Preclinical pharmacokinetic and in vitro metabolic stability study of lysosomotropic autophagy inhibitor, IITZ-01 in mice by using UPLC-MS/MS.

The present study evaluates the pharmacokinetics and metabolic stability of a novel lysosomotropic autophagy inhibitor, IITZ-01 using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). It is required as this lead molecule awaits pre-clinical studies for development because of significant therapeutic outcomes in triple-negative breast cancer and renal cancer. A bioanalytical method for the quantitative determination of IITZ-01 in the plasma of mice was developed using the UPLC-MS/MS technique. The UPLC-MS/MS method was validated according to US-FDA bioanalytical guidance and successfully applied to study the pharmacokinetics and metabolic stability. Separation of IITZ- 01 and ZSTK474 (IS) from endogenous components with high selectivity and sensitivity (0.5 ng/mL) was achieved using Waters Acquity BEH C-18 column (50 mm × 2.1 mm, 1.7 µm). A gradient mobile phase consisting of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile was applied at a flow rate of 0.2 mL/min. Electrospray ionization was employed in positive ion mode for detection, while quantification utilized the multiple reaction monitoring (MRM) mode. This involved using [M+H]+fragment ions at m/z 483.19 â†’ 235.09 for IITZ-01 and m/z 418 â†’ 138 for the internal standard (IS). The method was validated over the calibration range of 0.5-800 ng/mL. The LLOQ of IITZ-01 was 0.5 ng/mL in mice plasma. The method demonstrated good in terms of intra- and inter-day precision and accuracy. The matrix effect was found to be negligible, and the stability data were within acceptable limits. The validated technique supports suitability, reliability, reproducibility, and sensitivity for the pre-clinical investigation of IITZ-01 pharmacokinetics in mice and metabolic stability in human liver microsomes.

TLR4-A Pertinent Player in Radiation-Induced Heart Disease?

The heart is one of the organs that is sensitive to developing delayed adverse effects of ionizing radiation (IR) exposure. Radiation-induced heart disease (RIHD) occurs in cancer patients and cancer survivors, as a side effect of radiation therapy of the chest, with manifestation several years post-radiotherapy. Moreover, the continued threat of nuclear bombs or terrorist attacks puts deployed military service members at risk of exposure to total or partial body irradiation. Individuals who survive acute injury from IR will experience delayed adverse effects that include fibrosis and chronic dysfunction of organ systems such as the heart within months to years after radiation exposure. Toll-like receptor 4 (TLR4) is an innate immune receptor that is implicated in several cardiovascular diseases. Studies in preclinical models have established the role of TLR4 as a driver of inflammation and associated cardiac fibrosis and dysfunction using transgenic models. This review explores the relevance of the TLR4 signaling pathway in radiation-induced inflammation and oxidative stress in acute as well as late effects on the heart tissue and the potential for the development of TLR4 inhibitors as a therapeutic target to treat or alleviate RIHD.

Multifunctional Polymeric Nanoparticles for Chemo/Phototheranostics of Retinoblastoma.

Retinoblastoma (Rb) is the most critical and severe intraocular malignancy occurring in children. The clinical management of retinoblastoma is still challenging due to failure in early detection and control despite the advancements in medical strategies. Early-stage Rb tumors do not occupy major visual fields, so chemo/photothermal therapy (PTT) with biocompatible materials can be a practical approach. Herein, we report multifunctional polymeric nanoparticles (PNPs) entrapped with an FDA-approved anticancer drug, Palbociclib (PCB), and a near-infrared dye, IR820 (IR), as chemo/photothermal agents. These PCB/IR PNPs were evaluated for the combinational effect in the retinoblastoma cell line. Further, the in vivo photoacoustic imaging efficacy and acute toxicity profile of the PNPs were studied in a mice model. The results indicated that the PCB/IR PNPs exhibited a significant cytotoxic effect (86.5 ± 2.3%) in Y79 cell lines than the respective control groups upon exposure to NIR light. Qualitative and quantitative analyses indicated that PCB/IR PNPs with NIR light induction resulted in DNA damage followed by apoptosis. PCB/IR PNPs, when tested in vivo, showed optimal photoacoustic signals. Thus, the combination of PCB and PTT can emerge as a translational modality for retinoblastoma therapy.

Lipopolysaccharide exacerbates chronic restraint stress-induced neurobehavioral deficits: Mechanisms by redox imbalance, ASK1-related apoptosis, autophagic dysregulation.

Major depressive disorder (MDD) is the foremost leading psychiatric illness prevailing around the globe. It usually exists along with anxiety and other clinical conditions (cardiovascular, cancer, neurodegenerative diseases, and infectious diseases). Chronic restraint stress (RS) and LPS-induce neurobehavioral alterations in rodent models however their interaction studies in association with the pathogenesis of MDD are still unclear. Therefore, the current study was aimed to investigate the LPS influence on chronic RS mediated redox imbalance, apoptosis, and autophagic dysregulation in the hippocampus (HIP) and frontal cortex (FC) of mice brain. Male Balb/c mice were exposed to 28 days consecutive stress (6h/day) with a single-dose LPS challenge (0.83 mg/kg, i.p.) on the last day (Day 28). In addition, we also carried out separate study to understand physiological relevance, where we used the DSS (dextran sulfate sodium), a water soluble polysaccharide (negatively charged) and studied its influence on RS induced neurobehavioral and certain neurochemical anomalies. The obtained results in RS and RS + LPS animal groups showed significant immune dysfunction, depleted monoamines, lowered ATP & NAD level, elevated serum CORT level, serum and brain tissues IL-1ß/TNF-α/IL-6, SOD activity but reduced CAT activity. Furthermore, the redox perturbation was found where significantly upregulated P-NFκB p65, Keap-1, Prx-SO3 and downregulated Nrf2, Srx1, Prx2 protein expression was seen in RS + LPS mice. The apoptosis signaling (P-ASK1, P-p38 MAPK, P-SAPK/JNK, cleaved PARP, cleaved Caspase-3, Cyto-C), autophagic impairment (p62, LC3II/I) were noticed in HIP and FC of RS and RS + LPS grouped animals. Our new findings provide a complex interplay of chemical (LPS) and physical (RS) stressors where both single dose LPS challenge and 3% DSS in drinking water (for 7 days) exaggerated chronic RS-induced inflammation, lowered redox status, increased apoptosis and dysregulated autophagy leading drastic neurobehavioral alterations in the mice.

Quercetin and piperine enriched nanostructured lipid carriers (NLCs) to improve apoptosis in oral squamous cellular carcinoma (FaDu cells) with improved biodistribution profile.

Oral squamous cellular carcinoma (OSCC) is considered a life-threatening disease with detection in late stages, which forces us to opt for dangerous treatment with a combination of chemotherapy and radiotherapy. Herbal components such as piperine and quercetin are derived from edible sources, proving their anticancer potential against oral cancer cells in vitro. Encapsulation into lipid matrix-mediated nanostructured lipid carriers (NLCs) can make both drugs bio-accessible. NLCs were synthesised using the high shear homogenisation method and characterised for their physicochemical properties, followed by in vitro cellular evaluation in FaDu oral cancer cells. NLCs showed negatively charged particles smaller than 180 nm with a polydispersity index (PDI) of <0.3. Both drugs were found to encapsulate sufficiently, with >85% entrapment efficiency and an improved drug release profile compared to their pristine counterparts. Differential scanning calorimetry (DSC) thermograms showed conversion into an amorphous matrix in lyophilized NLCs, which was supported by X-ray diffraction (XRD) analysis. The cytotoxicity assay showed the IC50 concentration for dual drug-loaded NLCs, which was more effective than the pure drug solution. NLCs were found to be internalised in cells in a short time with an almost 95% co-localization rate. Dual drug-loaded NLCs showed maximum depolarisation of the mitochondrial membrane along with more apoptotic changes. Improved apoptosis was confirmed in NLCs using flow cytometry. The in vivo biodistribution of Coumarin-6 labelled NLCs in rats confirmed their efficient distribution in various parts of the oral cavity through oral administration. Optimised dual drug-loaded NLCs provide a better option for delivering both drugs through a single lipid matrix against oral cancer.

Lithocholic acid-tryptophan conjugate (UniPR126) based mixed micelle as a nano carrier for specific delivery of niclosamide to prostate cancer via EphA2 receptor.

Targeted delivery of chemotherapeutic agents is considered a prominent strategy for the treatment of cancer due to its site-specific delivery, augmented penetration, bioavailability, and improved therapeutic efficiency. In the present study, we employed UniPR126 as a carrier in a mixed nanomicellar delivery system to target and deliver anticancer drug NIC specifically to cancer cells via EphA2 receptors as these receptors are overexpressed in cancer cells but not in normal cells. The specificity of the carrier was confirmed from the significant enhancement in the uptake of coumarin-6 loaded mixed nanomicelle by EphA2 highly expressed PC-3 cells compared to EphA2 low expressed H4 cells. Further, niclosamide-loaded lithocholic acid tryptophan conjugate-based mixed nanomicelle has shown significant synergistic cytotoxicity in PC-3 but not in H4 cells. In vivo anticancer efficacy data in PC-3 xenograft revealed a significant reduction in the tumor volume (66.87%) with niclosamide-loaded lithocholic acid tryptophan conjugate nanomicelle, where pure niclosamide showed just half of the activity. Molecular signaling data by western blotting also indicated that niclosamide-loaded lithocholic acid tryptophan conjugate nanomicelle interfered with the EphA2 receptor signaling and inhibition of the Wnt/beta-catenin pathway and resulted in the synergistic anticancer activity compared to niclosamide pure drug.

Hispolon inhibits RANKL induced osteoclast differentiation in vitro.

Hispolon (HISP) is a bioactive compound isolated from Phellinu linteus. It has various pharmacological activities, including antioxidant, anti-inflammatory, and anti-cancer. However, its anti-osteoclastogenic activity has not yet been reported. Hence, in the current study, we have explored the anti-osteoclastogenic activity of HISP and elucidated the molecular mechanisms. HISP inhibited the RANKL induced differentiation of RAW 264.7 cells into osteoclasts in a dose-dependent manner. Mechanistic studies showed that HISP inhibited RANKL-mediated activation of NF-κB and MAPK signaling pathways in osteoclast precursors RAW 264.7 cells. In addition, Hispolon also downregulated the expression of master transcriptional factors essential for osteoclast differentiation, such as NFATc1 and c-FOS. In conclusion, these findings establish molecular mechanisms behind the anti-osteoclastogenic activity of HISP.

Potential role of TrkB agonist in neuronal survival by promoting CREB/BDNF and PI3K/Akt signaling in vitro and in vivo model of 3-nitropropionic acid (3-NP)-induced neuronal death.

Striatal neurons depends on an afferent supply of brain-derived neurotrophic factor-(BDNF) that explicitly interacts with tropomyosin receptor kinase B (TrkB) receptor and performs sundry functions including synaptic plasticity, neuronal differentiation and growth. Therefore, we aimed to scrutinize an active molecule that functions identical to BDNF in activating TrkB receptor and it's downstream targets for restoring neuronal survival in Huntington disease (HD). Data from in vitro Neuro-2a cell line showed that treatment with 7,8-dihydroxyflavone (7,8-DHF), improved 3-nitropropionic acid (3-NP) induced neuronal death by stabilizing the loss of mitochondrial membrane potential and transiently increased the activity of cAMP-response element-binding protein (CREB) and BDNF via TrkB receptor activation. Consistent with in vitro findings, our in vivo results stated that treatment with 7,8-DHF at a dose of 10 mg/kg body weight ameliorated various behavior alterations caused by 3-NP intoxication. Further histopathological and electron microscopy evidences from striatal region of 3-NP mice brain treated with 7,8-DHF showed more improved neurons with intact mitochondria and less autophagic vacuoles. Protein expression analysis of both in vitro and in vivo study showed that 7,8-DHF promotes neuronal survival through upregulation and phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt at serine-473/threonine-308). Akt phosphorylation additionally phosphorylates Bad at serine-136 and inhibits its translocation to mitochondria thereby promoting mitochondrial biogenesis, enhanced ATP production and inhibit apoptosis mediated neuronal death. These aforementioned findings help in strengthening our hypothesis and has come up with a novel neuroprotective mechanism of 7,8-DHF against 3-NP induced neuronal death.

Hesperidin alleviates chronic restraint stress and lipopolysaccharide-induced Hippocampus and Frontal cortex damage in mice: Role of TLR4/NF-κB, p38 MAPK/JNK, Nrf2/ARE signaling.

Stress and lipopolysaccharide (LPS) animal models are used for screening antidepressants and anxiolytic drugs. However, the lacunae for their combination (Restraint stress; RS and LPS) impacting inflammation, apoptosis and antioxidant signaling have not been explored. The present study investigated RS + LPS-induced neurobehavioral and neurochemical anomalies in hippocampus (HIP) and frontal cortex (FC) of mice. Furthermore, citrus-derived flavanone glycoside (Hesperidin; HSP) neuroprotective ability was also confirmed in this model. Male Balb/c mice were given RS (for 28 days) and LPS (single dose, 0.83 mg/kg, i.p.) on 28th day. RS + LPS challenge caused neurobehavioral deficits in mice as evaluated over elevated plus maze (EPM), open field test (OFT), light-dark box test, tail suspension test (TST), forced swim test (FST), sucrose preference test (SPT). Moreover, RS + LPS caused alteration via enhanced oxido-nitrosative stress, proinflammatory cytokines level (serum, HIP, FC), lower antioxidants (GSH, SOD, CAT), increased IBA-1, GFAP, TLR4/NF-κB, p38MAPK/JNK while decreased Nrf2/BDNF/HO-1 expression in HIP and FC of mice. The 21 days (8-28th day), HSP (50 and 100 mg/kg, p.o.) treatment significantly alleviated the anxiety and depressive-like behavior and reversed neurochemical, histopathological changes. HSP exerted the neuroprotective effect via its anti-inflammatory, anti-apoptotic, antioxidant and neurogenesis potential in treating psychiatric illness alone or associated with other diseases.

Photo-Electro Active Nanocomposite Silk Hydrogel for Spatiotemporal Controlled Release of Chemotherapeutics: An In Vivo Approach toward Suppressing Solid Tumor Growth.

Conventional systemic chemotherapeutic regimens suffer from challenges such as nonspecificity, shorter half-life, clearance of drugs, and dose-limiting toxicity. Localized delivery of chemotherapeutic drugs through noninvasive spatiotemporally controllable stimuli-responsive drug delivery systems could overcome these drawbacks while utilizing drugs approved for cancer treatment. In this regard, we developed photoelectro active nanocomposite silk-based drug delivery systems (DDS) exhibiting on-demand drug release in vivo. A functionally modified single-walled carbon nanotube loaded with doxorubicin (DOX) was embedded within a cross-linker free silk hydrogel. The resultant nanocomposite silk hydrogel showed electrical field responsiveness and near-infrared (NIR) laser-induced hyperthermal effect. The remote application of these stimuli in tandem or independent manner led to the increased thermal and electrical conductivity of nanocomposite hydrogel, which effectively triggered the intermittent on-demand drug release. In a proof-of-concept in vivo tumor regression study, the nanocomposite hydrogel was administered in a minimally invasive way at the periphery of the tumor by covering most of it. During the 21-day study, drastic tumor regression was recorded upon regular stimulation of nanocomposite hydrogel with simultaneous or individual external application of an electric field and NIR laser. Tumor cell death marker expression analysis uncovered the induction of apoptosis in tumor cells leading to its shrinkage. Heart ultrasound and histology revealed no cardiotoxicity associated with localized DOX treatment. To our knowledge, this is also the first report to show the simultaneous application of electric field and NIR laser in vivo for localized tumor therapy, and our results suggested that such strategy might have high clinical translational potential.

Nyctanthes arbor-tristis leaf extract ameliorates hyperlipidemia- and hyperglycemia-associated nephrotoxicity by improving anti-oxidant and anti-inflammatory status in high-fat diet-streptozotocin-induced diabetic rats.

Type 2 diabetes is a multifactorial disorder coupled with impaired glucose tolerance, diminished insulin sensitivity and hyperlipidemia. Incessant hyperglycemia and hyperlipidemia led a towering risk to develop cardiovascular hitches with end-stage renal failure. Leaves of Nyctanthes arbor-tristis L. (NAT) (family: Oleaceae) is traditionally used by tribes of Assam for various ailments without proper scientific validation and appropriate mechanism of action for its activity. Hence, we aimed to evaluate the mechanism involved in the hypoglycemic and hypolipidemic effects of NAT leaves in high-fat diet (HFD)-streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley rats were fed with in-house prepared high-fat diet (HFD) for a period of 4 weeks to create insulin resistance. Streptozotocin was injected intraperitoneally to these rats to cause ß-cell destructions to create a model of type 2 diabetes. Our results have shown that NAT extract has a dose-dependent hypoglycemic and hypolipidemic activity in controlling the early biochemical parameters of kidney and lipids. Moreover, the extract has anti-oxidant and anti-inflammatory activities which were more pronounced at a dose of 400 mg/kg body weight. NAT treatment group also restored the normal architecture of the kidney and aorta tissue. GC-MS data analysis revealed the presence of several active compounds which are directly or indirectly responsible for its anti-diabetic and anti-hyperlipidemic activity. The apparent mechanism of NAT for its nephroprotection may be due to the suppression of hyperglycemia-mediated oxidative stress and amelioration of inflammatory cascades allied with NF-kB activation.