as determined by optical near infrared molecular imaging (OsteoSense680) and histological analysis. In addition, niclosamide improved features of fatty liver, including decreased cholesterol levels along with decreased Crot expression, while plasma total cholesterol levels did not change. Proteomic analysis of aortic samples demonstrated that niclosamide affected wingless/integrated (Wnt) signaling pathway and decreased runt-related transcription factor 2 (Runx2) expression, an essential factor for calcification. Our target discovery strategy using a genetic perturbation database with existing drugs identified niclosamide, that in turn inhibited calcification in vivo and in vitro, indicating its potential for the treatment of vascular calcification.

Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H]5-hydroxymethyltolterodine, in the bladder and other tissues of rats.

In vitro and in vivo binding sites of [3H]-labeled 5-hydroxymethyltolterodine (5-HMT), a new radioligand for labeling muscarinic receptors in rat tissues were characterized. Specific [3H]5-HMT binding in rat tissues was saturable and of high affinity in each tissue. The dissociation constant (Kd) was significantly lower in bladder and heart than in submaxillary gland. Significant levels of in vivo specific [3H]5-HMT binding by intravenous injection of the radioligand were detected in tissues, except for cerebral cortex. Thus, [3H]5-HMT was shown to specifically label muscarinic receptors in rat tissues, suggesting a useful radioligand for labeling muscarinic receptors with high affinity.

Muscarinic receptor binding of fesoterodine, 5-hydroxymethyl tolterodine, and tolterodine in rat tissues after the oral, intravenous, or intravesical administration.

The present study aimed to characterize muscarinic receptor binding of fesoterodine, 5-hydroxymethyl tolterodine (5-HMT), and tolterodine in bladder and other tissues of rats after their oral, intravenous, or intravesical administration. Muscarinic receptors in tissues were measured by using [N-methyl-3H]scopolamine methyl chloride ([3H]NMS). The in vitro binding affinity for muscarinic receptors was the highest by 5-HMT, followed by tolterodine and fesoterodine. Fesoterodine exhibited lower affinity in rat submaxillary gland than in detrusor muscle and urothelium. Muscarinic binding affinities of 5-HMT and tolterodine were similar among tissues. The duration of binding of oral fesoterodine to muscarinic receptors was longer in bladder than in submaxillary gland, heart, and lung, and its binding was little observed in colon and cerebral cortex. Binding activity of intravenous 5-HMT to muscarinic receptors was significantly observed in all tissues, except cerebral cortex, with a longer duration in bladder. Significant binding of bladder detrusor and urothelial muscarinic receptors was observed following intravesical instillation of 5-HMT. This selectivity may be attributed to the direct blockade of bladder receptors by excreted urinary 5-HMT. Thus, fesoterodine may be efficacious as a treatment for patients with overactive bladder.

Urinary Excretion Contributes to Long-Lasting Blockade of Bladder Muscarinic Receptors by Imidafenacin: Effect of Bilateral Ureteral Ligation.

Imidafenacin is a potent and selective antagonist of M1 and M3 muscarinic receptors that is safe, efficacious, and well tolerated for controlling the symptoms of overactive bladder (OAB). However, the precise mechanisms responsible for the bladder-selective pharmacological effects of this agent remain unclear. The in vivo pharmacologic effects of imidafenacin result from receptor occupancy. Therefore, the present study was performed to characterize in vivo muscarinic receptor binding by tritium-labeled imidafenacin with high specific activity ([3H]imidafenacin) in the bladder and other tissues of mice, and to clarify the mechanisms underlying selective binding of imidafenacin to bladder muscarinic receptors. After intravenous injection of [3H]imidafenacin, its binding to muscarinic receptors in the bladder and other tissues of mice was assessed by a radioligand binding assay. [3H]Imidafenacin showed a significantly longer duration of binding to muscarinic receptors in the bladder than in other tissues, and muscarinic receptor binding of [3H]imidafenacin was markedly suppressed in the bladder alone after bilateral ligation of the ureters. After intravenous injection, the [3H]imidafenacin concentration was markedly higher in the urine than in the plasma, suggesting that urinary excretion may contribute significantly to the selective and long-lasting binding of imidafenacin to bladder muscarinic receptors. These findings suggest that the intravesicular concentration of an antimuscarinic agent and its active metabolites may have a substantial influence on its pharmacological effect and duration of action in patients with OAB. In addition, factors that modulate urine production may influence the efficacy and safety of antimuscarinic agents.

Characterization of muscarinic receptor binding by the novel radioligand, [(3)H]imidafenacin, in the bladder and other tissues of rats.

The present study aimed to directly characterize specific binding sites of tritium ([(3)H])-labeled imidafenacin, a new radioligand for labeling muscarinic receptors, in the bladder and other peripheral or central nervous tissues of rats. Muscarinic receptors in rat tissues were measured by radioligand binding assay using [(3)H]imidafenacin. Specific [(3)H]imidafenacin binding in rat tissues was saturable, reversible, and of high affinity. Estimated dissociation constants (Kd values) were significantly lower in submaxillary gland and prostate and higher in heart than in bladder, indicating lower Kd values in M1 and M3 subtype- than M2 subtype-dominating tissues. Unlabeled imidafenacin and clinically used antimuscarinic agents competed with [(3)H]imidafenacin for binding sites in bladder and other tissues in a concentration-dependent manner, which indicated pharmacological specificity of [(3)H]imidafenacin binding sites. Pretreatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), an irreversible inactivating agent of M3 subtype, significantly decreased the number of [(3)H]imidafenacin binding sites in bladder, submaxillary gland, and colon, but not in heart. [(3)H]imidafenacin labeled muscarinic receptors in M1 and M3 subtype-dominating tissues with higher affinity than [N-methyl-(3)H]scopolamine methyl chloride (NMS). [(3)H]imidafenacin is a useful radioligand to label muscarinic receptors in M1- and M3-dominating tissues with high affinity.

Muscarinic receptor binding of the novel radioligand, [3h]imidafenacin in the human bladder and parotid gland.

The aim of the current study was to demonstrate highly specific and direct binding activity of tritium ([(3)H])-labeled imidafenacin for labeling muscarinic receptors in human bladder and parotid gland. Specific binding of [(3)H]imidafenacin in human tissues was saturable, reversible, and of high affinity. The Kd value for specific [(3)H]imidafenacin binding in the human bladder was approximately 3 times higher than that in the parotid gland. Unlabeled imidafenacin as well as the clinically used antimuscarinic agents, oxybutynin, tolterodine, and solifenacin, competed with [(3)H]imidafenacin for binding sites in the human bladder and parotid gland in a concentrationdependent manner, which indicated pharmacological specificity of [(3)H]imidafenacin binding sites. The Ki for imidafenacin in the human bladder approximately corresponded to pharmacological potency for the competitive blockade of carbachol-induced contractions of bladder, indicating a close correlation between binding affinity of imidafenacin to bladder muscarinic receptors and its pharmacological effects in the bladder. In conclusion, the current study is the first to provide direct evidence to demonstrate that imidafenacin bound muscarinic receptors in the human bladder, supporting its clinical relevance as a therapeutic agent for overactive bladder. [(3)H]Imidafenacin may also be a useful radioligand for labeling the M3 subtype of muscarinic receptors with higher selectivity than other radioligands.

Fesoterodine, its active metabolite, and tolterodine bind selectively to muscarinic receptors in human bladder mucosa and detrusor muscle.

OBJECTIVE: To comparatively characterize the binding activity of fesoterodine, its active metabolite (5-hydroxymethyl tolterodine [5-HMT]), and tolterodine in the human bladder mucosa, detrusor muscle, and parotid gland. MATERIALS AND METHODS: Muscarinic receptors in the homogenates of human bladder mucosa, detrusor muscle, and parotid gland were measured by a radioligand binding assay using [N-methyl-(3)H] scopolamine methyl chloride. RESULTS: Fesoterodine, 5-HMT, and tolterodine competed with [N-methyl-(3)H] scopolamine methyl chloride for binding sites in the bladder mucosa, detrusor muscle, and parotid gland in a concentration-dependent manner. The affinity for muscarinic receptors of these agents was significantly greater in the bladder than in the parotid gland, suggesting pharmacologic selectivity for the bladder over the parotid gland. The bladder selectivity was larger for fesoterodine and 5-HMT than for tolterodine. Fesoterodine, 5-HMT, and tolterodine resulted in significantly increased (two- to five-fold) values of the apparent dissociation constant for specific [N-methyl-(3)H] scopolamine methyl chloride binding in the detrusor muscle and parotid gland, with little effect on the corresponding values of the maximal number of binding sites. This finding indicates that these agents bind to the human muscarinic receptors in a competitive and reversible manner. CONCLUSION: Fesoterodine and 5-HMT bind to the muscarinic receptors with greater affinity in the human bladder mucosa and detrusor muscle than in the parotid gland in a competitive and reversible manner.

Characterization of bladder selectivity of antimuscarinic agents on the basis of in vivo drug-receptor binding.

The in vivo muscarinic receptor binding of antimuscarinic agents (oxybutynin, solifenacin, tolterodine, and imidafenacin) used to treat urinary dysfunction in patients with overactive bladder is reviewed. Transdermal administration of oxybutynin in rats leads to significant binding of muscarinic receptors in the bladder without long-term binding in the submaxillary gland and the abolishment of salivation evoked by oral oxybutynin. Oral solifenacin shows significant and long-lasting binding to muscarinic receptors in mouse tissues expressing the M(3) subtype. Oral tolterodine binds more selectively to muscarinic receptors in the bladder than in the submaxillary gland in mice. The muscarinic receptor binding of oral imidafenacin in rats is more selective and longer-lasting in the bladder than in other tissues such as the submaxillary gland, heart, colon, lung, and brain, suggesting preferential muscarinic receptor binding in the bladder. In vivo quantitative autoradiography with (+)N-[(11)C]methyl-3-piperidyl benzilate in rats shows significant occupancy of brain muscarinic receptors with the intravenous injection of oxybutynin, solifenacin, and tolterodine. The estimated in vivo selectivity in brain is significantly greater for solifenacin and tolterodine than for oxybutynin. Imidafenacin occupies few brain muscarinic receptors. Similar findings for oral oxybutynin were observed with positron emission tomography in conscious rhesus monkeys with a significant disturbance of short-term memory. The newer generation of antimuscarinic agents may be advantageous in terms of bladder selectivity after systemic administration.

Muscarinic Receptor Binding of Imidafenacin in the Human Bladder Mucosa and Detrusor and Parotid Gland.

OBJECTIVES: The current study aimed to characterize comparatively the binding of imidafenacin to muscarinic receptors in the human bladder mucosa and detrusor muscle and parotid gland. METHODS: The muscarinic receptor in homogenates of human tissues (bladder mucosa and detrusor muscle and parotid gland) was measured using a radioligand binding assay with [N-methyl-(3) H]scopolamine methyl chloride ([(3) H]NMS). RESULTS: Imidafenacin competed with [(3) H]NMS for binding sites in the bladder mucosa and detrusor muscle and parotid gland, and its affinity was significantly (2.6-8.7 times) higher than that of oxybutynin. Also, the affinity of imidafenacin for muscarinic receptors was approximately two-fold higher in the parotid gland than bladder tissue. The affinity of imidafenacin in the mucosa was similar to that in the detrusor muscle, suggesting that this agent exhibits therapeutic effects by blocking muscarinic receptors in the mucosa as well as detrusor muscle. Scatchard analysis revealed that imidafenacin increased significantly (approximately four-fold) Kd values for [(3) H]NMS binding in the human detrusor muscle and parotid gland, with little effect on Bmax values. This observation indicates that imidafenacin binds to the muscarinic receptors in human tissues in a competitive and reversible manner. CONCLUSION: Imidafenacin binds to muscarinic receptors in the human bladder mucosa and detrusor muscle and parotid gland with high affinity. This agent was considered to exhibit therapeutic effects on the lower urinary tract symptoms due to an overactive bladder by blocking muscarinic receptors in the urothelium as well as detrusor muscle.