Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control

Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected low-copy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene-doping detection.     

EPO transgene detection in dried blood spots for antidoping application

The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-μl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-μl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-μl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.     

实时荧光定量PCR方法应用于药品无菌快速检测的研究

目的 建立应用实时荧光定量PCR进行无菌快速检测的方法。方法 选取金黄色葡萄球菌及大肠埃希菌,用裂解试剂盒抽提细菌基因组DNA,进行实时荧光定量PCR检测,并应用叠氮溴化丙锭（PMA）抑制样品中死菌基因组DNA的PCR扩增。结果 PMA能有效去除样品中死菌干扰,针对16S rRNA基因保守序列进行扩增的荧光定量PCR方法具有较高的灵敏度。在金黄色葡萄球菌和大肠埃希氏菌检测中,最低含菌量组与阴性对照组Ct值有明显差异（P〈0.05）,其最低检出限为2 CFU/PCR。在对人工污染药品的无菌检测中,该方法与药典检测方法结果一致。结论 进行无菌检查时,采用PMA去除样品中死菌基因组DNA干扰,以裂解试剂盒抽提细菌基因组后用荧光定量PCR分析样品中细菌污染,可将检测时间缩短到4 h左右,操作简单,灵敏性高,可应用于药品无菌检查的快速筛查。     

Towards a robust test to detect gene doping for anabolic enhancement in human athletes

Success in gene therapy in treating human disease makes this technology attractive to enhance athletic performance, creating the need for gene doping detection. In 2021, World Anti-Doping Agency (WADA) approved the first gene doping test. Here, we describe a new method to detect doping with four additional genes, follistatin, growth hormone 1, growth hormone–releasing hormone and insulin-like growth factor 1, that may improve performance by increasing muscle size and strength. The method utilises four hydrolysis probe-based polymerase chain reaction (PCR) assays that target the transgenes based on the coding sequence of the four endogenous genes. The assays are specific, reproducible and capable to detect five copies of transgene in the presence of very similar endogenous gene in 25,000 times excess. To underpin reliable and comparable routine method performance by doping testing laboratories, a synthetic reference material for the method was designed and generated following the ISO Guide 35. The complete method was validated in blood samples using plasma as extraction matrix and QIAamp DNA blood midi DNA extraction kit. All blood samples from different donors (n = 8) simulated to be negative or positive (1500 transgene copies spiked per millilitre of blood) for the transgenes were reported correctly. The new method that targets four additional genes will extend the capabilities of laboratories involved in doping control to protect athletes' health, fairness and equality.     

双重实时荧光定量 PCR 检测人维生素 D 受体的方法学构建

目的 建立单管检测人维生素 D 受体（VDR）及甘油醛-3-磷酸脱氢酶（GAPDH）基因的双重实时荧光定量聚合酶链反应 （dual real-time PCR） 的方法。方法 以 GAPDH 基因为内参, 采用 Primer Premier 5.0 软件设计特异性引物及 TaqMan 探针, 进行 PCR 扩增检测 VDR 基因。将 VDR 及 GAPDH 扩增产物片段纯化后克隆构建成重组质粒, 作为定量检测基因表达的标准品, 并用于分析该方法的灵敏度和重复性。结果 PCR 扩增产物经测序分析证实为 VDR 及 GAPDH 特异性片段; 该方法检测 VDR 与 GAPDH 灵敏度达 40 拷贝/μL; 线性范围为 4.00×101～4.00×105 拷贝/μL; 决定系数 R2分别为 0.998、 0.999; 扩增效率 E 分别为 96.10%、 85.15%; 批内变异系数（CV）分别为 0.09%~ 1.21%、 0.35%~0.88%; 批间 CV 分别为 0.17%~0.51%、 0.51%~2.46%。结论 成功建立了单管检测人 VDR 及 GAPDH 的双重实时荧光定量 PCR 方法, 且该方法特异性好、 灵敏度高、 可快速高通量检测 VDR 的相对表达量, 有效缩短时间, 减小实验误差。     

Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide

Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at −18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 10³ CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 10⁰ CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach.     

Detection of intra‐articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma

Gene therapy promotes the expression of missing or defective genes and can be curative following administration of a single dose. Gene therapy is prohibited in equine athletes by regulatory bodies due to the high potential for abuse and novel analytical methods are needed for detection. The goal of this study was to detect the administration of an experimental gene therapy: a recombinant adeno‐associated viral vector (rAAV) carrying a transgene for the anti‐inflammatory cytokine IL‐10 (rAAV‐IL10). Twelve horses were randomly assigned to receive an intra‐articular injection of rAAV‐IL10 or phosphate buffered saline (vehicle) into a middle carpal joint. Plasma and synovial fluid were collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Primer pairs were designed to detect two unique regions of rAAV. Using quantitative real time PCR, both sets of primers detected rAAV for 14–28 days in joints and up to 4 days in plasma, in all six treated horses. In synovial fluid, rAAV was detected on day 56 in 4/6 horses by both primer sets, and on day 84 in 1/6 horses by one primer set. In plasma, rAAV was detected for 7 days in 5/6 horses, 14 days in 2/6 horses, and 28 days in 1/6 horses by one primer set, and was detected for up to 14 days in 1/6 horses by the other primer set. This study is the first to validate that quantitative real time PCR can be used to systemically detect the local administration of a gene therapy product to horses.     

Inhibitory effects of sulfonylureas and non‐steroidal anti‐inflammatory drugs on in vitro metabolism of canagliflozin in human liver microsomes

Canagliflozin, used to treat type 2 diabetes mellitus (T2DM), is commonly co‐administered with sulfonylureas. The objective of the present study was to evaluate the possible inhibitory effect of sulfonylureas and non‐steroidal anti‐inflammatory drugs (NSAIDs) on canagliflozin metabolism in vitro. Three sulfonylurea derivatives were evaluated as inhibitors: chlorpropamide, glimepiride and gliclazide. Two other NSAIDs were used as positive control inhibitors: niflumic acid and diclofenac. The rate of formation of canagliflozin metabolites was determined by HPLC analysis of in vitro incubations of canagliflozin as a substrate with and without inhibitors, using human liver microsomes (HLMs). Among sulfonylureas, glimepiride showed the most potent inhibitory effect against canagliflozin M7 metabolite formation, with an IC₅₀ value of 88 μm , compared to chlorpropamide and gliclazide with IC₅₀ values of more than 500 μm . Diclofenac inhibited M5 metabolite formation more than M7, with IC₅₀ values of 32 μm for M5 and 80 μm for M7. Niflumic acid showed no inhibition activity against M5 formation, but had relatively selective inhibitory potency against M7 formation, which is catalysed by UGT1A9, with an IC₅₀ value of 1.9 μm and an inhibition constant value of 0.8 μm . A clinical pharmacokinetic interaction between canagliflozin and sulfonylureas is unlikely. However, a possible clinically important drug interaction between niflumic acid and canagliflozin has been identified.     

Administration and detection of gene therapy in horses: A systematic review

Gene therapy uses genetic modification of cells to produce a therapeutic effect. Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression. Gene therapy is currently prohibited in both human and equine athletes because of the potential to induce production of performance-enhancing proteins in the athlete's body, also referred to as “gene doping.” Detection of gene doping is challenging and necessitates development of creative, novel analytical methods for doping control. Methods for detection of gene doping must be specific to and will vary depending on the type of gene therapy. The purpose of this paper is to present the results of a systematic review of gene editing, gene therapy, and detection of gene doping in horses. Based on the published literature, gene therapy has been administered to horses in a large number of experimental studies and a smaller number of clinical cases. Detection of gene therapy is possible using a combination of PCR and sequencing technologies. This summary can provide a basis for discussion of appropriate and inappropriate uses for gene therapy in horses by the veterinary community and guide expansion of methods to detect inappropriate uses by the regulatory community.     

In‐vitro antileishmanial potential of peptide drug hirudin

Hirudin is clinically an important drug used for the treatment of cardiac diseases, but has never been elucidated for antileishmanial potential. This study was designed to determine the therapeutic utility of hirudin against leishmaniasis. Binding affinities of 28 potent proteinase inhibitors were screened computationally against leishmanolysin (GP63), out of which hirudin exhibited higher binding affinity with GP63 and good expected IC₅₀ values. Experimentally, hirudin showed most promising activity against promastigote and axenic amastigote forms of leishmanial parasites with IC₅₀ values of 0.60 ± 0.36 μg/mL and 0.43 ± 0.23 μg/mL, respectively, in a dose‐ and time‐dependent assay. The cytotoxicity assay revealed no adverse effects on human macrophages with LD₅₀ value of 860.11 ± 53.44 μg/mL. Hirudin caused leishmanial cell death mainly by apoptosis and membrane permeability. In spite of the basic knowledge obtained, hirudin mechanism is considerably less prone to the induction of resistance than classical drugs. Collectively, this study fosters further studies for the hirudin as new antileishmania lead with a new mode of action.     

Purification and characterization of a metalloproteinase, Porthidin-1, from the venom of Lansberg’s hog-nosed pitvipers (Porthidium lansbergii hutmanni)

Porthidium lansbergii hutmanni is a small pit viper found on Margarita Island, Venezuela. Local tissue damage is one of the most obvious characteristics of P. l. hutmanni envenomation, which can lead to diverse pathological effects, such as hemorrhage, edema, blistering, necrosis, lymphatic vessel damage and degradation of extracellular matrix. Metalloproteinases are one of the major components in venoms responsible for these effects. To date, very little is known or has been reported on P. l. hutmanni venom. Crude P. l. hutmanni venom had a LD₅₀ of 2.5 mg/kg and was considered very hemorrhagic (minimal hemorrhagic dose [MHD]: 0.98 μg) when compared to other hemorrhagic (Bothrops) venoms in Venezuela. Crude P. l. hutmanni venom also inhibited ADP-induced platelet aggregation. A metalloproteinase, Porthidin-1, from this venom was isolated by three chromatography steps (Sephadex G100, Superose 12 HR10/30 and Bioscale Q2). Porthidin-1 falls in the SVMP P-I class having a molecular weight of 23 kDa, verified by both SDS-PAGE and mass spectrometry. High-resolution mass spectrometry and a database search identified a peptide from Porthidin-1 (YNGDLDK) belonging to the SVMP family of proteins. Porthidin-1 contained hemorrhagic, fibrino(geno)lytic, caseinolytic and gelatinolytic activities, and these activities were capable of being neutralized by metalloproteinase inhibitors but not serine proteinase inhibitors. The peptide YNGDLDK shared similarities with five venom proteins with a BLAST e-value of <1. This work details the biochemical and pathophysiological effects that can result from envenomations, and highlights the importance and significance for characterizing unknown or poorly documented venoms from different geographical regions.     

Synthesis and biological evaluation of novel N‐aryl‐ω‐(benzoazol‐2‐yl)‐sulfanylalkanamides as dual inhibitors of α‐glucosidase and protein tyrosine phosphatase 1B

α‐Glucosidase is known to catalyze the digestion of carbohydrates and release free glucose into the digestive tract. Protein tyrosine phosphatase 1B (PTP1B) is engaged in the dephosphorylation of the insulin receptor and regulation of insulin sensitivity. Therefore, dual antagonists by targeting both α‐glucosidase and PTP1B may be potential candidates for type 2 diabetes therapy. In this work, three series of novel N‐aryl‐ω‐(benzoazol‐2‐yl)‐sulfanylalkanamides were synthesized and assayed for their α‐glucosidase and PTP1B inhibitory activities, respectively. Compound 3l , exhibiting the most effective α‐glucosidase inhibitory activity (IC₅₀ = 10.96 μm ( 3l ), IC₅₀ = 51.32 μm (Acarbose), IC₅₀ = 18.22 μm (Ursolic acid)) and potent PTP1B inhibitory activity (IC₅₀ = 13.46 μm ( 3l ), IC₅₀ = 14.50 μm (Ursolic acid)), was identified as a novel dual inhibitor of α‐glucosidase and PTP1B. Furthermore, 3l is a highly selective PTP1B inhibitor because no inhibition was showed by 3l at 100 μm against PTP‐MEG2, TCPTP, SHP2, or SHP1. Subsequent kinetic analysis revealed 3l inhibited α‐glucosidase in a reversible and mixed manner. Molecular docking study indicated that hydrogen bonds, van der Waals, charge interactions and Pi‐cation interactions all contributed to affinity between 3l and α‐glucosidase/PTP1B.     

N-acetylaspartylglutamate (NAAG) inhibits intravenous cocaine self-administration and cocaine-enhanced brain-stimulation reward in rats

Pharmacological activation of group II metabotropic glutamate (mGlu2 and mGlu3) receptors inhibits reward-seeking behavior and/or rewarding efficacy induced by drugs (cocaine, nicotine) or natural rewards (food, sucrose). In the present study, we investigated whether elevation of brain N-acetylaspartylglutamate (NAAG), an endogenous group II mGlu receptor agonist, by the NAAG peptidase inhibitor 2-PMPA attenuates cocaine's rewarding effects, as assessed by intravenous cocaine self-administration and intracranial electrical brain-stimulation reward (BSR) in rats. Systemic administration of 2-PMPA (10, 30, 100 mg/kg, i.p.) or intranasal administration of NAAG (100, 300 μg/10 μl/nostril) significantly inhibited intravenous cocaine self-administration under progressive-ratio (PR), but not under fixed-ratio 2 (FR2), reinforcement conditions. In addition, 2-PMPA (1, 10, 30 mg/kg, i.p) or NAAG (50, 100 μg/10 μl/nostril) significantly inhibited cocaine-enhanced BSR, but not basal BSR. Pretreatment with LY341495 (1 mg/kg, i.p.), a selective mGlu2/3 receptor antagonist, prevented the inhibitory effects produced by 2-PMPA or NAAG in both the self-administration and BSR paradigms. In vivo microdialysis demonstrated that 2-PMPA (10, 30, 100 mg/kg) dose-dependently attenuated cocaine-enhanced extracellular dopamine (DA) in the nucleus accumbens (NAc). 2-PMPA alone inhibited basal NAc DA release, an effect that was prevented by LY341495. These findings suggest that systemic administration of 2-PMPA or intranasal administration of NAAG inhibits cocaine's rewarding efficacy and cocaine-enhanced NAc DA - likely by activation of presynaptic mGlu2/3 receptors in the NAc. These data suggest a potential utility for 2-PMPA or NAAG in the treatment of cocaine addiction.     

Drug−drug Interaction between Losartan and Paclitaxel in Human Liver Microsomes with Different CYP2C8 Genotypes

The cytochrome P450 (CYP) 2C8*3 allele is associated with reduced metabolic activity of paclitaxel. This study was aimed to investigate the inhibitory effect of losartan on paclitaxel metabolism in human liver microsomes (HLMs) and to determine the impact of the CYP2C8*3 polymorphism. HLMs that contained the CYP2C8*1 homozygote (HL60) or CYP2C8*3 heterozygote (HL54) genotype were used for the inhibition study. Losartan, at a concentration of 50 μmol/L, significantly inhibited paclitaxel metabolism by 29% and 57% in the HL60 (p < 0.001) and HL54 (p < 0.01), respectively. When using HL60, losartan and the CYP3A4‐selective inhibitors, erythromycin and ketoconazole, caused a greater inhibition of the paclitaxel metabolism than quercetin, a CYP2C8‐selective inhibitor. This demonstrated that the paclitaxel metabolism was mainly catalysed by CYP3A4 in HL60. There were no significant differences found for the inhibitory effects caused by the four inhibitors of the paclitaxel metabolism in HL54, indicating that both CYP2C8 and CYP3A4 play important roles in paclitaxel metabolism in HL54. These findings suggest that 50 μmol/L of losartan inhibits both CYP2C8 and CYP3A4 in HLMs. In summary, losartan inhibited paclitaxel metabolism, with concentrations over 50 μmol/L in HLMs. The CYP2C8*3 allele carriers are likely susceptible to the interactions of losartan and CYP3A4 inhibitors to paclitaxel metabolism.     

分析仪器确认规范在实时荧光定量PCR仪确认中的实施

 目的  通过对实时荧光定量PCR（real-time fluorescent quantitative PCR,RTFQ-PCR）仪实施确认,了解分析仪器确认的具体步骤。方法  根据药品生产质量管理规范的要求,对该RTFQ-PCR仪分别进行安装确认、运行确认和性能确认,以及年度再确认。在性能确认中,通过定量PCR法和环介导恒温（loop-mediated isothermal amplification,LAMP）法检测分枝杆菌核酸来验证该仪器的准确性和重复性。结果  RTFQ-PCR仪的安装符合要求,各项测试操作均达到规定的标准。当采用定量PCR法检测TB进行RTFQ-PCR仪性能确认时,结核杆菌（tubercle bacillus,TB）核酸浓度的对数与循环阈值（cycle threshold,Ct）之间呈现良好的线性关系,相关系数绝对值达0.999 9;TB核酸回收率为97%～105%;对2个TB样品分别进行3次检测获得的Ct值的变异系数（CV）均小于5%。当采用LAMP法检测分枝杆菌进行RTFQ-PCR仪性能确认和再确认时,对分枝杆菌阳性对照和样品分别进行3次检测获得的Ct值的CV均小于5%。结论  该RTFQ-PCR仪得到成功确认,各方面均符合规定的要求。     

Glycyl-glutamine (β-endorphin30-31) inhibits morphine-induced dopamine efflux in the nucleus accumbens

Glycyl-glutamine (Gly-Gln) is an endogenous dipeptide that is synthesized from β-endorphin post-translationally. Previously, we showed that Gly-Gln prevents acquisition of morphine-conditioned place preference, a behavioral test of morphine reward, but does not interfere with morphine analgesia. In this study, we tested the hypothesis that Gly-Gln inhibits morphine reward by blocking morphine-induced dopamine efflux in the nucleus accumbens (NAc). Extracellular dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were sampled by microdialysis and analyzed by high-performance liquid chromatography with electrochemical detection. Guide cannulas were implanted in the right NAc and left lateral ventricle of male Sprague–Dawley rats stereotaxically. Approximately 24 h later, a microdialysis probe was inserted into the NAc and perfused at 1 μl/min. Gly-Gln (1, 3, 30, or 100 nmol/5 μl) or saline was administered intracerebroventricularly, morphine (2.5 mg/kg) was injected intraperitoneally (i.p.) 2 min later, and extracellular dopamine and DOPAC were sampled at 20-min intervals. Morphine administration increased extracellular dopamine concentrations by approximately 600% within 40 min. Gly-Gln pretreatment inhibited the rise in extracellular dopamine in a dose-related manner; the lowest significantly inhibitory dose was 1 nmol. Gly-Gln also inhibited the morphine-induced rise in extracellular DOPAC concentrations but did not affect extracellular dopamine or DOPAC in control animals. Gly-Gln (100 nmol/5 μl) prevented morphine-induced dopamine efflux in rats treated with morphine chronically (10 mg/kg, i.p. twice daily for 6 days), although it did not affect DOPAC concentrations significantly. These data support the hypothesis that Gly-Gln abolishes the rewarding effect of morphine by inhibiting the ability of morphine to stimulate dopamine release in the NAc.     

Studies on the interaction of five triazole fungicides with human renal transporters in cells

The widespread use of triazole fungicides in agricultural production poses a potential risk to human health. This study investigates the interaction of five triazole fungicides, i.e., tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole with human renal transporters, including OAT1, OAT3, OCT2, OCTN1, OCTN2, MATE1, MATE2-K, MRP2, MDR1, and BCRP, using transgenic cell models. For uptake transporters, triticonazole was the substrate of OAT1 and OAT3 and the inhibitor of OCT2. Tebuconazole and penconazole inhibited OCTN2 (100 μM), while tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole inhibited MATE1 (100 μM). Tebuconazole and hexaconazole inhibited MATE2-K (100 μM). All five triazole fungicides were not substrates or strong inhibitors of MRP2, MDR1, and BCRP efflux transporters. Penconazole inhibited OCT2 with IC₅₀ = 1.12 μM. Penconazole and uniconazole inhibited MATE1 with IC₅₀ = 0.94 μM and 0.87 μM. Tebuconazole and hexaconazole inhibited MATE2-K with IC₅₀ = 0.96 μM and 1.04 μM, indicating that triazole fungicides may inhibit renal drug transporter activity at low concentrations. Triticonazole was transported by OAT1 and OAT3, and the K_m values of triticonazole were 5.81 ± 1.75 and 47.35 ± 14.27, respectively. Tebuconazole and uniconazole were transported by OAT3, and the K_m values of tebuconazole and uniconazole were 30.28 ± 7.18 and 87.61 ± 31.70, respectively, which may induce nephrotoxicity.     

Monoamine oxidase inhibition by selected dye compounds

Monoamine oxidase (MAO) is an important drug target as the MAO isoforms play key roles in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease, as well as in neuropsychiatric diseases such as depression. Methylene blue is an inhibitor of MAO‐A, while azure B, the major metabolite of methylene blue, and various other structural analogues retain the ability to inhibit MAO‐A. Based on this, the present study evaluated 22 dyes, many of which are structurally related to methylene blue, as potential inhibitors of human MAO‐A and MAO‐B. The results highlighted three dye compounds as good potency competitive and reversible MAO inhibitors, and which exhibit higher MAO inhibition than methylene blue: acridine orange, oxazine 170 and Darrow red. Acridine orange was found to be a MAO‐A specific inhibitor (IC₅₀ = 0.017 μM), whereas oxazine 170 is a MAO‐B specific inhibitor (IC₅₀ = 0.0065 μM). Darrow red was found to be a non‐specific MAO inhibitor (MAO‐A, IC₅₀ = 0.059 μM; MAO‐B, IC₅₀ = 0.065 μM). These compounds may be advanced for further testing and preclinical development, or be used as possible lead compounds for the future design of MAO inhibitors.