Evaluation of mucosal immune profile associated with Zileuton nanocrystal-formulated BCS-II drug upon oral administration in Sprague Dawley rats.

Nanocrystal drug formulation involves several critical manufacturing procedures that result in complex structures to improve drug solubility, dissolution, bioavailability, and consequently the efficacy of poorly soluble Biopharmaceutics Classification System (BCS) II and IV drugs. Nanocrystal formulation of an already approved oral drug may need additional immunotoxic assessment due to changes in the physical properties of the active pharmaceutical ingredient (API). In this study, we selected Zileuton, an FDA-approved drug that belongs to BCS-II for nanocrystal formulation. To evaluate the efficacy and mucosal immune profile of the nanocrystal drug, 10-week-old rats were dosed using capsules containing either API alone or nanocrystal formulated Zileuton (NDZ), or with a physical mixture (PM) using flexible oral gavage syringes. Control groups consisted of untreated, or placebo treated animals. Test formulations were administrated to rats at a dose of 30 mg/kg body weight (bw) once a day for 15 days. The rats treated with NDZ or PM had approximately 4.0 times lower (7.5 mg/kg bw) API when compared to the micron sized API treated rats. At the end of treatment, mucosal (intestinal tissue) and circulating cytokines were measured. The immunological response revealed that NDZ decreased several proinflammatory cytokines in the ileal mucosa (Interleukin-18, Tumor necrosis Factor-α and RANTES [regulated upon activation, normal T cell expressed and secreted]). A similar pattern in the cytokine profile was also observed for the micron sized API and PM treated rats. The cytokine production revealed that there was a significant increase in the production of IL-1ß and IL-10 in the females in all experimental groups. Additionally, NDZ showed an immunosuppressive effect on proinflammatory cytokines both locally and systemically, which was similar to the response in micron sized API treated rats. These findings indicate that NDZ significantly decreased several proinflammatory cytokines and it displays less immunotoxicity, probably due to the nanocrystal formulation. Thus, the nanocrystal formulation is more suitable for oral drug delivery, as it exhibited better efficacy, safety, and reduced toxicity.

Eeyarestatin I, an inhibitor of the valosin-containing protein, exhibits potent virucidal activity against the flaviviruses.

Cellular responses to stress generally lead to the activation of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Several lines of study support that ERAD may be playing a proviral role during flaviviral infection. A key host factor in ERAD is the valosin-containing protein (VCP), an ATPase which ushers ubiquitin-tagged proteins to degradation by the proteasome. VCP exhibits different proviral activities, such as engaging in the biogenesis of viral replication organelles and facilitating flavivirus genome uncoating after the viral particle entry. To investigate the possible antiviral value of drugs targeting VCP, we tested two inhibitors: eeyarestatin I (EEY) and xanthohumol (XAN). Both compounds were highly effective in suppressing Zika virus (ZIKV) and Usutu virus (USUV) replication during infection in cell culture. Further analysis revealed an unexpected virucidal activity for EEY, but not for XAN. Preincubation of ZIKV or USUV with EEY before inoculation to cells resulted in significant decreases in infectivity in a dose- and time-dependent manner. Viral genomes in samples previously treated with EEY were more sensitive to propidium monoazide, an intercalating agent, with 10- to 100-fold decreases observed in viral RNA levels, supporting that EEY affects viral particle integrity. Altogether, these results support that EEY is a strong virucide against two unrelated flaviviruses, encouraging further studies to investigate its potential use as a broad-acting drug or the development of improved derivatives in the treatment of flaviviral infection.

Zileuton Alleviates Radiation-Induced Cutaneous Ulcers via Inhibition of Senescence-Associated Secretory Phenotype in Rodents.

Radiation-induced cutaneous ulcers are a challenging medical problem for patients receiving radiation therapy. The inhibition of cell senescence has been suggested as a prospective strategy to prevent radiation ulcers. However, there is no effective treatment for senescent cells in radiation ulcers. In this study, we investigated whether zileuton alleviated radiation-induced cutaneous ulcer by focusing on cell senescence. We demonstrate increased cell senescence and senescence-associated secretory phenotype (SASP) in irradiated dermal fibroblasts and skin tissue. The SASP secreted from senescent cells induces senescence in adjacent cells. In addition, 5-lipoxygenase (5-LO) expression increased in irradiated dermal fibroblasts and skin tissue, and SASP and cell senescence were regulated by 5-LO through p38 phosphorylation. Finally, the inhibition of 5-LO following treatment with zileuton inhibited SASP and mitigated radiation ulcers in animal models. Our results demonstrate that inhibition of SASP from senescent cells by zileuton can effectively mitigate radiation-induced cutaneous ulcers, indicating that inhibition of 5-LO might be a viable strategy for patients with this condition.

Zileuton, a 5-Lipoxygenase Inhibitor, Attenuates Haemolysate-Induced BV-2 Cell Activation by Suppressing the MyD88/NF-κB Pathway.

M1 microglia induce neuroinflammation-related neuronal death in animal models of spontaneous subarachnoid haemorrhage. Zileuton is a 5-lipoxygenase inhibitor that reduces the levels of downstream pro-inflammatory cytokines. This study aimed to investigate whether zileuton inhibits microglial activation and describe its underlying mechanisms. BV-2 cells were exposed to 1 mg/mL haemolysate for 30 min, followed by treatment with different concentrations (5, 10, 15, or 20 µM) of zileuton for 24 h. The cells were then assessed for viability, polarisation, and protein expression levels. Haemolysate increases the viability of BV-2 cells and induces M1 polarisation. Subsequent exposure to high concentrations of zileuton decreased the viability of BV-2 cells, shifted the polarisation to the M2 phenotype, suppressed the expression of 5-lipoxygenase, decreased tumour necrosis factor α levels, and increased interleukin-10 levels. Furthermore, high concentrations of zileuton suppressed the expression of myeloid differentiation primary response protein 88 and reduced the phosphorylated-nuclear factor-kappa B (NF-kB)/NF-kB ratio. Therefore, phenotype reversal from M1 to M2 is a possible mechanism by which zileuton attenuates haemolysate-induced neuroinflammation after spontaneous subarachnoid haemorrhage.

Long-term role of zileuton in the treatment of chronic rhinosinusitis in aspirin exacerbated respiratory disease.

BACKGROUND: Chronic rhinosinusitis (CRS) in the setting of Aspirin Exacerbated Respiratory Disease (AERD) have high rate of treatment failure and disease recurrence. OBJECTIVE: Evaluate the long-term effect of zileuton on sinonasal outcomes in patients with AERD. METHODS: AERD patients were reviewed and divided into two cohorts, depending if they were treated with zileuton during their clinical course. Demographic data, 22-item sinonasal outcome test (SNOT-22), Lund-Kennedy (LK) endoscopy score, duration of treatment, and number of sinus surgeries performed were collected. RESULTS: 40 AERD patients were included, with follow-up duration up to 10 years (avg of 5.2 years). All patients were treated with topical saline and budesonide irrigations, intranasal steroid spray, and montelukast. 19 patients had uncontrolled sinus disease requiring multiple steroid tapers and were switched from montelukast to zileuton (cohort 1, 47.5%) at some point in their treatment. 21 patients (cohort 2, 52.5%) never needed zileuton. The average duration of treatment with zileuton was 6 years. Patients who required zileuton had a worse SNOT-22 (32.1 vs 19, p = 0.117), worse LK score (8.1 vs 7.5, p = 0.504), and higher average number of surgeries (1.9 vs 1.6, p = 0.343). The outcomes in the zileuton cohort trended toward improvement, however these did not reach statistical significance with an improved SNOT-22 from 32.1 to 27.4 (p = 0.617) and LK score from 7.9 to 6.2 (p = 0.092); The addition of zileuton significantly lowered the number of surgeries needed to an average of 0.5 (p < 0.0001). CONCLUSION: Zileuton may help decrease the number of sinus surgeries needed in AERD.

Zileuton inhibits arachidonate-5-lipoxygenase to exert antitumor effects in preclinical cervical cancer models.

BACKGROUND: Inhibitors of arachidonate lipoxygenase 5 (ALOX5) exhibit anticancer activity. Zileuton is an FDA-approved drug for treating asthma and an ALOX5 inhibitor. This study evaluated the efficacy of zileuton in cervical cancer, determined the molecular mechanism of action, and assessed ALOX5 expression in cervical cancer patients. METHODS: The effects of zileuton were evaluated using cervical cancer cell lines and xenograft mouse models. Loss-of-function analysis of ALOX5 was performed using siRNA. The levels of ALOX5 and 5-HETE were determined using immunohistochemistry and ELISA. RESULTS: Zileuton resulted in cell proliferation inhibition and apoptosis induction in a dose-dependent manner, regardless of cellular origin or HPV infection. In two independent cervical cancer xenograft mouse models, zileuton at nontoxic doses significantly prevented tumor formation and decreased tumor growth. Zileuton acts on cervical cancer cells by inhibiting the ALOX5-5-HETE axis. Of note, ALOX5-5-HETE was significantly upregulated in cervical cancer compared with normal tissue. Inhibition of ALOX5 via the siRNA approach mimics the inhibitory effects of zileuton and confirms the roles of ALOX5 in cervical cancer. CONCLUSIONS: Our work demonstrates that the ALOX5-5-HETE axis is activated in cervical cancer, with important roles in growth and survival, and this can be therapeutically targeted by zileuton. Our findings also provide preclinical evidence to assess the efficacy of zileuton in cervical cancer in clinical settings.

Inhibition of Lipoxygenases Showed No Benefit for the Musculoskeletal System in Estrogen Deficient Rats.

Background: In previous studies, we reported the beneficial impact of two lipoxygenase-inhibitors, Baicalein and Zileuton, on osteoporotic bone in a postmenopausal rat model. Whereas subcutaneous Baicalein predominantly improved cortical bone, Zileuton enhanced vertebral and femoral trabecular bone. In this study, we aimed to reveal whether the oral administration of Baicalein caused similar effects on bone and whether a combined administration of Baicalein and Zileuton could act synergistically to ameliorate the formerly reported effects in the musculoskeletal system. Methods: We treated ovariectomized (OVX) female Sprague-Dawley rats either with Baicalein (10mg/kg BW), Zileuton (10mg/kg BW) or a combination of both (each 10mg/kg BW) for 13 weeks and compared with untreated OVX and NON-OVX groups (n=12-16 rats per group). Lumbar vertebral bodies and femora were analyzed. Tibiae were osteotomized, plate-stabilized (at week 8 after OVX) and likewise analyzed by biomechanical, histological, micro-computed tomographical and ashing tests. The skeletal muscle structure was analyzed. Results: Oral administration of Baicalein did not confirm the reported favorable cortical effects in neither vertebra nor femur. Zileuton showed a beneficial effect on trabecular vertebra, while the femur was negatively affected. Callus formation was enhanced by all treatments; however, its density and biomechanical properties were unaltered. Lipoxygenase inhibition did not show a beneficial effect on skeletal muscle. The combination therapy did not ameliorate OVX-induced osteoporosis but induced even more bone loss. Conclusions: The preventive anti-osteoporotic treatments with two lipoxygenase inhibitors applied either alone or in combination showed no benefit for the musculoskeletal system in estrogen deficient rats.

Development of polymer-based nanoparticles for zileuton delivery to the lung: PMeOx and PMeOzi surface chemistry reduces interactions with mucins.

In this paper, two amphiphilic graft copolymers were synthesized by grafting polylactic acid (PLA) as hydrophobic chain and poly(2-methyl-2-oxazoline) (PMeOx) or poly(2-methyl-2-oxazine) (PMeOzi) as hydrophilic chain, respectively, to a backbone of α,ß-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA). These original graft copolymers were used to prepare nanoparticles delivering Zileuton in inhalation therapy. Among various tested methods, direct nanoprecipitation proved to be the best technique to prepare nanoparticles with the smallest dimensions, the narrowest dimensional distribution and a spherical shape. To overcome the size limitations for administration by inhalation, the nano-into-micro strategy was applied, encapsulating the nanoparticles in water-soluble mannitol-based microparticles by spray-drying. This process has allowed to produce spherical microparticles with the proper size for optimal lung deposition, and, once in contact with fluids mimicking the lung district, able to dissolve and release non-aggregated nanoparticles, potentially able to spread through the mucus, releasing about 70% of the drug payload in 24 h.

ALOX5-5-HETE promotes gastric cancer growth and alleviates chemotherapy toxicity via MEK/ERK activation.

BACKGROUND: Recent studies highlight the regulatory role of arachidonate lipoxygenase5 (Alox5) and its metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) in cancer tumorigenesis and progression. In this study, we analyzed the expression, biological function and the downstream signaling of Alox5 in gastric cancer. METHODS: Alox5 protein levels were measured using IHC and ELISA. Growth, migration and survival assays were performed. Phosphorylation of molecules involved in growth and survival signaling were analyzed by WB. Analysis of variance and t-test were used for statistic analysis. RESULTS: Alox5 and 5-HETE levels were upregulated in gastric cancer patients. ALOX5 overexpression or 5-HETE addition activates gastric cancer cells and reduces chemotherapy's efficacy. In contrast, ALOX5 inhibition via genetic and pharmacological approaches suppresses gastric cancer cells and enhances chemotherapy's efficacy. In addition, Alox5 inhibition led to suppression of ERK-mediated signaling pathways whereas ALOX5-5-HETE activates ERK-mediated signaling in gastric cancer cells. CONCLUSIONS: Our work demonstrates the critical role of ALOX5-5-HETE in gastric cancer and provides pre-clinical evidence to initialize clinical trial using zileuton in combination with chemotherapy for treating gastric cancer.

5-Lipoxygenase inhibition reduces inflammation and neuronal apoptosis via AKT signaling after subarachnoid hemorrhage in rats.

Early brain injury (EBI) is a major contributor to the high mortality and morbidity after subarachnoid hemorrhage (SAH). Inflammatory responses and neuronal apoptosis are important causes of EBI. Because 5- lipoxygenase (5-LOX) is known to be involved various central nervous system diseases, we investigated the effects of 5-LOX inhibition during EBI after SAH. Zileuton and LY294002 were used to inhibit expression of 5-LOX and Akt, respectively. We found that 5-LOX expression was significantly increased in the cytoplasm of cortical neurons after SAH and was accompanied by upregulated expression of the inflammatory factors LTB4, TNF-α, IL-1ß and IL-6; upregulation of the pro-apoptotic factor Bax; downregulation of the anti-apoptotic factor Bcl-2; and an increased apoptosis rate. Gastric Zileuton administration significantly suppressed all of those effects and improved neurological function. Zileuton also upregulated activated (phosphorylated) AKT levels, and these beneficial effects of Zileuton were abolished by intracerebroventricular infusion of the PI3K inhibitor LY294002. Taken together, these findings indicate that 5-LOX mediates pro-inflammatory and pro-apoptotic effects that contribute to EBI after SAH and that those effects are suppressed by activation of PI3K/Akt signaling. This suggests targeting 5-LOX may be an effective approach to treating EBI after SAH.

Inhibition of ERAD synergizes with FTS to eradicate pancreatic cancer cells.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancers, is driven by oncogenic KRAS mutations. Farnesyl thiosalicylic acid (FTS), also known as salirasib, is a RAS inhibitor that selectively dislodges active RAS proteins from cell membrane, inhibiting downstream signaling. FTS has demonstrated limited therapeutic efficacy in PDAC patients despite being well tolerated. METHODS: To improve the efficacy of FTS in PDAC, we performed a genome-wide CRISPR synthetic lethality screen to identify genetic targets that synergize with FTS treatment. Among the top candidates, multiple genes in the endoplasmic reticulum-associated protein degradation (ERAD) pathway were identified. The role of ERAD inhibition in enhancing the therapeutic efficacy of FTS was further investigated in pancreatic cancer cells using pharmaceutical and genetic approaches. RESULTS: In murine and human PDAC cells, FTS induced unfolded protein response (UPR), which was further augmented upon treatment with a chemical inhibitor of ERAD, Eeyarestatin I (EerI). Combined treatment with FTS and EerI significantly upregulated the expression of UPR marker genes and induced apoptosis in pancreatic cancer cells. Furthermore, CRISPR-based genetic ablation of the key ERAD components, HRD1 and SEL1L, sensitized PDAC cells to FTS treatment. CONCLUSION: Our study reveals a critical role for ERAD in therapeutic response of FTS and points to the modulation of UPR as a novel approach to improve the efficacy of FTS in PDAC treatment.

Targeting Neutrophils for Enhanced Cancer Theranostics.

Improving tumor accumulation and delivery efficiency is an important goal of nanomedicine. Neutrophils play a vital role in both chemically mediating inflammatory response through myeloperoxidase (MPO) and biologically promoting metastasis during inflammation triggered by the primary tumor or environmental stimuli. Herein, a novel theranostic nanomedicine that targets both the chemical and biological functions of neutrophils in tumor is designed, facilitating the enhanced retention and sustained release of drug cargos for improved cancer theranostics. 5-hydroxytryptamine (5-HT) is equipped onto nanoparticles (NPs) loaded with photosensitizers and Zileuton (a leukotriene inhibitor) to obtain MPO and neutrophil targeting NPs, denoted as HZ-5 NPs. The MPO targeting property of 5-HT modified NPs is confirmed by noninvasive positron emission tomography imaging studies. Furthermore, photodynamic therapy is used to initiate the inflammatory response which further mediated the accumulation and retention of neutrophil targeting NPs in a breast cancer model. This design renders a greatly improved theranostic nanomedicine for efficient tumor suppression, and more importantly, inhibition of neutrophil-mediated lung metastasis via the sustained release of Zileuton. This work presents a novel strategy of targeting neutrophils for improved tumor theranostics, which may open up new avenues in designing nanomedicine through exploiting the tumor microenvironment.

Repurposing Zileuton as a Depression Drug Using an AI and In Vitro Approach.

Repurposing drugs to target M1 macrophages inflammatory response in depression constitutes a bright alternative for commonly used antidepressants. Depression is a significant type of mood disorder, where patients suffer from pathological disturbances associated with a proinflammatory M1 macrophage phenotype. Presently, the most commonly used antidepressants such as Zoloft and Citalopram can reduce inflammation, but suffer from dangerous side effects without offering specificity toward macrophages. We employed a new strategy for drug repurposing based on the integration of RNA-seq analysis and text mining using deep neural networks. Our system employs a Google semantic AI universal encoder to compute sentences embedding. Sentences similarity is calculated using a sorting function to identify drug compounds. Then sentence relevance is computed using a custom-built convolution differential network. Our system highlighted the NRF2 pathway as a critical drug target to reprogram M1 macrophage response toward an anti-inflammatory profile (M2). Using our approach, we were also able to predict that lipoxygenase inhibitor drug zileuton could modulate NRF2 pathway in vitro. Taken together, our results indicate that reorienting zileuton usage to modulate M1 macrophages could be a novel and safer therapeutic option for treating depression.

Effect of 5-lipoxygenase inhibitor, VIA-2291 (Atreleuton), on epicardial fat volume in patients with recent acute coronary syndrome.

BACKGROUND AND AIMS: The association between inflammation and atherosclerosis has been well described in the literature. There is now mounting evidence in support of adipose tissue as a reservoir for inflammatory markers. Epicardial adipose tissue (EAT) has been shown to associate with coronary atherosclerosis and found to be a predictor of future adverse cardiovascular events. This study, VIA-EAT, assesses the change in epicardial fat volume (EFV) after treatment with an investigational anti-inflammatory agent (VIA-2291) in a cohort of post-acute coronary syndrome (ACS) patients. METHODS: This study is derived from a post-hoc analysis of a previously conducted randomized clinical trial (NCT00358826). Patients were recruited for a prospective, double-blind, multi-center randomized trial of a 5-lipooxygenase inhibitor or placebo in a 3:1 randomization, including doses of placebo, and 25 mg, 50 mg and 100 mg of active treatment. Cardiac computed tomography was performed at baseline and at 24 weeks after treatment with VIA-2291. EAT and pericardial adipose tissue (PAT) were measured using previously published methodology. A Pearson correlation test was used to determine the relationship between change in epicardial fat and change in plaque composition. RESULTS: We analyzed 54 pre- and post-treatment scans. There were no major differences between traditional cardiovascular risk factors among the 4 randomized study arms. There was a significant decrease in EAT and PAT in patients in the treatment arms vs. placebo, -3.0 ± 8.2mm3 and -3.9 ± 10.9mm3 vs. 1.7 ± 7.5mm3 and 1.4 ± 10.7mm3 (p = 0.001), respectively. The changes in EAT and PAT were more pronounced in patients taking 100 mg of the drug vs. placebo: 4.2 ± 9.6mm3, -7.6 ± 8.5mm3, p = 0.0001, respectively. In a subgroup analysis, reduction in epicardial fat volume correlated with reduction in total atherosclerotic plaque volume across all VIA treatment groups, r = 0.52 (p = 0.004). CONCLUSIONS: After adjustment for traditional cardiovascular risk factors including age, gender, body mass index, dyslipidemia and smoking, VIA-2291 decreases EAT and PAT in individuals with recent ACS. Treatment with the drug also appears to alter plaque volume and composition.

Zileuton ameliorates depressive-like behaviors, hippocampal neuroinflammation, apoptosis and synapse dysfunction in mice exposed to chronic mild stress.

Our previous study has found that zileuton, a selective 5-lipoxygenase (5LO) inhibitor, abrogated lipopolysaccharide-induced depressive-like behaviors and hippocampal neuroinflammation. Herein, we further extended our curiosity to investigate effects of zileuton on stress-induced depressive-like behaviors. Our data indicated that zileuton significantly ameliorated depressive-like behaviors in mice subjected to chronic mild stress (CMS), as shown in the tail suspension test, forced swimming test and novelty-suppressed feeding test. The further studies indicated that zileuton suppressed hippocampal neuroinflammation, evidenced by lower levels of TNF-α, IL-1ß and nuclear NF-κB p65 as well as decreased number of Iba1-positive cells. It also significantly ameliorated hippocampal apoptosis, indicated by deceased number of TUNEL-positive cells, deceased ratio of cleaved caspase-3/procaspase-3 and increased ratio of Bcl-2/Bax. More importantly, zileuton increased the level of synaptic proteins PSD-95 and SYN and the number of NeuN+/BrdU+ cells in the hippocampus. Over all, zileuton alleviated CMS-induced depressive-like behaviors, neuroinflammatory and apoptotic responses, abnormalities of synapse and neurogenesis in the hippocampus, suggesting that it might has beneficial effects on depression.

Pharmaceutical analysis combined with in-silico therapeutic and toxicological profiling on zileuton and its impurities to assist in modern drug discovery.

The obligatory testing of drug molecules and their impurities to protect users against toxic compounds seems to provide interesting opportunities for new drug discovery. Impurities, which proved to be non-toxic, may be explored for their own therapeutic potential and thus be a part of future drug discovery. The essential role of pharmaceutical analysis can thus be extended to achieve this purpose. The present study examined these objectives by characterizing the major degradation products of zileuton (ZLT), a 5-lipoxygenase (5-LOX) inhibitor being prevalently used to treat asthma. The drug sample was exposed to forced degradation and found susceptible to hydrolysis and oxidative stress. The obtained Forced Degradation Products (FDP's) were resolved using an earlier developed and validated Ultra-High-Pressure Liquid Chromatography Photo-Diode-Array (UHPLC-PDA) protocol. ZLT, along with acid-and alkali-stressed samples, were subjected to Liquid-chromatography Mass-spectrometry Quadrupole Time-of-flight (LC/MS-QTOF) studies. Major degradation products were isolated using Preparative TLC and characterized using Q-TOF and/or Proton nuclear magnetic resonance (1HNMR) studies. The information obtained was assembled for structural conformation. Toxicity Prediction using Komputer Assisted Technology (TOPKAT) toxicity analyses indicated some FDP's as non-toxic when compared to ZLT. Hence, these non-toxic impurities may have bio-affinity and can be explored to interact with other therapeutic targets, to assist in drug discovery. The drug molecule and the characterized FDP's were subjected to 3-Dimensional Extra Precision (3D-XP)-molecular docking to explore changes in bio-affinity for the 5-LOX enzyme (PDB Id: 3V99). One FDP was found to have a higher binding affinity than the drug itself, indicating it may be a suitable antiasthmatic. The possibility of being active at other sites cannot be neglected and this is evaluated to a reasonable extent by Prediction of Activity Spectra for Substances (PASS). Besides being antiasthmatic, some FDP's were predicted antineoplastic, antiallergic and inhibitors of Complement Factor-D.

Zileuton™ loaded in polymer micelles effectively reduce breast cancer circulating tumor cells and intratumoral cancer stem cells.

Tumor recurrence, metastatic spread and progressive gain of chemo-resistance of advanced cancers are sustained by the presence of cancer stem cells (CSCs) within the tumor. Targeted therapies with the aim to eradicate these cells are thus highly regarded. However, often the use of new anti-cancer therapies is hampered by pharmacokinetic demands. Drug delivery through nanoparticles has great potential to increase efficacy and reduce toxicity and adverse effects. However, its production has to be based on intelligent design. Likewise, we developed polymeric nanoparticles loaded with Zileuton™, a potent inhibitor of cancer stem cells (CSCs), which was chosen based on high throughput screening. Its great potential for CSCs treatment was subsequently demonstrated in in vitro and in in vivo CSC fluorescent models. Encapsulated Zileuton™ reduces amount of CSCs within the tumor and effectively blocks the circulating tumor cells (CTCs) in the blood stream and metastatic spread.

Participation of 5-lipoxygenase and LTB4 in liver regeneration after partial hepatectomy.

Regeneration is the unmatched liver ability for recovering its functional mass after tissue lost. Leukotrienes (LT) are a family of eicosanoids with the capacity of signaling to promote proliferation. We analyzed the impact of blocking LT synthesis during liver regeneration after partial hepatectomy (PH). Male Wistar rats were subjected to two-third PH and treated with zileuton, a specific inhibitor of 5-lipoxygenase (5-LOX). Our first find was a significant increment of intrahepatic LTB4 during the first hour after PH together with an increase in 5-LOX expression. Zileuton reduced hepatic LTB4 levels at the moment of hepatectomy and also inhibited the increase in hepatic LTB4. This inhibition produced a delay in liver proliferation as seen by decreased PCNA and cyclin D1 nuclear expression 24 h post-PH. Results also showed that hepatic LTB4 diminution by zileuton was associated with a decrease in NF-ĸB activity. Additionally, decreased hepatic LTB4 levels by zileuton affected the recruitment of neutrophils and macrophages. Non-parenchymal cells (NPCs) from zileuton-treated PH-rats displayed higher apoptosis than NPCs from PH control rats. In conclusion, the present work provides evidences that 5-LOX activation and its product LTB4 are involved in the initial signaling events for liver regeneration after PH and the pharmacological inhibition of this enzyme can delay the initial time course of the phenomenon.

The 5-Lipoxygenase Inhibitor Zileuton Protects Pressure Overload-Induced Cardiac Remodeling via Activating PPARα.

Zileuton has been demonstrated to be an anti-inflammatory agent due to its well-known ability to inhibit 5-lipoxygenase (5-LOX). However, the effects of zileuton on cardiac remodeling are unclear. In this study, the effects of zileuton on pressure overload-induced cardiac remodeling were investigated and the possible mechanisms were examined. Aortic banding was performed on mice to induce a cardiac remodeling model, and the mice were then treated with zileuton 1 week after surgery. We also stimulated neonatal rat cardiomyocytes with phenylephrine (PE) and then treated them with zileuton. Our data indicated that zileuton protected mice from pressure overload-induced cardiac hypertrophy, fibrosis, and oxidative stress. Zileuton also attenuated PE-induced cardiomyocyte hypertrophy in a time- and dose-dependent manner. Mechanistically, we found that zileuton activated PPARα, but not PPARγ or PPARθ, thus inducing Keap and NRF2 activation. This was confirmed with the PPARα inhibitor GW7647 and NRF2 siRNA, which abolished the protective effects of zileuton on cardiomyocytes. Moreover, PPARα knockdown abolished the anticardiac remodeling effects of zileuton in vivo. Taken together, our data indicate that zileuton protects against pressure overload-induced cardiac remodeling by activating PPARα/NRF2 signaling.

Zileuton, a 5-Lipoxygenase Inhibitor, Exerts Anti-Angiogenic Effect by Inducing Apoptosis of HUVEC via BK Channel Activation.

The arachidonic acid metabolism through 5-lipoxygenase (5-LO) pathways is involved in modulating both tumorigenesis and angiogenesis. Although anti-carcinogenic activities of certain 5-LO inhibitors have been reported, the role of zileuton, a well known 5-LO inhibitor, on the endothelial cell proliferation and angiogenesis has not been fully elucidated. Here, we report that zileuton has an anti-angiogenic effect, and the underlying mechanisms involved activation of the large-conductance Ca2+-activated K+ (BK) channel. Our results show that zileuton significantly prevented vascular endothelial growth factor (VEGF)-induced proliferation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as in vivo. However, such anti-angiogenic effect of zileuton was abolished by iberiotoxin (IBTX), a BK channel blocker, suggesting zileuton-induced activation of BK channel was critical for the observed anti-angiogenic effect of zileuton. Furthermore, the anti-angiogenic effect of zileuton was, at least, due to the activation of pro-apoptotic signaling cascades which was also abolished by IBTX. Additionally, zileuton suppressed the expression of VCAM-1, ICAM-1, ETS related gene (Erg) and the production of nitric oxide (NO). Taken together, our results show that zileuton prevents angiogenesis by activating the BK channel dependent-apoptotic pathway, thus highlighting its therapeutic capacity in angiogenesis-related diseases, such as cancer.