Development of novel mucoadhesive gels containing nanoparticle for buccal administration of dexamethasone

Abstract This study aimed to develop promising and innovative mucoadhesive gel systems containing dexamethasone-loaded nanoparticle to increase the effectiveness of treatment for oral precancerous lesions and to reduce side effects. In this respect, a dexamethasone-loaded nanoparticle formulation was prepared by using emulsification/solvent evaporation method. The nanoparticle has high zeta potential (-10.3±0.5 mV), low particle size (218.42±2.1), low polydispersity index (0.070±0.014) and high encapsulation efficiency (95.018±2.982%). To improve the mucosal retention time, the dexamethasone-loaded nanoparticle was dispersed in mucoadhesive gel using gellan gum. The developed gels offered appropriate pH value, high drug content, suitable mechanical and mucoadhesive performance and appropriate viscosity for mucosal administration. All formulations exhibited plastic flow and typical gel-type mechanical spectra after the determined frequency value. The developed formulations exhibited extended drug release as intended for these systems. Cytotoxicity was tested by MTT assay in human epithelioid carcinoma cell (HeLa) in vitro. The MTT assay showed that the blank formulations were non-toxic to cells. It was observed that the bioactivity of the free dexamethasone was potentiated by mucoadhesive gels containing dexamethasone-loaded nanoparticle in HeLa cells. Results from this study indicate that mucoadhesive gels are effective for the local treatment of precancerous lesions. Our findings showed that the developed formulations were worthy of further studies.

Clinical evidence of an interferon-glucocorticoid therapeutic synergy in COVID-19.

Synthetic glucocorticoid dexamethasone is the first trial-proven drug that reduces COVID-19 mortality by suppressing immune system. In contrast, interferons are a crucial component of host antiviral immunity and can be directly suppressed by glucocorticoids. To investigate whether therapeutic interferons can compensate glucocorticoids-induced loss of antiviral immunity, we retrospectively analyzed a cohort of 387 PCR-confirmed COVID-19 patients with quasi-random exposure to interferons and conditional exposure to glucocorticoids. Among patients receiving glucocorticoids, early interferon therapy was associated with earlier hospital discharge (adjusted HR 1.68, 95% CI 1.19-2.37) and symptom relief (adjusted HR 1.48, 95% CI 1.06-2.08), while these associations were insignificant among glucocorticoids nonusers. Early interferon therapy was also associated with lower prevalence of prolonged viral shedding (adjusted OR 0.24, 95% CI 0.10-0.57) only among glucocorticoids users. Additionally, these associations were glucocorticoid cumulative dose- and timing-dependent. These findings reveal potential therapeutic synergy between interferons and glucocorticoids in COVID-19 that warrants further investigation.

C­Myc inhibitor 10058­F4 increases the efficacy of dexamethasone on acute lymphoblastic leukaemia cells.

The long­term survival rate in paediatric acute lymphoblastic leukaemia (ALL) exceeds 80%; however, the outcome of adult ALL remains to be poor. Glucocorticoids (GCs) are the preferred drugs in the traditional treatment of ALL patients. In the anti­leukaemia molecular mechanisms of GCs, c­Myc inhibition serves a critical role. In the present study, a c­Myc inhibitor that increased the sensitivity to GCs in NALM6 cells of the B­cell­ALL cell line and CEM cells of the T­cell­ALL cell line was investigated. The data demonstrated that 10058­F4, a c­Myc inhibitor, increased the growth inhibition, G0/G1 phase arrest and apoptosis of the NALM6 and CEM cells as induced by dexamethasone (DXM), a type of GC. Additionally, 10058­F4 reinforced the decreased expressions of c­Myc, cyclin­dependent kinase (CDK)­4 and CDK6 in the NALM6 and CEM cells treated with DXM. These findings indicated that DXM in combination with the c­Myc inhibitor 10058­F4 may be a novel, potent therapeutic strategy for the treatment of ALL.

Early postnatal treatment with soluble epoxide hydrolase inhibitor or 15-deoxy-Δ(12,14)-prostagandin J2 prevents prenatal dexamethasone and postnatal high saturated fat diet induced programmed hypertension in adult rat offspring.

Prenatal dexamethasone (DEX) exposure, postnatal high-fat (HF) intake, and arachidonic acid pathway are closely related to hypertension. We tested whether a soluble epoxide hydrolase (SEH) inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) or 15-deoxy-Δ(12,14)-prostagandin J2 (15dPGJ2) therapy can rescue programmed hypertension in the DEX+HF two-hit model. Four groups of Sprague Dawley rats were studied: control, DEX+HF, AUDA, and 15dPGJ2. Dexamethasone (0.1mg/kg body weight) was intraperitoneally administered to pregnant rats from gestational day 16-22. Male offspring received high-fat diet (D12331, Research Diets) from weaning to 4 months of age. In AUDA group, mother rats received 25mg/L in drinking water during lactation. In the 15dPGJ2 group, male offspring received 15dPGJ2 1.5mg/kg BW by subcutaneous injection once daily for 1 week after birth. We found postnatal HF diet aggravated prenatal DEX-induced programmed hypertension, which was similarly prevented by early treatment with AUDA or 15dPGJ2. The beneficial effects of AUDA and 15d-PGJ2 therapy include inhibition of SEH, increases of renal angiotensin converting enzyme-2 (ACE2) and angiotensin II type 2 receptor (AT2R) protein levels, and restoration of nitric oxide bioavailability. Better understanding of the impact of arachidonic acid pathway in the two-hit model will help prevent programmed hypertension in children exposed to corticosteroids and postnatal HF intake.

Lipid raft- and protein kinase C-mediated synergism between glucocorticoid- and gonadotropin-releasing hormone signaling results in decreased cell proliferation.

Cross-talk between the glucocorticoid receptor (GR) and other receptors is emerging as a mechanism for fine-tuning cellular responses. We have previously shown that gonadotropin-releasing hormone (GnRH) ligand-independently activates the GR and synergistically modulates glucocorticoid-induced transcription of an endogenous gene in LßT2 pituitary gonadotrope precursor cells. Here, we investigated GR and GnRH receptor (GnRHR) cross-talk that involves co-localization with lipid rafts in LßT2 cells. We report that the GnRHR and a small population of the GR co-localize with the lipid raft protein flotillin-1 (Flot-1) at the plasma membrane and that the GR is present in a complex with Flot-1, independent of the presence of ligands. We found that the SGK-1 gene is up-regulated by Dex and GnRH alone, whereas a combination of both ligands resulted in a synergistic increase in SGK-1 mRNA levels. Using siRNA-mediated knockdown and antagonist strategies, we show that the gene-specific synergistic transcriptional response requires the GR, GnRHR, and Flot-1 as well as the protein kinase C pathway. Interestingly, although several GR cofactors are differentially recruited to the SGK-1 promoter in the presence of Dex and GnRH, GR levels remain unchanged compared with Dex treatment alone, suggesting that lipid raft association of the GR has a role in enhancing its transcriptional output in the nucleus. Finally, we show that Dex plus GnRH synergistically inhibit cell proliferation in a manner dependent on SGK-1 and Flot-1. Collectively the results support a mechanism whereby GR and GnRHR cross-talk within Flot-1-containing lipid rafts modulates cell proliferation via PKC activation and SGK-1 up-regulation.

Decreased hydrocortisone sensitivity of T cell function in multiple sclerosis-associated major depression.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS with a high prevalence of depression. Both MS and depression have been linked to elevated cortisol levels and inflammation, indicating disturbed endocrine-immune regulation. An imbalance in mineralocorticoid versus glucocorticoid signaling in the CNS has been proposed as a pathogenetic mechanism of depression. Intriguingly, both receptors are also expressed in lymphocytes, but their role for 'escape' of the immune system from endocrine control is unknown. Using steroid sensitivity of T cell function as a read-out system, we here investigate a potential role of mineralocorticoid receptor (MR) versus glucocorticoid receptor (GR) regulation in the immune system as a biological mechanism underlying MS-associated major depression. Twelve female MS patients meeting diagnostic criteria for current major depressive disorder (MDD) were compared to twelve carefully matched MS patients without depression. We performed lymphocyte phenotyping by flow cytometry. In addition, steroid sensitivity of T cell proliferation was tested using hydrocortisone as well as MR (aldosterone) and GR (dexamethasone) agonists. Sensitivity to hydrocortisone was decreased in T cells from depressed MS patients. Experiments with agonists suggested disturbed MR regulation, but intact GR function. Importantly, there were no differences in lymphocyte composition and frequency of T cell subsets, indicating that the differences in steroid sensitivity are unlikely to be secondary to shifts in the immune compartment. To our knowledge, this study provides first evidence for altered steroid sensitivity of T cells from MS patients with comorbid MDD possibly due to MR dysregulation.

Dexamethasone enhances 1alpha,25-dihydroxyvitamin D3 effects by increasing vitamin D receptor transcription.

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner.

[Influence of polyoxidonium on IL-1 beta, TNF-alpha and IL-6 production by mononuclears and monocytes under the dexamethasone effect].

It was established that in a mononuclear fraction polyoxidonium inhibited the TNF-alpha production and stimulated the IL-1 beta and IL-6 production. In LPS-induced mononuclear cultures polyoxidonium inhibited the IL-6 production and had no statistically significant effect on the synthesis of TNF-alpha and IL-1 beta. Polyoxidonium had no effect on TNF-alpha, IL-6 and IL-1 beta production by purified monocytes. The addition of polyoxidonium with dexamethasone to the cultures only in monocyte fraction enhanced the IL-1 beta production as compared with the effect of dexamethasone alone. Data obtained allow suggesting that under certain conditions polyoxidonium could alleviate pronounced suppressive influence of glucocorticoids on a secretory activity of effectors of innate immunity.

Bisphenol-A can bind to human glucocorticoid receptor as an agonist: an in silico study.

Bisphenol-A (BPA) is a primary monomer in polycarbonate plastics and epoxy resins. BPA may be released into the environment following its formation via hydrolysis of ester bonds of the polymers. It has been detected in human plasma, placenta, amniotic fluid, amniotic chord, urine and saliva. BPA disrupts normal cell function by acting as an estrogen agonist as well as an androgen antagonist. The present study was carried out to investigate whether BPA can bind to human glucocorticoid receptor (GR) and elucidate its mode of interaction. BPA has been successfully docked in silico into the ligand binding site of GR using the program Discovery Studio 2.0. The structure has been compared with other agonist and antagonist bound structures of GR. It is found that the mode of interactions and binding energy of BPA were similar to that of DEXA and cortisol, two known agonists of GR. This reveals that BPA can bind to GR as an agonist. Hence, BPA may produce biological effects similar to that produced by glucocorticoids.

Conformational changes of the glucocorticoid receptor ligand binding domain induced by ligand and cofactor binding, and the location of cofactor binding sites determined by hydrogen/deuterium exchange mass spectrometry.

HXMS (hydrogen/deuterium exchange mass spectrometry) of the glucocorticoid receptor ligand-binding domain (GR LBD) complexed with the agonist dexamethasone and the antagonist RU-486 is described. Variations in the rates of exchange were observed in regions consistent with the published crystal structures of GR LBD complexed with RU-486 when compared with the GR dexamethasone complex. We also report the HXMS results for agonist-bound GR LBD with the coactivator transcriptional intermediary factor 2 (TIF2) and anatagonist-bound GR LBD with nuclear receptor corepressor (NCoR). Alterations in exchange rates observed for agonist-bound GR LBD with TIF2 present were consistent with the published crystal structural contacts for the complex. Alterations in exchange rates observed for antagonist-bound GR LBD with NCoR were a subset of those observed with TIF2 binding, suggesting a common or overlapping binding site for coactivator and corepressor.

Effects of an HMG-CoA reductase inhibitor, simvastatin, on human myeloma cells.

To look for new candidates for agents to use in maintenance therapy for myeloma patients, the growth inhibitory effects of a 3-hydroxy-3-mehtylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin), simvastatin, was analyzed using human myeloma cell lines. Several investigations have indicated growth reduction in certain lineages of cancer cells including one report on myeloma, and inhibitory effects of statins on GTPases and involving MAP-kinases. Most (12 out of 13) myeloma lines examined showed growth inhibition when cultured with various concentrations (1-30 microM) of simvastatin in a dose-dependent manner. Simvastatin in combination with other biological response modifiers such as ATRA or DEX had additional effects on growth. In addition, anti-oxides prevented the simvastatin-induced growth inhibition and apoptosis. Furthermore, myeloma cells treated with simvastatin clearly showed inactivation of various MAP-kinase pathways such as ERK1/2, MEK1/2, JNK, and p38. Based on these findings, statins may be suitable for clinical usage in maintenance therapy for myeloma patients.

IFN-beta1b augments glucocorticoid-induced suppression of tumor necrosis factor-alpha production by increasing the number of glucocorticoid receptors on a human monocytic cell line.

We studied the effect of recombinant interferon-beta1b (IFN-beta1b) on the sensitivity to glucocorticoids (GC) and on the number of GC receptors (GCR) in the human monocytic cell line THP-1. We found that IFN-beta1b augments the suppressive effect that dexamethasone has on the stimulated production of tumor necrosis factor-alpha (TNF-alpha), most likely related to the increased number of GCR observed after exposure to IFN-beta1b. This provides a possible clue to the mechanism of action of IFN-beta in multiple sclerosis.