Knockdown of Hyaluronan synthase 2 suppresses liver fibrosis in mice via induction of transcriptomic changes similar to 4MU treatment.

Hepatic fibrosis remains a significant clinical challenge due to ineffective treatments. 4-methylumbelliferone (4MU), a hyaluronic acid (HA) synthesis inhibitor, has proven safe in phase one clinical trials. In this study, we aimed to ameliorate liver fibrosis by inhibiting HA synthesis. We compared two groups of mice with CCl4-induced fibrosis, treated with 4-methylumbelliferone (4MU) and hyaluronan synthase 2 (HAS2) targeting siRNA (siHAS2). The administration of 4MU and siHAS2 significantly reduced collagen and HA deposition, as well as biochemical markers of hepatic damage induced by repeated CCl4 injections. The transcriptomic analysis revealed converging pathways associated with downstream HA signalling. 4MU- and siHAS2-treated fibrotic livers shared 405 upregulated and 628 downregulated genes. These genes were associated with xenobiotic and cholesterol metabolism, mitosis, endoplasmic reticulum stress, RNA processing, and myeloid cell migration. The functional annotation of differentially expressed genes (DEGs) in siHAS2-treated mice revealed attenuation of extracellular matrix-associated pathways. In comparison, in the 4MU-treated group, DEGs were related to lipid and bile metabolism pathways and cell cycle. These findings confirm that HAS2 is an important pharmacological target for suppressing hepatic fibrosis using siRNA.

Novel Dihydrocoumarins Induced by Radiolysis as Potent Tyrosinase Inhibitors.

A representative naturally occurring coumarin, 4-methylumbelliferone (5), was exposed to 50 kGy of gamma ray, resulting in four newly generated dihydrocoumarin products 1-4 induced by the gamma irradiation. The structures of these new products were elucidated by interpretation of spectroscopic data (NMR, MS, [α]D, and UV). The unusual bisdihydrocoumarin 4 exhibited improved tyrosinase inhibitory capacity toward mushroom tyrosinase with IC50 values of 19.8 ± 0.5 µM as compared to the original 4-methylumbelliferone (5). A kinetic analysis also exhibited that the potent metabolite 4 had non-competitive modes of action. Linkage of the hydroxymethyl group in the C-3 and C-4 positions on the lactone ring probably enhances the tyrosinase inhibitory effect of 4-methylumbelliferone (5). Thus, the novel coumarin analog 4 is an interesting new class of tyrosinase inhibitory candidates that requires further examination.

Low oral dose of 4-methylumbelliferone reduces glial scar but is insufficient to induce functional recovery after spinal cord injury.

Spinal cord injury (SCI) induces the upregulation of chondroitin sulfate proteoglycans (CSPGs) at the glial scar and inhibits neuroregeneration. Under normal physiological condition, CSPGs interact with hyaluronan (HA) and other extracellular matrix on the neuronal surface forming a macromolecular structure called perineuronal nets (PNNs) which regulate neuroplasticity. 4-methylumbelliferone (4-MU) is a known inhibitor for HA synthesis but has not been tested in SCI. We first tested the effect of 4-MU in HA reduction in uninjured rats. After 8 weeks of 4-MU administration at a dose of 1.2 g/kg/day, we have not only observed a reduction of HA in the uninjured spinal cords but also a down-regulation of CS glycosaminoglycans (CS-GAGs). In order to assess the effect of 4-MU in chronic SCI, six weeks after Th8 spinal contusion injury, rats were fed with 4-MU or placebo for 8 weeks in combination with daily treadmill rehabilitation for 16 weeks to promote neuroplasticity. 4-MU treatment reduced the HA synthesis by astrocytes around the lesion site and increased sprouting of 5-hydroxytryptamine fibres into ventral horns. However, the current dose was not sufficient to suppress CS-GAG up-regulation induced by SCI. Further adjustment on the dosage will be required to benefit functional recovery after SCI.

Inhibition of hyaluronan synthesis prevents ß-cell loss in obesity-associated type 2 diabetes.

Pancreatic ß-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low concentrations of HA were present in healthy pancreatic islets. However, HA substantially accumulated in cadaveric islets of T2D patients and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the main HA receptor CD44, preserved glycemic control and insulin concentrations in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserved glycemic control in other settings of ß-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we found that 4-MU increased the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on ß-cells such that caspase 3 activation did not result in ß-cell apoptosis. These data indicated a role for HA accumulation in diabetes pathogenesis and suggested that it may be a viable target to ameliorate ß-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

4-Methylumbelliferone enhances the effects of chemotherapy on both temozolomide-sensitive and resistant glioblastoma cells.

Glioblastoma (GBM) is the most frequent malignant primary tumor of the CNS in adults, with a median survival of 14.6 months after diagnosis. The effectiveness of GBM therapies remains poor, highlighting the need for new therapeutic alternatives. In this work, we evaluated the effect of 4-methylumbelliferone (4MU), a coumarin derivative without adverse effects reported, in combination with temozolomide (TMZ) or vincristine (VCR) on U251, LN229, U251-TMZ resistant (U251-R) and LN229-TMZ resistant (LN229-R) human GBM cells. We determined cell proliferation by BrdU incorporation, migration through wound healing assay, metabolic and MMP activity by XTT and zymography assays, respectively, and cell death by PI staining and flow cytometry. 4MU sensitizes GBM cell lines to the effect of TMZ and VCR and inhibits metabolic activity and cell proliferation on U251-R cells. Interestingly, the lowest doses of TMZ enhance U251-R and LN229-R cell proliferation, while 4MU reverts this and even sensitizes both cell lines to TMZ and VCR effects. We showed a marked antitumor effect of 4MU on GBM cells alone and in combination with chemotherapy and proved, for the first time, the effect of 4MU on TMZ-resistant models, demonstrating that 4MU would be a potential therapeutic alternative for improving GBM therapy even on TMZ-refractory patients.

4-Methylumbelliferone-Functionalized Polyphosphazene and Its Assembly into Biocompatible Fluorinated Nanocoatings with Selective Antiproliferative Activity.

Advanced multifunctional biomaterials are increasingly relying on clinically dictated patterns of selectivity against various biological targets. Integration of these frequently conflicting features into a single material surface may be best achieved by combining various complementary methodologies. Herein, a drug with a broad spectrum of activity, i.e., 4-methylumbelliferone (4-MU), is synthetically multimerized into water-soluble anionic macromolecules with the polyphosphazene backbone. The polymer structure, composition, and solution behavior are studied by 1H and 31P NMR spectroscopy, size-exclusion chromatography, dynamic light scattering, and UV and fluorescence spectrophotometry. To take advantage of the clinically proven hemocompatibility of fluorophosphazene surfaces, the drug-bearing macromolecule was then nanoassembled onto the surface of selected substrates in an aqueous solution with fluorinated polyphosphazene of the opposite charge using the layer-by-layer (LbL) technique. Nanostructured 4-MU-functionalized fluoro-coatings exhibited a strong antiproliferative effect on vascular smooth muscle cells (VSMCs) and fibroblasts with no cytotoxicity against endothelial cells. This selectivity pattern potentially provides the opportunity for highly desirable fast tissue healing while preventing the overgrowth of VSMCs and fibrosis. Taken together with the established in vitro hemocompatibility and anticoagulant activity, 4-MU-functionalized fluoro-coatings demonstrate potential for applications as restenosis-resistant coronary stents and artificial joints.

4-Methylumbeliferone Treatment at a Dose of 1.2 g/kg/Day Is Safe for Long-Term Usage in Rats.

4-methylumbelliferone (4MU) has been suggested as a potential therapeutic agent for a wide range of neurological diseases. The current study aimed to evaluate the physiological changes and potential side effects after 10 weeks of 4MU treatment at a dose of 1.2 g/kg/day in healthy rats, and after 2 months of a wash-out period. Our findings revealed downregulation of hyaluronan (HA) and chondroitin sulphate proteoglycans throughout the body, significantly increased bile acids in blood samples in weeks 4 and 7 of the 4MU treatment, as well as increased blood sugars and proteins a few weeks after 4MU administration, and significantly increased interleukins IL10, IL12p70 and IFN gamma after 10 weeks of 4MU treatment. These effects, however, were reversed and no significant difference was observed between control treated and 4MU-treated animals after a 9-week wash-out period.

4-Methylumbelliferone Targets Revealed by Public Data Analysis and Liver Transcriptome Sequencing.

4-methylumbelliferone (4MU) is a well-known hyaluronic acid synthesis inhibitor and an approved drug for the treatment of cholestasis. In animal models, 4MU decreases inflammation, reduces fibrosis, and lowers body weight, serum cholesterol, and insulin resistance. It also inhibits tumor progression and metastasis. The broad spectrum of effects suggests multiple and yet unknown targets of 4MU. Aiming at 4MU target deconvolution, we have analyzed publicly available data bases, including: 1. Small molecule library Bio Assay screening (PubChemBioAssay); 2. GO pathway databases screening; 3. Protein Atlas Database. We also performed comparative liver transcriptome analysis of mice on normal diet and mice fed with 4MU for two weeks. Potential targets of 4MU public data base analysis fall into two big groups, enzymes and transcription factors (TFs), including 13 members of the nuclear receptor superfamily regulating lipid and carbohydrate metabolism. Transcriptome analysis revealed changes in the expression of genes involved in bile acid metabolism, gluconeogenesis, and immune response. It was found that 4MU feeding decreased the accumulation of the glycogen granules in the liver. Thus, 4MU has multiple targets and can regulate cell metabolism by modulating signaling via nuclear receptors.

A highly selective red-emitting fluorescent probe and its micro-nano-assembly for imaging endogenous peroxynitrite (ONOO-) in living cells.

Endogenous peroxynitrite plays a very important role in the regulation of life activities. However, validated tools for ONOO- tests are currently insufficient. We designed a fluorescent probe TPA-F-NO2 with a low fluorescence background in water based on the D-π-A structure for the imaging of endogenous ONOO- in living cells. TPA-F-NO2 can realize the naked eye detection of ONOO- due to the obvious color change. TPA-F-NO2 has the advantages of large stokes shift, high signal-to-noise ratio, high selectivity and sensitivity. The quantitative detection can be achieved in the range of 0-14 µM ONOO-. Due to its solvatochromic characteristics, TPA-F-NO2 has the potential to be used in OLEDs and other fields. In addition, 4-methylumbelliferone has a wide range of anticancer effects as an inhibitor of hyaluronic acid. We prepared TPA-MU-NPs by assembling TPA-F-NO2 and 4-methylumbelliferone. It also endows TPA-MU-NPs with ONOO- imaging function and anti-proliferation effect on breast cancer cells and other cells. This 'probe-drug' assembly strategy provides ideas for the design and optimization of dual-functional probes.

4-Methylumebelliferone Enhances Radiosensitizing Effects of Radioresistant Oral Squamous Cell Carcinoma Cells via Hyaluronan Synthase 3 Suppression.

Radioresistant (RR) cells are poor prognostic factors for tumor recurrence and metastasis after radiotherapy. The hyaluronan (HA) synthesis inhibitor, 4-methylumbelliferone (4-MU), shows anti-tumor and anti-metastatic effects through suppressing HA synthase (HAS) expression in various cancer cells. We previously reported that the administration of 4-MU with X-ray irradiation enhanced radiosensitization. However, an effective sensitizer for radioresistant (RR) cells is yet to be established, and it is unknown whether 4-MU exerts radiosensitizing effects on RR cells. We investigated the radiosensitizing effects of 4-MU in RR cell models. This study revealed that 4-MU enhanced intracellular oxidative stress and suppressed the expression of cluster-of-differentiation (CD)-44 and cancer stem cell (CSC)-like phenotypes. Interestingly, eliminating extracellular HA using HA-degrading enzymes did not cause radiosensitization, whereas HAS3 knockdown using siRNA showed similar effects as 4-MU treatment. These results suggest that 4-MU treatment enhances radiosensitization of RR cells through enhancing oxidative stress and suppressing the CSC-like phenotype. Furthermore, the radiosensitizing mechanisms of 4-MU may involve HAS3 or intracellular HA synthesized by HAS3.

Fluorescence and electrochemical integrated dual-signal sensor for the detection of iron ions in water based on an ITO substrate.

A novel type of fluorescent and electrochemical dual-signal sensor was constructed for the sensitive and selective detection of iron ions (Fe3+) based on a fluorescent material (Chi-FITC-4MU), which was synthesized by combining the organic dye 4-methylumbelliferone (4-MU), chitosan, and fluorescein isothiocyanate (FITC) in a simple step process. The 4-MU could bind to Fe3+ to form a complex, and clearly improved the selectivity of Chi-FITC-4MU for Fe3+ detection. FITC showed excellent fluorescence performance and chitosan was beneficial to the curing of the material. By solidifying the fluorescent material on an ITO surface, the dual-signal detection of Fe3+ could be realized with excellent selectivity, stability, and anti-interference ability. Based on the unique fluorescence properties of this sensor, the concentration of Fe3+ could be visualized in the linear range of 0.1-100 µM based on the degree of fluorescence quenching. Moreover, the highly sensitive and rapid analysis of low concentrations of Fe3+ was achieved through the electrochemical properties of the ITO sensor. The limit of detection (LOD) and the corresponding linear range were 0.0184 nM and 0.1-500 nM, respectively. Furthermore, this dual-signal sensor was effectively used for the detection of Fe3+ in actual water.

Quantitative morphokinetic parameters identify novel dynamics of oocyte meiotic maturation and cumulus expansion†.

Meiotic maturation and cumulus expansion are essential for the generation of a developmentally competent gamete, and both processes can be recapitulated in vitro. We used a closed time-lapse incubator (EmbryoScope+™) to establish morphokinetic parameters of meiotic progression and cumulus expansion in mice and correlated these outcomes with egg ploidy. The average time to germinal vesicle breakdown (GVBD), time to first polar body extrusion (PBE), and duration of meiosis I were 0.91 ± 0.01, 8.82 ± 0.06, and 7.93 ± 0.06 h, respectively. The overall rate of cumulus layer expansion was 0.091 ± 0.002 µm/min, and the velocity of expansion peaked during the first 8 h of in vitro maturation (IVM) and then slowed. IVM of oocytes exposed to Nocodazole, a microtubule disrupting agent, and cumulus oocyte complexes (COCs) to 4-methylumbelliferone, a hyaluronan synthesis inhibitor, resulted in a dose-dependent perturbation of morphokinetics, thereby validating the system. The incidence of euploidy following IVM was >90% for both denuded oocytes and intact COCs. No differences were observed between euploid and aneuploid eggs with respect to time to GVBD (0.90 ± 0.22 vs. 0.97 ± 0.19 h), time to PBE (8.89 ± 0.98 vs. 9.10 ± 1.42 h), duration of meiosis I (8.01 ± 0.91 vs. 8.13 ± 1.38 h), and overall rate and kinetics of cumulus expansion (0.089 ± 0.02 vs 0.088 ± 0.03 µm/min) (P > 0.05). These morphokinetic parameters provide novel quantitative and non-invasive metrics for the evaluation of meiotic maturation and cumulus expansion and will enable screening compounds that modulate these processes.

Cell-Type-Specific Expression of Hyaluronan Synthases HAS2 and HAS3 Promotes Goblet Cell Hyperplasia in Allergic Airway Inflammation.

Allergic rhinitis (AR) is a multifactorial airway disease characterized by basal and goblet cell hyperplasia. Hyaluronic acid (HA) is a major component of extracellular matrix and a critical contributor to tissue repair and remodeling after injury. We previously demonstrated that the intermediate progenitor cell (IPC) surface marker CD44v3 is upregulated in the basal and suprabasal layers of well-differentiated primary human nasal epithelial (HNE) cells after stimulation with the Th2 (T-helper cell type 2) cytokine IL-4, and an antibody blocking the CD44v3-HA interaction suppressed IL-4-induced goblet cell hyperplasia. We now show that the expression of HA and two HA synthases, HAS2 and HAS3, was upregulated in both the nasal surface epithelium of subjects with AR and IL-4-stimulated HNE cells. Inhibition of HA synthesis by 4-methylumbelliferone suppressed IL-4-induced goblet cell hyperplasia. Moreover, HAS2 and HAS3 were expressed in IPCs depending on the differentiation events, as follows: the rapid, transient upregulation of HAS2 induced basal IPC proliferation and basal-to-suprabasal transition, whereas the delayed upregulation of HAS3 promoted the transition of suprabasal IPCs to a goblet cell fate. 4-methylumbelliferone treatment in a house dust mite-induced murine AR model attenuated goblet cell metaplasia. Last, HA concentrations in nasal epithelial lining fluids from patients with AR positively correlated with the concentrations of mediators causing allergic inflammation. These data suggest that HA produced after the sequential upregulation of HAS2 and HAS3 contributes to goblet cell hyperplasia in allergic airway inflammation and modulates disease progression.

Oral hymecromone decreases hyaluronan in human study participants.

BACKGROUNDHyaluronan (HA), an extracellular matrix glycosaminoglycan, has been implicated in the pathophysiology of COVID-19 infection, pulmonary hypertension, pulmonary fibrosis, and other diseases, but is not targeted by any approved drugs. We asked whether hymecromone (4-methylumbelliferone [4-MU]), an oral drug approved in Europe for biliary spasm treatment that also inhibits HA in vitro and in animal models, could be repurposed as an inhibitor of HA synthesis in humans.METHODSWe conducted an open-label, single-center, dose-response study of hymecromone in healthy adults. Subjects received hymecromone at 1200 (n = 8), 2400 (n = 9), or 3600 (n = 9) mg/d divided into 3 doses daily, administered orally for 4 days. We assessed safety and tolerability of hymecromone and analyzed HA, 4-MU, and 4-methylumbelliferyl glucuronide (4-MUG; the main metabolite of 4-MU) concentrations in sputum and serum.RESULTSHymecromone was well tolerated up to doses of 3600 mg/d. Both sputum and serum drug concentrations increased in a dose-dependent manner, indicating that higher doses lead to greater exposures. Across all dose arms combined, we observed a significant decrease in sputum HA from baseline after 4 days of treatment. We also observed a decrease in serum HA. Additionally, higher baseline sputum HA levels were associated with a greater decrease in sputum HA.CONCLUSIONAfter 4 days of exposure to oral hymecromone, healthy human subjects experienced a significant reduction in sputum HA levels, indicating this oral therapy may have potential in pulmonary diseases where HA is implicated in pathogenesis.TRIAL REGISTRATIONClinicalTrials.gov NCT02780752.FUNDINGStanford Medicine Catalyst, Stanford SPARK, Stanford Innovative Medicines Accelerator program, NIH training grants 5T32AI052073-14 and T32HL129970.

Inverting glucuronidation of hymecromone in situ by catalytic nanocompartments.

Glucuronidation is a metabolic pathway that inactivates many drugs including hymecromone. Adverse effects of glucuronide metabolites include a reduction of half-life circulation times and rapid elimination from the body. Herein, we developed synthetic catalytic nanocompartments able to cleave the glucuronide moiety from the metabolized form of hymecromone in order to convert it to the active drug. By shielding enzymes from their surroundings, catalytic nanocompartments favor prolonged activity and lower immunogenicity as key aspects to improve the therapeutic solution. The catalytic nanocompartments (CNCs) consist of self-assembled poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) diblock copolymer polymersomes encapsulating ß-glucuronidase. Insertion of melittin in the synthetic membrane of these polymersomes provided pores for the diffusion of the hydrophilic hymecromone-glucuronide conjugate to the compartment inside where the encapsulated ß-glucuronidase catalyzed its conversion to hymecromone. Our system successfully produced hymecromone from its glucuronide conjugate in both phosphate buffered solution and cell culture medium. CNCs were non-cytotoxic when incubated with HepG2 cells. After being taken up by cells, CNCs produced the drug in situ over 24 hours. Such catalytic platforms, which locally revert a drug metabolite into its active form, open new avenues in the design of therapeutics that aim at prolonging the residence time of a drug.

A Connecting Link between Hyaluronan Synthase 3-Mediated Hyaluronan Production and Epidermal Function.

Hyaluronan (HA), an essential component of the extracellular matrix of the skin, is synthesized by HA synthases (HAS1-3). To date, epidermal HA has been considered a major player in regulating cell proliferation and differentiation. However, a previous study reported that depletion of epidermal HA by Streptomyces hyaluronidase (St-HAase) has no influence on epidermal structure and function. In the present study, to further explore roles of epidermal HA, we examined effects of siRNA-mediated knockdown of HAS3, as well as conventional HA-depletion methods using St-HAase and 4-methylumbelliferone (4MU), on epidermal turnover and architecture in reconstructed skin or epidermal equivalents. Consistent with previous findings, HA depletion by St-HAase did not have a substantial influence on the epidermal architecture and turnover in skin equivalents. 4MU treatment resulted in reduced keratinocyte proliferation and epidermal thinning but did not seem to substantially decrease the abundance of extracellular HA. In contrast, siRNA-mediated knockdown of HAS3 in epidermal equivalents resulted in a significant reduction in epidermal HA content and thickness, accompanied by decreased keratinocyte proliferation and differentiation. These results suggest that HAS3-mediated HA production, rather than extracellularly deposited HA, may play a role in keratinocyte proliferation and differentiation, at least in the developing epidermis in reconstructed epidermal equivalents.

Hymecromone: a clinical prescription hyaluronan inhibitor for efficiently blocking COVID-19 progression.

Currently, there is no effective drugs for treating clinically COVID-19 except dexamethasone. We previously revealed that human identical sequences of SARS-CoV-2 promote the COVID-19 progression by upregulating hyaluronic acid (HA). As the inhibitor of HA synthesis, hymecromone is an approved prescription drug used for treating biliary spasm. Here, we aimed to investigate the relation between HA and COVID-19, and evaluate the therapeutic effects of hymecromone on COVID-19. Firstly, HA was closely relevant to clinical parameters, including lymphocytes (n = 158; r = -0.50; P < 0.0001), C-reactive protein (n = 156; r = 0.55; P < 0.0001), D-dimer (n = 154; r = 0.38; P < 0.0001), and fibrinogen (n = 152; r = 0.37; P < 0.0001), as well as the mass (n = 78; r = 0.43; P < 0.0001) and volume (n = 78; r = 0.41; P = 0.0002) of ground-glass opacity, the mass (n = 78; r = 0.48; P < 0.0001) and volume (n = 78; r = 0.47; P < 0.0001) of consolidation in patient with low level of hyaluronan (HA < 48.43 ng/mL). Furthermore, hyaluronan could directly cause mouse pulmonary lesions. Besides, hymecromone remarkably reduced HA via downregulating HAS2/HAS3 expression. Moreover, 89% patients with hymecromone treatment had pulmonary lesion absorption while only 42% patients in control group had pulmonary lesion absorption (P < 0.0001). In addition, lymphocytes recovered more quickly in hymecromone-treated patients (n = 8) than control group (n = 5) (P < 0.05). These findings suggest that hymecromone is a promising drug for COVID-19 and deserves our further efforts to determine its effect in a larger cohort.

Dual targeting nanoparticles based on hyaluronic and folic acids as a promising delivery system of the encapsulated 4-Methylumbelliferone (4-MU) against invasiveness of lung cancer in vivo and in vitro.

Lung cancer is the most common cause of cancer death worldwide. Thereby, new treatment strategies as targeting nano-therapy present promising possibilities to control the aggressiveness of lung cancer. Dual CD44 and folate receptors targetable nanocapsule based on folic-polyethylene glycol-hyaluronic (FA-PEG-HA) were fabricated to improve the therapeutic activity of 4-Methylumbelliferone (4-MU) toward lung cancer. In this study, we fabricate 4-MU Nps as a hybrid polymeric (protamine) protein (albumin) nanocapsule, then functionalized by targeting layer to form 4-MU@FA-PEG-HA Nps with encapsulation efficacy 96.15%. The in vitro study of free 4-MU, 4-MU Nps and 4-MU@FA-PEG-HA Nps on A549 lung cancer cells reveal that the 4-MU Nps and 4-MU@FA-PEG-HA Nps were more cytotoxic than free 4-MU on A549 cells. The observed therapeutic activity of 4-MU@FA-PEG-HA Nps on urethane-induced lung cancer model, potentiality revealed a tumor growth inhibition via apoptotic mechanisms and angiogenesis inhibition. The results were supported by Enzyme-linked immunosorbent assay (ELIZA) of transforming growth factors (TGFß1) and serum HA, histopathological analysis as well as immunohistochemical Ki67, CD44, Bcl-2 and caspace-3 staining. Moreover, 4-MU@FA-PEG-HA Nps exhibited a promising safety profile. Hence, it is expected that our developed novel nano-system can be used for potential application on tumor therapy for lung cancer.

Oral treatment of 4-methylumbelliferone reduced perineuronal nets and improved recognition memory in mice.

Hyaluronan (HA) is a core constituent of perineuronal nets (PNNs) that surround subpopulations of neurones. The PNNs control synaptic stabilization in both the developing and adult central nervous system, and disruption of PNNs has shown to reactivate neuroplasticity. We investigated the possibility of memory prolongation by attenuating PNN formation using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis. Adult C57BL/6 mice were fed with chow containing 5% (w/w) 4-MU for 6 months, at a dose ~6.7 mg/g/day. The oral administration of 4-MU reduced the glycosaminoglycan level in the brain to 72% and the spinal cord to 50% when compared to the controls. Spontaneous object recognition test (SOR) performed at 2, 3, 6 and 7 months showed a significant increase in SOR score in the 6-months treatment group 24 h after object presentation. The effect however did not persist in the washout group (1-month post treatment). Immunohistochemistry confirmed a reduction of PNNs, with shorter and less arborization of aggrecan staining around dendrites in hippocampus after 6 months of 4-MU treatment. Histopathological examination revealed mild atrophy in articular cartilage but it did not affect the motor performance as demonstrated in rotarod test. In conclusion, systemic oral administration of 4-MU for 6 months reduced PNN formation around neurons and enhanced memory retention in mice. However, the memory enhancement was not sustained despite the reduction of PNNs, possibly due to the lack of memory enhancement training during the washout period. Our results suggest that 4-MU treatment might offer a strategy for PNN modulation in memory enhancement.

Glycosylated 4-methylumbelliferone as a targeted therapy for hepatocellular carcinoma.

BACKGROUND & AIMS: Reaching efficacious drug delivery to target cells/tissues represents a major obstacle in the current treatment of solid malignancies including hepatocellular carcinoma (HCC). In this study, we developed a pipeline to selective add complex-sugars to the aglycone 4-methylumbelliferone (4MU) to help their bioavailability and tumour cell intake. METHODS: The therapeutic efficacy of sugar-modified rutinosyl-4-methylumbelliferone (4MUR) and 4MU were compared in vitro and in an orthotopic HCC model established in fibrotic livers. The mechanistic bases of its selective target to liver tumour cells were evaluated by the interaction with asialoglycoprotein receptor (ASGPR), the mRNA expression of hyaluronan synthases (HAS2 or HAS3) and hyaluronan deposition. RESULTS: 4MUR showed a significant antiproliferative effect on liver tumoural cells as compared to non-tumoural cells in a dose-dependent manner. Further analysis showed that 4MUR is incorporated mostly into HCC cells by interaction with ASGPR, a receptor commonly overexpressed in HCC cells. 4MUR-treatment decreased the levels of HAS2 and HAS3 and the cytoplasmic deposition of hyaluronan. Moreover, 4MUR reduced CFSC-2G activation, hence reducing the fibrosis. In vivo efficacy showed that 4MUR treatment displayed a greater tumour growth inhibition and increased survival in comparison to 4MU. 4MUR administration was associated with a significant reduction of liver fibrosis without any signs of tissue damage. Further, 60% of 4MUR treated mice did not present macroscopically tumour mass post-treatment. CONCLUSION: Our results provide evidence that 4MUR may be used as an effective HCC therapy, without damaging non-tumoural cells or other organs, most probably due to the specific targeting.