Topical Semisolid Drug Product Critical Quality Attributes with Relevance to Cutaneous Bioavailability and Pharmacokinetics: Part I-Bioequivalence of Acyclovir Topical Creams.

PURPOSE: To develop a toolkit of test methods for characterizing potentially critical quality attributes (CQAs) of topical semisolid products and to evaluate how CQAs influence the rate and extent of active ingredient bioavailability (BA) by monitoring cutaneous pharmacokinetics (PK) using an In Vitro Permeation Test (IVPT). METHODS: Product attributes representing the physicochemical and structural (Q3) arrangement of matter, such as attributes of particles and globules, were assessed for a set of test acyclovir creams (Aciclostad® and Acyclovir 1A Pharma) and compared to a set of reference acyclovir creams (Zovirax® US, Zovirax® UK and Zovirax® Australia). IVPT studies were performed with all these creams using heat-separated human epidermis, evaluated with both, static Franz-type diffusion cells and a flow through diffusion cell system. RESULTS: A toolkit developed to characterize quality and performance attributes of these acyclovir topical cream products identified certain differences in the Q3 attributes and the cutaneous PK of acyclovir between the test and reference sets of products. The cutaneous BA of acyclovir from the set of reference creams was substantially higher than from the set of test creams. CONCLUSIONS: This research elucidates how differences in the composition or manufacturing of product formulations can alter Q3 attributes that modulate myriad aspects of topical product performance. The results demonstrate the importance of understanding the Q3 attributes of topical semisolid drug products, and of developing appropriate product characterization tests. The toolkit developed here can be utilized to guide topical product development, and to mitigate the risk of differences in product performance, thereby supporting a demonstration of bioequivalence (BE) for prospective topical generic products and reducing the reliance on comparative clinical endpoint BE studies.

In vitro and in vivo protective potential of quercetin-3-glucuronide against lipopolysaccharide-induced pulmonary injury through dual activation of nuclear factor-erythroid 2 related factor 2 and autophagy.

In vitro and in vivo models of lipopolysaccharide (LPS)-induced pulmonary injury, quercetin-3-glucuronide (Q3G) has been previously revealed the lung-protective potential via downregulation of inflammation, pyroptotic, and apoptotic cell death. However, the upstream signals mediating anti-pulmonary injury of Q3G have not yet been clarified. It has been reported that concerted dual activation of nuclear factor-erythroid 2 related factor 2 (Nrf2) and autophagy may prove to be a better treatment strategy in pulmonary injury. In this study, the effect of Q3G on antioxidant and autophagy were further investigated. Noncytotoxic doses of Q3G abolished the LPS-caused cell injury, and reactive oxygen species (ROS) generation with inductions in Nrf2-antioxidant signaling. Moreover, Q3G treatment repressed Nrf2 ubiquitination, and enhanced the association of Keap1 and p62 in the LPS-treated cells. Q3G also showed potential in inducing autophagy, as demonstrated by formation of acidic vesicular organelles (AVOs) and upregulation of autophagy factors. Next, the autolysosomes formation and cell survival were decreased by Q3G under pre-treatment with a lysosome inhibitor, chloroquine (CQ). Furthermore, mechanistic assays indicated that anti-pulmonary injury effects of Q3G might be mediated via Nrf2 signaling, as confirmed by the transfection of Nrf2 siRNA. Finally, Q3G significantly alleviated the development of pulmonary injury in vivo, which may result from inhibiting the LPS-induced lung dysfunction and edema. These findings emphasize a toxicological perspective, providing new insights into the mechanisms of Q3G's protective effects on LPS-induced pulmonary injury and highlighting its role in dual activating Nrf2 and autophagy pathways.

Analysis of non-mutagenic substances in the context of drug impurity assessment - Few are potent toxicants.

ICH Q3A/B guidelines provide qualification thresholds for impurities or degradation products in new drug substances and products. However, the guidelines note that certain impurities/degradation products may warrant further safety evaluation for being unusually potent or toxic. The purpose of this study was to confirm that especially toxic non-mutagenic compounds are rare and to identify classes of compounds that could warrant lower qualification thresholds. A total of 2815 compounds were evaluated, of which 2213 were assessed as non-mutagenic. For the purpose of this analysis, compounds were considered potent when the point of departure was ≤0.2 mg/kg/day based on the qualification threshold (1 mg/day or 0.02 mg/kg/day for a 50 kg human) in a new drug substance, with an additional 10-fold margin. Only 54 of the entire set (2.4%) would be considered potent based on this conservative potency analysis, confirming that the existing ICH Q3A/B qualification thresholds are appropriate for the majority of impurities. If the Q3A/B threshold, without the additional 10-fold margin is used, 14 compounds (0.6%) are considered "highly potent". Very few non-mutagenic structural classes were identified, including organothiophosphates and derivatives, polychlorinated benzenes and polychlorinated polycyclic aliphatics, that correlate with potential high potency, consistent with prior publications.

Deriving acceptable limits for non-mutagenic impurities in medicinal products - Durational adjustments.

ICH Q3A/B guidelines are not intended for application during the clinical research phase of development and durationally adjusted qualification thresholds are not included. A central tenet of ICH Q3A is that lifetime exposure to 1 mg/day of an unqualified non-mutagenic impurity (NMI) is not a safety concern. An analysis of in vivo toxicology data from 4878 unique chemicals with established NO(A)ELs was conducted to determine whether durationally adjusted qualification limits can be supported. Although not recommended in ICH Q3A/B, a conservative approach was taken by using allometric scaling in the analysis. Following allometric scaling of the 5th percentile of the distribution of NO(A)ELs from available chronic toxicology studies, it was reconfirmed that there is a safety basis for the 1 mg/day qualification threshold in ICH Q3A. Additionally, allometric scaling of the 5th percentile of the distribution of NO(A)ELs from sub-acute and sub-chronic toxicology studies could support acceptable limits of 20 and 5 mg/day for an unqualified NMI for dosing durations of less than or greater than one month, respectively. This analysis supports durationally adjusted NMI qualification thresholds for pharmaceuticals that protect patient safety and contribute to 3Rs efforts for qualifying impurities using new approach methods.

New limits proposed for the management of non-mutagenic impurities.

Multiple international guidelines exist that describe both quality and safety considerations for the control of the broad spectrum of impurities inherent to drug substance and product manufacturing processes. However, regarding non-mutagenic impurities (NMI) the most relevant ICH Q3A/B guidelines are not applicable during early phases of drug development leading to confusion about acceptable limits at this stage. Thus, there is need for more flexible approaches that ensure that patient safety remains paramount, while taking into consideration the limited duration of exposure. An EFPIA survey, which collected quantitative data from different types of studies applied to qualify impurities in accordance with ICH Q3A, shows that no toxicities could be attributed to any of the 467 impurities at any tested level in vivo. This data combined with earlier published toxicological datasets encompassing drug substances and intermediates, food related substances and chemicals provide convincing evidence that for NMIs, the application of a generic 5 mg/day limit for an exposure duration <6 months, and a 1 mg/day generic limit for life-long exposure, provides sufficient margins to ensure patient safety. Hence, application of these absolute limits to trigger qualification studies (instead of the relative limits described in Q3A/B), is considered warranted. This approach will prevent conduct of unnecessary dedicated impurity qualification studies and the resulting use of animals.

Non-mutagenic impurities - Recent industry experience of using dose durational limits in drug development.

Absence of clear guidance on the qualification threshold for non-mutagenic impurities during clinical development is a source of inconsistency in both sponsor qualification approaches and health authority requests. A survey was conducted in March 2020 with 6 member companies of the European Federation of Pharmaceutical Industries and Associations (EFPIA). Thirteen examples were gathered of where non-International Council for Harmonisation (ICH) limits have been used in regulatory submissions for various indications and stages of development, together with the regulatory outcomes. As expected, few challenges were faced in early clinical development, with health authorities generally commenting that sponsors should work towards ICH Q3A and Q3B guideline specification limits as development progresses. However, inconsistent health authority requests were noted even for early phase clinical trials in late-stage oncology patients. For an optimised use of resources, consistent approaches would have the benefit of supporting faster access of safe medicines to patients while including Replacement, Reduction and Refinement (the 3Rs) considerations with respect to animal testing.

Combination LSD1 and HOTAIR-EZH2 inhibition disrupts cell cycle processes and induces apoptosis in glioblastoma cells.

Glioblastoma (GBM) is the most common primary central nervous system tumor and has a poor prognosis, with a median survival time of only 14 months from diagnosis. Abnormally expressed long noncoding RNAs (lncRNAs) are important epigenetic regulators of chromatin modification and gene expression regulation in tumors, including GBM. We previously showed that the lncRNA HOTAIR is related to the cell cycle progression and can be used as an independent predictor in GBM. Lysine-specific demethylase 1 (LSD1), binding to 3' domain of HOTAIR, specifically removes mono- and di-methyl marks from H3 lysine 4 (H3K4) and plays key roles during carcinogenesis. In this study, we combined a HOTAIR-EZH2 disrupting agent and an LSD1 inhibitor, AC1Q3QWB (AQB) and GSK-LSD1, respectively, to block the two functional domains of HOTAIR and potentially provide therapeutic benefit in the treatment of GBM. Using an Agilent Human ceRNA Microarray, we identified tumor suppressor genes upregulated by AQB and GSK-LSD1, followed by Chromatin immunoprecipitation (ChIP) assays to explore the epigenetic mechanisms of genes activation. Microarray analysis showed that AQB and GSK-LSD1 regulate cell cycle processes and induces apoptosis in GBM cell lines. Furthermore, we found that the combination of AQB and GSK-LSD1 showed a powerful effect of inhibiting cell cycle processes by targeting CDKN1A, whereas apoptosis promoting effects of combination therapy were mediated by BBC3 in vitro. ChIP assays revealed that GSK-LSD1 and AQB regulate P21 and PUMA, respectively via upregulating H3K4me2 and downregulating H3K27me3. Combination therapy with AQB and GSK-LSD1 on tumor malignancy in vitro and GBM patient-derived xenograft (PDX) models shows enhanced anti-tumor efficacy and appears to be a promising new strategy for GBM treatment through its effects on epigenetic regulation.

Massive pulmonary embolism presenting with hemoptysis and S1Q3T3 ECG findings.

BACKGROUND: Venous thromboembolism clinically presenting with a deep vein thrombosis or pulmonary embolism is among the most commonly seen cardiovascular syndromes. The aim of this case presentation is to emphasise the typical electrocardiographic findings that are detected with massive pulmonary embolism along with the electrocardiographic S1Q3 and S1Q3T3 accompanied by T negativity at the D3 derivation based on prevalent T negativity. CASE PRESENTATION: We present the case of an adult male who presented with a massive pulmonary embolism that was associated with tachycardia, haemoptysis and typical S1Q3T3 electrocardiographic findings. Tomographic findings showed filling defects in the two main pulmonary artery lumens, which were found to be compatible with a massive embolism. Intravenous heparin was injected (5000 IU), and low molecule weight heparin (LMWH) treatment was initiated. After two days of observation and treatment in the coronary intensive care unit, the patient was discharged for outpatient care. DISCUSSION: Massive pulmonary embolism is an urgent life-threatening clinical situation that is frequently confused with acute ST elevation myocardial infarction. The definitive diagnosis of massive pulmonary embolism was made with a computed tomography pulmonary angiogram. Electrocardiographic findings and hypoxic hypercarbia in the blood gas analysis are typical. Early diagnosis with laboratory and imaging investigations is vital in the treatment and prognosis of pulmonary embolism. CONCLUSIONS: Ventricular overload signs accompanied by ST segment elevation in electrocardiography and S1Q3 and prevalent T negativity are crucial features in terms of distinguishing between pulmonary embolism and myocardial infarction and selecting effective treatments for patients admitted to the emergency department.

Greater radial tuberosity size is associated with distal biceps tendon rupture: a quantitative 3-D CT case-control study.

PURPOSE: During pronation, the distal biceps tendon and radial tuberosity internally rotate into the radioulnar space, reducing the linear distance between the radius and ulna by approximately 50%. This leaves a small space for the distal biceps tendon to move in and could possibly cause mechanical impingement or rubbing of the distal biceps tendon. Hypertrophy of the radial tuberosity potentially increases the risk of mechanical impingement of the distal biceps tendon. The purpose of our study was to determine if radial tuberosity size is associated with rupturing of the distal biceps tendon. METHODS: Nine patients with a distal biceps tendon rupture who underwent CT were matched 1:2 to controls without distal biceps pathology. A quantitative 3-dimensional CT technique was used to calculate the following radial tuberosity characteristics: 1) volume in mm3, 2) surface area in mm2, 3) maximum height in mm and 4) location (distance in mm from the articular surface of the radial head). RESULTS: Analysis of the 3-dimensional radial tuberosity CT-models showed larger radial tuberosity volume and maximum height in the distal biceps tendon rupture group compared to the control group. Mean radial tuberosity volume in the rupture-group was 705 mm3 (SD: 222 mm3) compared to 541 mm3 (SD: 184 mm3) in the control group (p = 0.033). Mean radial tuberosity maximum height in the rupture-group was 4.6 mm (SD: 0.9 mm) compared to 3.7 mm (SD: 1.1 mm) in the control group, respectively (p = 0.011). There was no statistically significant difference in radial tuberosity surface area (ns) and radial tuberosity location (ns). CONCLUSION: Radial tuberosity volume and maximum height were significantly greater in patients with distal biceps tendon ruptures compared to matched controls without distal biceps tendon pathology. This supports the theory that hypertrophy of the radial tuberosity plays a role in developing distal biceps tendon pathology. LEVEL OF EVIDENCE: Level III.

Qualification of impurities based on metabolite data.

Regulatory Guidance documents ICH Q3A (R2) and ICH Q3B (R2) state that "impurities that are also significant metabolites present in animal and/or human studies are generally considered qualified". However, no guidance is provided regarding data requirements for qualification, nor is a definition of the term "significant metabolite" provided. An opportunity is provided to define those categories and potentially avoid separate toxicity studies to qualify impurities. This can reduce cost, animal use and time, and avoid delays in drug development progression. If the concentration or amount of a metabolite, in animals or human, is similar to that of the known, structurally identical impurity (arising from the administered test material), the qualification of the impurity on the grounds of it also being a metabolite is justified. We propose two complementary approaches to support conclusions to this effect: 1) demonstrate that the impurity is formed by metabolism in animals and/or man, based preferably on plasma exposures or, alternatively, amounts excreted in urine, and, where appropriate, 2) show that animal exposure to (or amount of) the impurity/metabolite is equal or greater in animals than in humans. An important factor of both assessments is the maximum theoretical concentration (or amount) (MTC or MTA) of the impurity/metabolite achievable from the administered dose and recommendations on the estimation of the MTC and MTA are presented.

ICH Q3D based elemental impurities study in liquid pharmaceutical dosage form with high daily intake - comparative analysis by ICP-OES and ICP-MS.

Guideline for Elemental Impurities-Q3D of the International Conference on Harmonisation represents a new paradigm in the control of elemental impurities (EIs) in pharmaceuticals. It changes the approach toward control of EIs from the historical 'heavy metals test', to a scientific-based risk assessment and testing by modern analytical instrumentation such as inductively coupled plasma-optical emission spectroscopy (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS). Management of EIs related to all finished drug products must be implemented in strict compliance with the regulatory requirements of pharmaceutical industry due to their quality and safety concerns. Testing for presence of EIs from Class 1 and Class 2a in methadone hydrochloride 1 mg/ml oral solution with recommended daily intake of 150 mg methadone hydrochloride was initially performed on ICP-OES using in-house validated method according to the requirements of pharmacopoeias, in line with Q3D. During the procedure, it became apparent that ICP-OES has its own limitations, especially when it comes to testing arsenic and lead in low concentrations. ICP-MS in-house validated method was developed and employed for determination of trace concentrations of arsenic and lead, providing resourceful information that were compared and correlated to the data obtained by ICP-OES analysis. Sample preparation using microwave digestion technique was applied for the analyses by both techniques. Although the applied ICP-OES in-house method is suitable for determination of Hg, Cd, Co, V, and Ni, more sensitive technique such as ICP-MS is required for accurate determination of As and Pb concerning pharmaceuticals with high daily intakes.

Demand for larger Y-STR reference databases in ethnic melting-pot countries: Argentina as a test case.

The Y chromosome behaves as a single locus. Its genetic information is useful in forensic casework, deficiency kinship testing, and population genetics studies. Continuous increases of loci number within commercial kits forced modification of worldwide reference databases. In Pan American countries, like Argentina, diverse parental ethnic groups contributed to the extant admixed urban populations. We report 509 additional haplotypes of 23 Y-STRs from donors inhabiting urban areas of six Argentinean provinces: Buenos Aires, Santiago del Estero, Santa Cruz, Rio Negro, Santa Fe, and Formosa. To better understand the demographic landscape of the admixed urban paternal lineages, structural analysis was performed using published data from other Argentinean provinces. AMOVA by Rst distance and inferred haplogroups by two predictive online software methods based on haplotypes yielded complementary results with respect to detected population structure, probably due to the different proportions of the Native American Q3-M3 haplogroup in the studied samples. This situation, which is common to most North, Meso, and South American countries, underscores the need for the additional step of typing specific SNPs for haplogroup diagnosis. We propose organizing Y-STR haplotype reference databases according to the most frequent haplogroups detected in a given admixed population.

Flavonoid metabolism: the interaction of metabolites and gut microbiota.

Several dietary flavonoids exhibit anti-oxidative, anti-inflammatory, and anti-osteoporotic activities relevant to prevention of chronic diseases, including lifestyle-related diseases. Dietary flavonoids (glycoside forms) are enzymatically hydrolyzed and absorbed in the intestine, and are conjugated to their glucuronide/sulfate forms by phase II enzymes in epithelial cells and the liver. The intestinal microbiota plays an important role in the metabolism of flavonoids found in foods. Some specific products of bacterial transformation, such as ring-fission products and reduced metabolites, exhibit enhanced properties. Studies on the metabolism of flavonoids by the intestinal microbiota are crucial for understanding the role of these compounds and their impact on our health. This review focused on the metabolic pathways, bioavailability, and physiological role of flavonoids, especially metabolites of quercetin and isoflavone produced by the intestinal microbiota.

Q3: A Compact Device for Quick, High Precision qPCR.

An accurate and easy-to-use Q3 system for on-chip quantitative real-time Polymerase Chain Reaction (qPCR) is hereby demonstrated, and described in detail. The qPCR reactions take place inside a single-use Lab-on-a-Chip with multiple wells, each with 5 to 15 µL capacity. The same chip hosts a printed metal heater coupled with a calibrated sensor, for rapid and accurate temperature control inside the reaction mixture. The rest of the system is non-disposable and encased in a 7 × 14 × 8.5 (height) cm plastic shell weighing 300 g. Included in the non-disposable part is a fluorescence read-out system featuring up to four channels and a self-contained control and data storage system, interfacing with an external user-friendly software suite. Hereby, we illustrate the engineering details of the Q3 system and benchmark it with seamlessly ported testing protocols, showing that Q3 equals the performance of standard commercial systems. Overall, to the best of our knowledge, this is one of the most mature general-purpose systems for on-chip qPCR currently available.

Pharmacophore modeling, 3D-QSAR, and in silico ADME prediction of N-pyridyl and pyrimidine benzamides as potent antiepileptic agents.

Biological mechanism attributing mutations in KCNQ2/Q3 results in benign familial neonatal epilepsy (BFNE), a rare form of epilepsy and thus neglected. It offers a potential target for antiepileptic drug discovery. In the present work, a pharmacophore-based 3D-QSAR model was generated for a series of N-pyridyl and pyrimidine benzamides possessing KCNQ2/Q3 opening activity. The pharmacophore model generated contains one hydrogen bond donor (D), one hydrophobic (H), and two aromatic rings (R). They are the crucial molecular write-up detailing predicted binding efficacy of high affinity and low affinity ligands for KCNQ2/Q3 opening activity. Furthermore, it has been validated by using a biological correlation between pharmacophore hypothesis-based 3D-QSAR variables and functional fingerprints of openers responsible for the receptor binding and also by docking of these benzamides into the validated homology model. Excellent statistical computational tools of QSAR model such as good correlation coefficient (R2 > 0.80), higher F value (F > 39), and excellent predictive power (Q2 > 0.7) with low standard deviation (SD <0.3) strongly suggest that the developed model could be used for prediction of antiepileptic activity of newer analogs. A preliminary pharmacokinetic profile of these derivatives was also performed on the basis of QikProp predictions.

Inducing optimal substitution between antibiotics under open access to the resource of antibiotic susceptibility.

This paper designs a bio-economic model to examine the use of substitute antibiotic drugs (analogs) sold by an industry that has open access to the resource of the antibiotic class's susceptibility (treatment effectiveness). Antibiotics are characterized by different expected recovery rates and production costs, which in conjunction with the class's treatment susceptibility determines their relative effectiveness. Our analysis reveals that the high-quality antibiotic drug loses its comparative advantage over time making the low-quality drug the treatment of last resort in the market equilibrium and the social optimum when antibiotic susceptibility cannot replenish. However, when antibiotic susceptibility is renewable, both antibiotics may be used in the long run, and the comparative advantage of the high-quality drug may be restored in the social optimum that allows lowering infection in the long run. We develop the optimal tax/subsidy scheme that would induce antibiotic producers under open access to behave optimally and account for the social cost of infection and value of antibiotic susceptibility. We show that the welfare loss associated with the uncorrected open-access allocation is highest; when the resource of antibiotic susceptibility is non-renewable, high morbidity costs are incurred by individuals, and low social discount rates apply. Copyright © 2016 John Wiley & Sons, Ltd.

Management of organic impurities in small molecule medicinal products: Deriving safe limits for use in early development.

Management of organic non-mutagenic impurities (NMIs) in medicinal products is regulated by the ICH Q3A, B and C guidelines that are applicable at late stages of clinical development (Phase III onwards) and as a consequence there is no guidance for the assessment and control of NMIs in early clinical trials. An analysis of several key in vivo toxicology databases supports the ICH Q3A defined concept that a lifetime dose to 1 mg/day of a NMI would not represent a safety concern to patients. In conjunction with routine (Q)SAR approaches, this 1 mg/day value could be used as a universal qualification threshold for a NMI during any stage of clinical development. This analysis also proposes that modification of this 1 mg/day dose using an established methodology (i.e. Modified Haber's Law) could support 5 mg/day or 0.7% (whichever is lower) as an acceptable limit for a NMI in a drug substance or product in early clinical studies (<6 months). Given the controlled nature of clinical development and the knowledge that most toxicities are dose and duration dependent, these proposed NMI limits provide assurance of patient safety throughout clinical development, without the requirement to commission dedicated in vivo toxicology impurity qualification studies.

QSAR and docking studies of anthraquinone derivatives by similarity cluster prediction.

Forty anthraquinone derivatives have been downloaded from PubChem database and investigated in a quantitative structure-activity relationships (QSAR) study. The models describing log P and LD50 of this set were built up on the hypermolecule scheme that mimics the investigated receptor space; the models were validated by the leave-one-out procedure, in the external test set and in a new version of prediction by using similarity clusters. Molecular docking approach using Lamarckian Genetic Algorithm was made on this class of anthraquinones with respect to 3Q3B receptor. The best scored molecules in the docking assay were used as leaders in the similarity clustering procedure. It is demonstrated that the LD50 data of this set of anthraquinones are related to the binding energies of anthraquinone ligands to the 3Q3B receptor.

Involvement of the lesser sigmoid notch in elbow fracture dislocations.

BACKGROUND: This study addressed the primary null hypothesis that there is no difference in the articular surface area of the lesser sigmoid notch involved among Mayo classes. Secondarily, we analyzed the fracture line location and the pattern of lesser sigmoid notch articular surface involvement among Mayo classes. METHODS: Using quantitative 3-dimensional computed tomography, we reconstructed and analyzed fractures involving the lesser sigmoid notch articular surface in 52 patients. Further, we assessed the surface area involved in the fracture, the number of fracture fragments, and the location and direction of the fracture lines. Coronoid fractures were classified according to Mayo types. RESULTS: There was no significant difference between Mayo types 1 and 2 in any characteristic of the involvement of the lesser sigmoid notch articular surface, whereas Mayo type 3 was significantly different from both Mayo types 1 and 2 in the area involved in the fracture (42% in Mayo type 3 vs. 9% in Mayo types 1 and 2), the number of articular fragments (>3 fragments in type 3 vs. 2 fragments in types 1 and 2), and the direction of fracture line (both horizontal and vertical lines in type 3 vs. only horizontal line in types 1 and 2). CONCLUSION: Mayo type III results in a more complex fracture, which might need to be addressed directly or indirectly during open reduction with internal fixation of olecranon fracture dislocations because changes in the geometry of lesser sigmoid notch may affect the radioulnar joint if it remains incongruent.

Comparison of the Antioxidant Effects of Quercitrin and Isoquercitrin: Understanding the Role of the 6″-OH Group.

The role of the 6″-OH (ω-OH) group in the antioxidant activity of flavonoid glycosides has been largely overlooked. Herein, we selected quercitrin (quercetin-3-O-rhamnoside) and isoquercitrin (quercetin-3-O-glucoside) as model compounds to investigate the role of the 6″-OH group in several antioxidant pathways, including Fe2+-binding, hydrogen-donating (H-donating), and electron-transfer (ET). The results revealed that quercitrin and isoquercitrin both exhibited dose-dependent antioxidant activities. However, isoquercitrin showed higher levels of activity than quercitrin in the Fe2+-binding, ET-based ferric ion reducing antioxidant power, and multi-pathways-based superoxide anion-scavenging assays. In contrast, quercitrin exhibited greater activity than isoquercitrin in an H-donating-based 1,1-diphenyl-2-picrylhydrazyl radical-scavenging assay. Finally, in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl assay based on an oxidatively damaged mesenchymal stem cell (MSC) model, isoquercitrin performed more effectively as a cytoprotector than quercitrin. Based on these results, we concluded that (1) quercitrin and isoquercitrin can both indirectly (i.e., Fe2+-chelating or Fe2+-binding) and directly participate in the scavenging of reactive oxygen species (ROS) to protect MSCs against ROS-induced oxidative damage; (2) the 6″-OH group in isoquercitrin enhanced its ET and Fe2+-chelating abilities and lowered its H-donating abilities via steric hindrance or H-bonding compared with quercitrin; and (3) isoquercitrin exhibited higher ROS scavenging activity than quercitrin, allowing it to improve protect MSCs against ROS-induced oxidative damage.