Isolation and Quantification of Palmatine from Fibraurea tinctoria Lour: In Vitro Antioxidant Activity and In Silico Antidiabetic Activity Evaluation.

Purpose: This study aimed to isolate and characterize palmatine from Fibraurea tinctoria Lour stems, quantify its content, and determine its antioxidant and antidiabetic activities. Patients and Methods: Palmatine was isolated from the methanol extract of Fibraurea tinctoria Lour stems by silica gel column chromatography. Structural elucidation of the isolated compounds was performed using spectral data analysis and comparison with the literature. High-Performance Liquid Chromatography (HPLC) was used to quantitatively determine palmatine in the crude methanol extract and fractions. The DPPH and non-enzymatic SOD mimic methods were used to assess the antioxidant activity of the methanol extract, fractions, and isolated compounds. The antidiabetic activity was evaluated in silico by the molecular docking method of alpha-glucosidase and DPP-IV enzymes. Palmatine was used as a test ligand and was compared with berberine and its native ligand or standard compounds. Results: The isolated compound was identified as palmatine. Quantification of palmatine compound by HPLC showed that palmatine was found in the extract and all fractions. In the in vitro antioxidant activity test using the DPPH method, fraction 4 showed the highest activity, with an IC50 value of 91 ppm. In contrast, using the non-enzymatic SOD mimic method, the methanol extract, fraction 5, and isolated compound (palmatine) exhibited very strong antioxidant activity, with IC50 values of 18, 20, and 28 ppm, respectively. The in silico antidiabetic activity of palmatine is thought to have the potential to inhibit these two enzymes. Conclusion: These results showed that Fibraurea tinctoria Lour stems have potential as an antioxidant and antidiabetic agent. Further research on phytochemical and pharmacological is required to validate the use of this plant species for the treatment of various diseases, especially diabetes mellitus.

Exploring allocryptopine as a neuroprotective agent against oxidative stress-induced neural apoptosis via Akt/GSK-3ß/tau pathway modulation.

Alzheimer's disease (AD) is characterized by neuronal loss due to hyperphosphorylated proteins induced by oxidative stress. AD remains a formidable challenge in the medical field, as current treatments focusing on single biomarkers have yielded limited success. Hence, there's a burgeoning interest in investigating novel compounds that can target mechanisms, offering alternative therapeutic approaches. The aim of this study is to investigate the effects of allocryptopine, an isoquinoline alkaloid, on mechanisms related to AD in order to develop alternative treatment strategies. In this study, the in vitro AD cell model was obtained by inducing nerve growth factor (NGF)-differentiated PC12 (dPC12) cells to oxidative stress with H2O2, and also the effect mechanism of different allocryptopine concentrations on the in vitro AD cell model was studied. The treatments' antioxidative effects at the ROS level and their regulation of the cell cycle were assessed through flow cytometry, while their anti-apoptotic effects were evaluated using both flow cytometry and qRT-PCR. Additionally, the phosphorylation levels of Akt, GSK-3ß, and tau proteins were analyzed via western blot, and the interactions between Akt, GSK-3ß, CDK5 proteins, and allocryptopine were demonstrated through molecular docking. Our study's conclusive results revealed that allocryptopine effectively suppressed intracellular ROS levels, while simultaneously enhancing the Akt/GSK-3ß signaling pathway by increasing p-Akt and p-GSK-3ß proteins. This mechanism played a critical role in inhibiting neural cell apoptosis and preventing tau hyperphosphorylation. Moreover, allocryptopine demonstrated its ability to regulate the G1/S cell cycle progression, leading to cell cycle arrest in the G1 phase, and facilitating cellular repair mechanisms, potentially contributing to the suppression of neural apoptosis. The in silico results of allocryptopine were shown to docking with the cyclin-dependent kinase 5 (CDK 5) playing a role in tau phosphorylation Akt and GSK-3ß from target proteins. Therefore, the in silico study results supported the in vitro results. The results showed that allocryptopine can protect dPC12 cells from oxidative stress-induced apoptosis and hyperphosphorylation of the tau protein by regulating the Akt/GSK-3ß signaling pathway. Based on these findings, it can be suggested that allocryptopine, with its ability to target biomarkers and its significant effects on AD-associated mechanisms, holds promise as a potential candidate for drug development in the treatment of AD. Further research and clinical trials are recommended in the future.

Discovery of natural AMPK activator from the fruits of Xanthium sibiricum Patr.: Xanthiumine A, protoberberine alkaloid with unique C28 skeleton.

Two protoberberine alkaloids with a unique C28 skeleton, named xanthiumines A (1) and B (2), respectively, were isolated from the fruits of Xanthium sibiricum Patr. Their structures including absolute configurations were unequivocally established by the comprehensive NMR and MS spectroscopic data analysis together with gauge-independent atomic orbital (GIAO) NMR calculations, and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are the first examples of natural protoberberine alkaloid with a phenolic acid group at C-13a. Their plausible biosynthetic pathway was proposed on the basis of the coexisting alkaloid monomer as the precursor. Furthermore, the effects and related molecular mechanism of compound 1 on hepatic lipid accumulation were also investigated in oleic acid (OA)-treated HepG2 cells.

Protopine and Allocryptopine Interactions with Plasma Proteins.

A comprehensive study of the interactions of human serum albumin (HSA) and α-1-acid glycoprotein (AAG) with two isoquinoline alkaloids, i.e., allocryptopine (ACP) and protopine (PP), was performed. The UV-Vis spectroscopy, molecular docking, competitive binding assays, and circular dichroism (CD) spectroscopy were used for the investigations. The results showed that ACP and PP form spontaneous and stable complexes with HSA and AAG, with ACP displaying a stronger affinity towards both proteins. Molecular docking studies revealed the preferential binding of ACP and PP to specific sites within HSA, with site 2 (IIIA) being identified as the favored location for both alkaloids. This was supported by competitive binding assays using markers specific to HSA's drug binding sites. Similarly, for AAG, a decrease in fluorescence intensity upon addition of the alkaloids to AAG/quinaldine red (QR) complexes indicated the replacement of the marker by the alkaloids, with ACP showing a greater extent of replacement than PP. CD spectroscopy showed that the proteins' structures remained largely unchanged, suggesting that the formation of complexes did not significantly perturb the overall spatial configuration of these macromolecules. These findings are crucial for advancing the knowledge on the natural product-protein interactions and the future design of isoquinoline alkaloid-based therapeutics.

Discovery of palmatine derivatives as potent neuroprotective agents.

Alzheimer's disease is a neurodegenerative disorder characterized by the presence of neurodegenerative lesions and cognitive impairment. In this study, a series of novel palmatine derivatives were designed and synthesized through the introduction of a heteroatom using carbodiimide-mediated condensation. The synthesized compounds were then screened for toxicity and potency, leading to the identification of compound 2q, which exhibited low toxicity and high potency. Our findings demonstrated that compound 2q displayed significant neuroprotective activity in vitro, emerging as a promising candidate for Alzheimer's disease treatment.

Insights into the interaction and inhibitory action of palmatine on lysozyme fibrillogenesis: Spectroscopic and computational studies.

Interaction under amyloidogenic condition between naturally occurring protoberberine alkaloid palmatine and hen egg white lysozyme was executed by adopting spectrofluorometric and theoretical molecular docking and dynamic simulation analysis. In spetrofluorometric method, different types of experiments were performed to explore the overall mode and mechanism of interaction. Intrinsic fluorescence quenching of lysozyme (Trp residues) by palmatine showed effective binding interaction and also yielded different binding parameters like binding constant, quenching constant and number of binding sites. Synchronous fluorescence quenching and 3D fluorescence map revealed that palmatine was able to change the microenvironment of the interacting site. Fluorescence life time measurements strongly suggested that this interaction was basically static in nature. Molecular docking result matched with fluorimetric experimental data. Efficient drug like interaction of palmatine with lysozyme at low pH and high salt concentration prompted us to analyze its antifibrillation potential. Different assays and microscopic techniques were employed for detailed analysis of lysozyme amyloidosis.Thioflavin T(ThT) assay, Congo Red (CR) assay, 8-anilino-1-naphthalenesulfonic acid (ANS) assay, Nile Red (NR) assay, anisotropy and intrinsic fluorescence measurements confirmed that palmatine successfully retarded and reduced lysozyme fibrillation. Dynamic light scattering (DLS) and atomic force microscopy (AFM) further reiterated the excellent antiamyloidogenic potency of palmatine.

Compound-compound interaction analysis of baicalin and berberine derivatives in aqueous solution.

Baicalin and berberine are biologically active constituents of the crude drugs Scutellaria root and Coptis rhizome/Phellodendron bark, respectively. Baicalin and berberine are reported to combine together as a 1:1 complex that forms yellow precipitates by electrostatic interaction in decoctions of Kampo formulae containing these crude drugs. However, the structural basis and mechanism for the precipitate formation of this compound-compound interaction in aqueous solution remains unclarified. Herein, we searched for berberine derivatives in the Coptis rhizome that interact with baicalin and identified the chemical structures involved in the precipitation formation. Precipitation assays showed that baicalin formed precipitates with berberine and coptisine but not with palmatine and epiberberine. Thus, the 2,3-methylenedioxy structure may be crucial to the formation of the precipitates, and electrostatic interaction is necessary but is not sufficient. In this multicomponent system experiment, palmatine formed a dissociable complex with baicalin and may competitively inhibit the formation of berberine and coptisine precipitation with baicalin. Therefore, the precipitation formed by berberine and baicalin was considered to be caused by the aggregation of the berberine-baicalin complex, and the 2,3-methylenedioxy structure is likely crucial to the aggregation of the complex.

Studies on crystal structures, optical, dyeing and biological properties of protoberberine alkaloids and their supramolecular salts.

The novel Palmatine (PLT)-based supramolecular salt palmatine-sulfosalicylic acid (PLT-SSA) was designed and synthesized, and its structures was determined by the single crystal X-ray diffraction. It is found that PLT-SSA exhibited enhancing thermodynamic stability, fluorescence intensity and emission lifetime in crystal state, which indicated that these structures and aromatic rings may give more overlap between the host-guest units and give rise to a long-lived charge-separated state. In addition, the dyeing properties and toxicity of these protoberberine alkaloid (BBC and PLTCl) and their supramolecular salts will be developed in this work used as yellow dyes for development multifunctional fabrics.

Discovery and development of palmatine analogues as anti-NASH agents by activating farnesoid X receptor (FXR).

Sixty-one palmatine (PMT) derivatives, of which twenty-eight were new, were synthesized and evaluated for their anti-fibrogenic activities via collagen type I α 1 (COL1A1)-promoter based luciferase model in LX-2 cells, taking 2,3,10-trimethoxy-9-p-isopropyloxyprotopalmatine bromide (1) as the lead. Among them, compound 3a exerted the highest potency with the IC50 value of 8.19 µmol/L and SI value of 8.59, and reduced the expressions of multiple fibrogenic biomarkers, including COL1A1, TGF-ß1, α-SMA and TIMP1 in a dose-dependent manner. In addition, it significantly reduced liver steatosis and inflammation, and especially attenuated the degree of liver fibrosis in choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD)-induced NASH mice model in vivo. Mechanism study indicated that it significantly ameliorated liver injury by activating farnesoid X receptor (FXR). BDL-induced fibrosis rats model further verified its liver-protective and anti-fibrosis activities. Therefore, PMT derivatives constituted a new family of non-steroidal FXR agonists as anti-NASH candidates, with the advantage of good safety profile, and are worthy for further investigation.

Entropy-Driven Inclusion of Natural Protoberberine Alkaloids in Sulfobutylether-ß-Cyclodextrin.

The understanding of the relationship between molecular structure and the thermodynamics of host-guest binding is essential for the rational design of the applications of inclusion complexes. To obtain insight into the factors governing the driving force of complex formation in aqueous solutions, the encapsulation of five pharmaceutically important protoberberine alkaloids was studied in sulfobutylether-ß-cyclodextrin having on average 6.4 degrees of substitution (SBE6.4ßCD). Spectrophotometric, fluorescence spectroscopic, and isothermal calorimetric measurements showed 1:1 complexation in dilute solutions. From 1.92 × 104 M−1, about an eight-fold decrease of the association constant was observed in the series of berberine ≈ coptisine >> palmatine > epiberberine > dehydrocorydaline. The embedment of these alkaloids in the SBE6.4ßCD cavity was entropy-controlled with mildly negative enthalpy contributions. These findings suggest that the stabilization of the examined complexes arises primarily from the hydrophobic interaction between the constituents. The more than three orders of magnitude smaller association constants of protoberberine alkaloids with SBE6.4ßCD than with cucurbit[7]uril, a host having similar cavity size, originates from the much smaller exothermicity of the confinement in the former macrocycle.

Chemometrics-enhanced high-performance liquid chromatography-diode array detection strategy to quantify protoberberine alkaloids in varying Coptidis Rhizoma-related medicines.

Quantitation of protoberberine alkaloids is an essential guarantee for efficacy control and medication safety of Coptidis Rhizoma (CR) related medicines. Traditional univariate chromatography faced challenges with co-elution, unknown interferences, and retention time shift when analyzing isomeric analytes in varying sample matrices. We presented a chemometrics-enhanced high-performance liquid chromatography-diode array detection (HPLC-DAD) strategy for simultaneous quantification of six protoberberine alkaloids and processed multi-channels chromatographic-spectral data with four second-order calibration algorithms. Chromatographic conditions were firstly optimized. Four groups of predicted samples were modeled individually with the designed calibration set. Mathematical resolutions were then obtained, and pseudo-univariate regression gave the quantitative concentration of each analyte. Four models were scored on fit, linearity, recovery, and robustness, where alternating trilinear decomposition assisted multivariate curve resolution (ATLD-MCR) exhibited an optimal and stable performance. Besides, the resolved spectra presented high consistency with the actual spectra (r≥0.9993). Limits of quantification (LOQ) fully met the pharmacopoeia stipulation and were 0.17, 0.60, 0.19, 0.74, 0.15, and 0.38 µg mL-1 for columbamine, epiberberine, jatrorrhizine, coptisine, palmatine, and berberine, respectively. The importance of this strategy is to exploit collinearity resolution and additional selectivity that permit accurate quantitation at poor chromatographic resolutions, avoiding individual pretreatment and HPLC optimizations for different samples. This study provides a universal alternative for routine quality assessment of protoberberine alkaloids in CR-related medicines.

Immobilization of peroxisome proliferator-activated receptor gamma and the application in screening modulators of the receptor from herbal medicine.

Screening and identification of potential compounds from herbal medicine is a prevailing way to find a lead for the development of innovative drugs. This promotes the development of new methods that are feasible in complex matrices. Here, we described a one-step reversible methodology to immobilize nuclear peroxisome proliferator-activated receptor gamma (PPARγ) onto amino microsphere coated with a DNA strand specifically binding to the receptor. The specific interaction allowed us to achieve the immobilization of PPARγ by mixing the DNA modified microspheres with E. coli lysates expressing the receptor. Characterization of the immobilized receptor was carried out by morphology and binding specificity analysis. Feasibility of immobilized PPARγ in the drug-receptor interaction analysis was performed by an injection amount-dependent method. Besides, immobilized PPARγ was also applied in screening modulators of the receptor from Coptidis Rhizoma extract. The binding of the screened compounds to PPARγ was examined by time-resolved fluorescence resonance energy transfer assay. The results showed that immobilized PPARγ was stable for thirty days with a high-specificity of ligand recognition at the subtype receptor level. Berberine and palmatine were the bioactive compounds of Coptidis Rhizoma specifically binding to PPARγ. The two compounds exhibited half maximal inhibitory concentrations of 4.11 and 2.98 µM during their binding to the receptor. We concluded that the current method is possible to become a common strategy for the immobilization of nuclear receptors, and the immobilized receptor is a high throughput method for recognizing and separating the receptor modulators from complex matrices including herbal medicine.

Spatial Distribution and Stability of Cholinesterase Inhibitory Protoberberine Alkaloids from Papaver setiferum.

During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehydroprotoberberine alkaloids (1 and 2) and nine known compounds (3-11) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range (1-4, 6, 7), offering new lead compounds for the development of cholinesterase inhibitory drugs.

Study of the interaction of the palmatine alkaloid with hybrid G-quadruplex/duplex and i-motif/duplex DNA structures.

There is an increasing interest in the study of guanine or cytosine-rich sequences that may fold into G-quadruplex (G4) or i-motif (iM) structures showing a short hairpin (or stem-loop) stabilized by Watson-Crick base pairs. These hybrid spatial arrangements may be target of ligands that have been shown to interact strongly with B-DNA. In this work, the interaction of the palmatine alkaloid with several sequences forming different G4s, iMs, and hybrid structures has been studied by means of spectroscopic and separation techniques, as well as multivariate data analysis methods. At the experimental conditions used in this work, the results have shown that this ligand strongly stabilizes parallel G4 structures, whereas a weaker interaction was observed with the antiparallel G4 adopted by the thrombin-binding aptamer or iMs. The presence of hairpins within the loops scarcely affects the affinity of this ligand for the hybrid G4/duplex or iM/duplex structures. Fluorescence measurements have provided evidence of a certain interaction with iMs at pH 5.1, despite the absence of thermal stabilization effects.

In situ label-free and sensitive detection assay for cell apoptosis via polyadenosine-coralyne fluorescence enhancement strategy.

Cell apoptosis detection is vital for biological analysis and clinical application; some detection assays are already commercially available. However, it is still far from perfect and needs further improvement for less cost, time-consuming and operation demanding. TUNEL, a high market share cell apoptosis assay, depends on adulteration fluorescent labelling dUTP by terminal deoxynucleotidyl transferase(TdT) which randomly adds deoxyribonucleoside triphosphates (dNTPs) at the 3'-OH terminal of ssDNA with a template-free manner. Based on our previous work, we adopted a label-free strategy to reduce the cost and operation maintenance of TUNEL and developed a facile, rapid, convenient and in-situ assay for cell apoptosis.

Chemoenzymatic Cascades toward Methylated Tetrahydroprotoberberine and Protoberberine Alkaloids.

Tetrahydroprotoberberine and protoberberine alkaloids are a group of biologically active natural products with complex molecular scaffolds. Isolation from plants is challenging and stereoselective synthetic routes, particularly of methylated compounds are limited, reducing the potential use of these compounds. In this work, we describe chemoenzymatic cascades toward various 13-methyl-tetrahydroprotoberberbine scaffolds using a stereoselective Pictet-Spenglerase, regioselective catechol O-methyltransferases and selective chemical Pictet-Spengler reactions. All reactions could be performed sequentially, without the workup or purification of any synthetic intermediates. Moreover, the naturally occurring alkaloids have the (+)-configuration and importantly here, a strategy to the (-)-isomers was developed. A methyl group at C-8 was also introduced with some stereocontrol, influenced by the stereochemistry at C-13. Furthermore, a single step reaction was found to convert tetrahydroprotoberberine alkaloids into the analogous protoberberine scaffold, avoiding the use of harsh oxidizing conditions or a selective oxidase. This work provides facile, selective routes toward novel analogues of bioactive alkaloids.

A new strategy for quality evaluation and control of Chinese patent medicine based on chiral isomer ratio analysis: With Yuanhuzhitong tablet as an example.

Chiral compounds commonly exist in traditional Chinese medicine (TCM), but little research on the quality control of TCM has been conducted. In this study, a new strategy is proposed, taking Yuanhuzhitong tablet [YHZT, consisting of Radix Angelicae Dahuricae and Rhizoma Corydalis (Yan Hu Suo, YHS)] for example, which is based on chiral isomer ratio analysis to monitor the production process of Chinese patent medicine companies. In the process of content determination for tetrahydropalmatine (THP) in YHZT from different companies, noticeable differences were observed in their chromatographic behaviors. It is known that THP has two enantiomers, naturally coexisting in YHS as a racemic mixture, so we prepared THP twice and subsequently performed chiral separation analysis using supercritical fluid chromatography. As a result, the peak area ratios of two enantiomers from different companies varied remarkably, demonstrating that some companies did not probably manufacture YHZT products in accordance with the prescription proportion, used inferior or extracted YSH crude materials in the production process, and added raw chemical medicine in the production to reach the standard and lower the costs. In conclusion, the peak area ratio of chiral isomers could be taken as a key quality index.

Screening of differential components of Gegenqinlian decoction and their comparative pharmacokinetics in normal and pyrexia rats using UHPLC-FT-ICR-MS and UHPLC-MS/MS.

UHPLC combined with Fourier-transform ion cyclotron resonance MS metabonomic approach was employed to screen the differential components between normal rats and yeast-induced pyrexia rats after an oral administration of Gegenqinlian decoction (GQLD). Nine compounds, namely puerarin, daidzein, baicalin, wogonoside, wogonin, berberine, palmatine, jateorhizine, and coptisine, were identified as differential components in the plasma. A rapid, sensitive, selective, and accurate UHPLC-MS method was developed and fully validated for the simultaneous determination of the screened components in rat plasma after an oral administration of GQLD. The values for the limit of quantification ranged from 0.025 to 5.0 ng/mL. The inter- and intra-day precision of all analytes was ≤10.7%, with an accuracy of ≤10.5%. Good extraction recovery and matrix effects were also obtained. The method was successfully applied to a comparative pharmacokinetic study of GQLD in normal and pyrexia rats. The results showed that the pharmacokinetic behavior of the analytes was changed in pyrexia rats compared to normal rats. These results could provide beneficial guidance for clinical applications of GQLD.

Design, synthesis, and biological evaluation of novel tetrahydroprotoberberine derivatives to reduce SREBPs expression for the treatment of hyperlipidemia.

Statins play an important role in the treatment of hyperlipidemia, but drug resistance and adverse effects greatly limits their application. To discover new lipid-lowering drugs, three different series of tetrahydroprotoberberine derivatives (THPBs) were designed and synthesized. These compounds were first tested for their effects on viability of HepG2 cells and 21 compounds with the percent of cell viability over 90% were further screened to evaluate their ability to reduce total cholesterol (TC) and triglyceride (TG) levels. Among these derivatives, two compounds displayed significant down-regulation both intracellular of TC and TG content, especially compound 49 exhibited the greatest efficacy. Mechanistically, compound 49 promoted proteasomal degradation of SREBPs. Importantly, compound 49 displayed superior bioavailability (F = 65.1%) and obvious efficacy in the treatment of high fat diet induced obesity in vivo. Therefore, compound 49 is a promising candidate to develop new treatment of hyperlipidemia.

Antimetastatic and Antitumor Activities of Orally Administered NAX014 Compound in a Murine Model of HER2-Positive Breast Cancer.

The natural isoquinoline alkaloid Berberine (BBR) has been shown to possess several therapeutic effects, including anticancer activity. Different BBR derivatives have been designed and synthesized in order to obtain new compounds with enhanced anticancer efficacy. We previously showed that intraperitoneal (IP) administration of the BBR-derived NAX014 compound was able to counteract HER-2 overexpressing mammary tumors onset and progression in transgenic mice. However, the IP administration was found to induce organ toxicity at doses higher than 2.5 mg/Kg. In this study, we evaluated the effect of intragastric (IG) administration of 20 mg/kg of NAX014 on both safety and anticancer efficacy in HER-2/neu transgenic mice. Furthermore, cancer cell dissemination and migration, tumor cell senescence and immunological changes were examined. Our results demonstrated that IG NAX014 administration delayed the onset of mammary tumors with no negative effects on health and survival. NAX014 reduced HER-2 overexpressing BC cells migration in vitro and the frequency of lung metastasis in HER-2/neu transgenic mice. A statistically significant increase of senescence-associated p16 expression was observed in tumors from NAX014-treated mice, and the induction of cell senescence was observed in HER-2 overexpressing BC cells after in vitro treatment with NAX014. Although NAX014 did not modulate the presence of tumor-infiltrating lymphocytes, the level of circulating TNF-α and VEGF was found to be reduced in NAX014-treated mice. The overall results address the NAX014 compound as potential tool for therapeutic strategies against HER-2 overexpressing breast cancer.