Facile Rebridging Conjugation Approach to Attain Monoclonal Antibody-Targeted Nanoparticles with Enhanced Antigen Binding and Payload Delivery.

Adopting conventional conjugation approaches to construct antibody-targeted nanoparticles (NPs) has demonstrated suboptimal control over the binding orientation and the structural stability of monoclonal antibodies (mAbs). Hitherto, the developed antibody-targeted NPs have shown proof of concept but lack product homogeneity, batch-to-batch reproducibility, and stability, precluding their advancement toward the clinic. To circumvent these limitations and advance toward clinical application, herein, a refined approach based on site-specific construction of mAb-immobilized NPs will be appraised. Initially, the conjugation of atezolizumab (anti-PDL1 antibody, Amab) with polymeric NPs was developed using bis-haloacetamide (BisHalide) rebridging chemistry, followed by click chemistry (NP-Fab BisHalide Ab and NP-Fc BisHalide Ab). For comparison purposes, mAb-immobilized NPs developed utilizing conventional conjugation methods, namely, N-hydroxysuccinimide (NHS) coupling and maleimide chemistry (NP-NHS Ab and NP-Mal Ab), were included. Next, flow cytometry and confocal microscopy experiments evaluated the actively targeted NPs (loaded with fluorescent dye) for cellular binding and uptake. Our results demonstrated the superior and selective binding and uptake of NP-Fab BisHalide Ab and NP-Fc BisHalide Ab into EMT6 cells by 19-fold and 13-fold, respectively. To evaluate the PDL1-dependent cell uptake and the selectivity of the treatments, a blocking step of the PDL1 receptor with Amab was performed prior to incubation with NP-Fab BisHalide Ab and NP-Fc BisHalide Ab. To our delight, the binding and uptake of fluorescent NPs were reduced significantly by 3-fold for NP-Fab BisHalide Ab, demonstrating the PDL1-mediated uptake. Moreover, NP-Fab BisHalide Ab and NP-Fc BisHalide Ab were entrapped with the paclitaxel payload, and their cytotoxicity was evaluated. They showed significant enhancements compared to free paclitaxel and NP-NHS Ab. Overall, this work will provide a facile conjugation method that could be implemented to actively target NPs with a plethora of therapeutic mAbs approved for various malignancies.

Encapsulation of gingerol into nanoliposomes: Evaluation of in vitro anti-inflammatory and anti-cancer activity.

Nanoliposomes (NLs) are ideal carriers for delivering complex molecules and phytochemical products, but ginger by-products, despite their therapeutic benefits, have poor bioavailability due to their low water solubility and stability. Crude ginger extracts (CGEs) and 6-gingerol were individually encapsulated within NLs for in vitro activity assessment. In vitro evaluation of anti-proliferative and anti-inflammatory properties of encapsulated 6-gingerol and CGE was performed on healthy human periodontal ligament (PDL) fibroblasts and MDA-MB-231 breast cancer cells. Encapsulation efficiency and loading capacity of 6-gingerol reached 25.23% and 2.5%, respectively. NLs were found stable for up to 30 days at 4°C with a gradual load loss of up to 20%. In vitro cytotoxic effect of encapsulated 6-gingerol exceeded 70% in the MDA-MB-231 cell line, in a comparable manner with non-encapsulated 6-gingerol and CGE. The effect of CGE with an IC50 of 3.11 ± 0.39, 7.14 ± 0.80, and 0.82 ± 0.55 µM and encapsulated 6-gingerol on inhibiting IL-8 was evident, indicating its potential anti-inflammatory activity. Encapsulating 6-gingerol within NLs enhanced its stability and facilitated its biological activity. All compounds, including vitamin C, were equivalent at concentrations below 2 mg/mL, with a slight difference in antioxidant activity. The concentrations capable of inhibiting 50% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) substrate were comparable.

The Effect of Inflammatory Bowel Disease and Irritable Bowel Syndrome on Pravastatin Oral Bioavailability: In vivo and in silico evaluation using bottom-up wbPBPK modeling.

The common disorders irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) can modify the drugs' pharmacokinetics via their induced pathophysiological changes. This work aimed to investigate the impact of these two diseases on pravastatin oral bioavailability. Rat models for IBS and IBD were used to experimentally test the effects of IBS and IBD on pravastatin pharmacokinetics. Then, the observations made in rats were extrapolated to humans using a mechanistic whole-body physiologically-based pharmacokinetic (wbPBPK) model. The rat in vivo studies done herein showed that IBS and IBD decreased serum albumin (> 11% for both), decreased PRV binding in plasma, and increased pravastatin absolute oral bioavailability (0.17 and 0.53 compared to 0.01) which increased plasma, muscle, and liver exposure. However, the wbPBPK model predicted muscle concentration was much lower than the pravastatin toxicity thresholds for myotoxicity and rhabdomyolysis. Overall, IBS and IBD can significantly increase pravastatin oral bioavailability which can be due to a combination of increased pravastatin intestinal permeability and decreased pravastatin gastric degradation resulting in higher exposure. This is the first study in the literature investigating the effects of IBS and IBD on pravastatin pharmacokinetics. The high interpatient variability in pravastatin concentrations as induced by IBD and IBS can be reduced by oral administration of pravastatin using enteric-coated tablets. Such disease (IBS and IBD)-drug interaction can have more drastic consequences for narrow therapeutic index drugs prone to gastric degradation, especially for drugs with low intestinal permeability.

Potential biomarkers and metabolomics of acetaminophen-induced liver injury during alcohol consumption: A preclinical investigation on C57/BL6 mice.

The toxic effects of alcohol consumption on population health are significant worldwide and the synergistic toxic effects of concurrent intake of Acetaminophen and alcohol is of clinical concern. The understanding of molecular mechanisms beneath such synergism and acute toxicity may be enhanced through assessing underlying metabolomics changes. The molecular toxic activities of the model hereby, is assessed though metabolomics profile with a view to identifying metabolomics targets which could aid in the management of drug-alcohol interactions. In vivo exposure of C57/BL6 mice to APAP (70 mg/kg), single dose of ethanol (6 g/kg of 40%) and APAP after alcohol consumption was employed. Plasma samples were prepared and subjected to biphasic extraction for complete LC-MS profiling, and tandem mass MS2 analysis. Among the detected ions, 174 ions had significant (VIP scores >1 and FDR <0.05) changes between groups and were selected as potential biomarkers and significant variables. The presented metabolomics approach highlighted several affected metabolic pathways, including nucleotide and amino acid metabolism; aminoacyl-tRNA biosynthesis as well as bioenergetics of TCA and Krebs cycle. The impact of APAP on the concurrent administration of alcohol showed great biological interactions in the vital ATP and amino acid producing processes. The metabolomics changes show distinct metabolites which are altered to alcohol-APAP consumption while presenting several unneglectable risks on the vitality of metabolites and cellular molecules which shall be concerned.

Transcriptional profiling of drug-induced liver injury biomarkers: association of hepatic Srebf1/Pparα signaling and crosstalk of thrombin, alcohol dehydrogenase, MDR and DNA damage regulators.

Cell stress transcribing genes provide a diverse platform of molecular mediators that vary in response to toxicity. Common drug-induced liver injury (DILI) biomarkers are usually expressed in mild toxicity and limited to confirming it rather than categorizing its intensity. Thus, new parametric biomarkers are needed to be explored. Classifying the toxicological response based on the dose-level and severity of stimuli will aid in the evaluation and approach against drug exposure. The present research explored the involvement of gene expression of potential biomarkers as a severity-specific hallmark in different acetaminophen (APAP)-induced hepatotoxicity levels in C57BL/6 mice. The differentially expressed genes were annotated and analyzed using bioinformatics tools to predict canonical pathways altered by DILI. The results revealed alteration in genes encoding for antioxidant enhancement; Slc7a11, bile efflux; MDR4, fatty acid metabolism and transcriptional factors namely Srebf1 and Pparα. Potential APAP toxicity biomarkers included Adh1 and thrombin, and other DNA damage and stress chaperones which were changed at least fourfold between control and the three tested severity models. The current investigation demonstrates a dose-mediated association of several hallmark genes in APAP-induced liver damage and addressed the involvement of uncommonly studied molecular responses. Such biomarkers can be further developed into predictive models, translated for risk assessment against drug exposure and guide in building theragnostic targets.

Deep Eutectic Liquids as a Topical Vehicle for Tadalafil: Characterisation and Potential Wound Healing and Antimicrobial Activity.

Deep eutectic solvents (DESs) and ionic liquids (ILs) offer novel opportunities for several pharmaceutical applications. Their tunable properties offer control over their design and applications. Choline chloride (CC)-based DESs (referred to as Type III eutectics) offer superior advantages for various pharmaceutical and therapeutic applications. Here, CC-based DESs of tadalafil (TDF), a selective phosphodiesterase type 5 (PDE-5) enzyme inhibitor, were designed for implementation in wound healing. The adopted approach provides formulations for the topical application of TDF, hence avoiding systemic exposure. To this end, the DESs were chosen based on their suitability for topical application. Then, DES formulations of TDF were prepared, yielding a tremendous increase in the equilibrium solubility of TDF. Lidocaine (LDC) was included in the formulation with TDF to provide a local anaesthetic effect, forming F01. The addition of propylene glycol (PG) to the formulation was attempted to reduce the viscosity, forming F02. The formulations were fully characterised using NMR, FTIR and DCS techniques. According to the obtained characterisation results, the drugs were soluble in the DES with no detectable degradation. Our results demonstrated the utility of F01 in wound healing in vivo using cut wound and burn wound models. Significant retraction of the cut wound area was observed within three weeks of the application of F01 when compared with DES. Furthermore, the utilisation of F01 resulted in less scarring of the burn wounds than any other group including the positive control, thus rendering it a candidate formula for burn dressing formulations. We demonstrated that the slower healing process associated with F01 resulted in less scarring potential. Lastly, the antimicrobial activity of the DES formulations was demonstrated against a panel of fungi and bacterial strains, thus providing a unique wound healing process via simultaneous prevention of wound infection. In conclusion, this work presents the design and application of a topical vehicle for TDF with novel biomedical applications.

The Formulation and Evaluation of Deep Eutectic Vehicles for the Topical Delivery of Azelaic Acid for Acne Treatment.

The current work was aimed at the development of a topical drug delivery system for azelaic acid (AzA) for acne treatment. The systems tested for this purpose were deep eutectic systems (DESs) prepared from choline chloride (CC), malonic acid (MA), and PEG 400. Three CC to MA and eight different MA: CC: PEG400 ratios were tested. The physical appearance of the tested formulations ranged from solid and liquid to semisolid. Only those that showed liquid formulations of suitable viscosity were considered for further investigations. A eutectic mixture made from MA: CC: PEG400 1:1:6 (MCP 116) showed the best characteristics in terms of viscosity, contact angle, spreadability, partition coefficient, and in vitro diffusion. Moreover, the MCP116 showed close rheological properties to the commercially available market lead acne treatment product (Skinorin®). In addition, the formula showed synergistic antibacterial activity between the MA moiety of the DES and the AzA. In vitro diffusion studies using polyamide membranes demonstrated superior diffusion of MCP116 over the pure drug and the commercial product. No signs of skin irritation and edema were observed when MCP116 was applied to rabbit skin. Additionally, the MCP116 was found to be, physically and chemically, highly stable at 4, 25, and 40 °C for a one-month stability study.

Nrf2/ARE axis signalling in hepatocyte cellular death.

The Nrf2-ARE transcriptional pathway plays an important role amongst cellular defence systems regulating and ensuring adequacy of redox responses and oxidant signalling factors. Hepatocyte cellular death and injury is a prominent feature underlying liver pathologies. Diverse endogenous molecules and targets contribute to the outcome of cell survival and the consequent mode of cell death. Several research efforts focused on the confirmation of Nrf2 presence in cell death and its vital necessity against cell compromise, however, little they comprehend of such participation. Hepatocyte cell death modes discussed in this review including autophagy, apoptosis, necrosis, ferroptosis, pyroptosis, fibrosis and others, vary in response of the stimuli burdened. The current review presents a handful of highlights and crosstalk involved in the communication of Nrf2 signalling network with the "up to date" reported hepatocyte cell death modes and their underling mechanisms, and addressing key cellular networks of hepatocyte fate, through a perspective of Nrf2 as a critical transcriptional factor. Collectively, labelling the cross-transduction of Nrf2-ARE axis with key cell execution pathways could provide insights to therapeutic interventions and better research outcomes.

HPLC with Fluorescence and Photodiode Array Detection for Quantifying Capmatinib in Biological Samples: Application to In Vivo and In Vitro Studies.

Capmatinib, a recently approved tyrosine kinase inhibitor, is used for the treatment of non-small cell lung cancer. We describe two new HPLC methods for capmatinib quantification in vivo and in vitro. HPLC with a fluorescence detection method was used to quantify capmatinib in plasma for the first time. The method was successfully applied in a pharmacokinetic study following a 10 mg/kg oral dose of capmatinib given to rats. The chromatographic separation was performed using a Eurospher II 100-3 C18H (50 × 4 mm, 3 µm) column and a mobile phase containing 10 mM of ammonium acetate buffer (pH 5.5): acetonitrile (70:30, v/v), at a flow rate of 2.0 mL min-1. The study also describes the use of HPLC-PDA for the first time for the determination of capmatinib in human liver microsomes and describes its application to study its metabolic stability in vitro. Our results were in agreement with those reported using LC-MS/MS, demonstrating the reliability of the method. The study utilized a Gemini-NX C18 column and a mobile phase containing methanol: 20 mM ammonium formate buffer pH 3.5 (53:47, v/v), delivered at a flow rate of 1.1 mL min-1. These methods are suitable for supporting pharmacokinetic studies, particularly in bioanalytical labs lacking LC-MS/MS capabilities.

Chitosan-biotin topical film: preparation and evaluation of burn wound healing activity.

A new composite film from chitosan (CS) and biotin (BIO) was developed to enhance burn wound healing. The film was prepared by electrostatic interaction between CS and BIO. Four different ratios of CS to BIO v/v (4:1, 3:2, 2:3, and 1:4) were prepared. The films were comprehensively characterized using FTIR, DSC, and AFM. The in-vitro release studies showed that the most promising formula with the highest release behavior was CS to BIO 1: 4. The ex vivo adhesion times were reported as 0.50 ± 0.30 min for CS film compared to 6.2 ± 0.30, 8.4 ± 0.40, 11.2 ± 0.50, and 13.83 ± 1.04 min for CS to BIO films v/v (1:4, 2:3, 3:2 and 4:1), respectively. Most importantly, the skin healing activities of CS/BIO film in the excision wound model in mice and skin burn model in rats showed faster rates of healing compared to CS and placebo. Furthermore, skin stretching and burn wound contraction behavior treated with CS/BIO were higher than that of CS treated skin. In conclusion, the results obtained revealed that CS/BIO films possessed superior burn wound healing activity compared to CS.

Changes in public knowledge and perceptions about antibiotic use and resistance in Jordan: a cross-sectional eight-year comparative study.

BACKGROUND: Resistance to antibiotics is a growing problem, worldwide and particularly in developing countries like Jordan. Raising public awareness on appropriate antibiotic use is crucial to combat this problem. The current study describes the change in public Knowledge and attitudes towards the use of antibiotics over a period of 8 years. METHODS: Two cross-sectional studies were performed 8 years apart on Jordanians of different age groups, and social settings, residing in Amman, Jordan. Convenience non-probability sampling techniques were used. In 2010, a questionnaire was distributed in paper form, whereas in 2018 snowball sampling was used to disseminate an identical electronic questionnaire. Chi-square test and post hoc analysis were done using the z-test to compare column proportions, adjustment for multiple testing using the Bonferroni method. Multiple logistic regression was used to adjust for case mix for each survey. Comparisons were made across the two studies and within each study. RESULTS: A total of 711 participants in 2010 and 436 participants in 2018 were surveyed. Over the 8-year period, there was a significant improvement in the beliefs regarding the use of antibiotics such as disagreeing to keeping left over antibiotics for later use from 57 to 70% (p < 0.05) and disagreeing to buying antibiotics without physicians' consent increased from 80 to 89% (P value < 0.001). There was no significant change in the beliefs that support self-medication such as: using antibiotics from a friend (72 to 77%) buying antibiotics without a prescription (42 to 45%), and getting information about medication use from leaflet without referring to a health care professional (60 to 63%). There were some areas of confusion regarding antibiotic range of effectiveness, and origin of resistance. Agreement about antibiotic resistance being a problem in Jordan increased significantly from 44 to 60% (p < 0.001). In addition, there was a significant increase in the percentage of participants who said that they don't request antibiotics from physicians (56 to 75% (P ≤ 0.001) and who said they would trust physicians' decisions about the necessity of antibiotics (70 to 83% P < 0.05). CONCLUSION: Findings indicate the need for better suited, and more inclusive, public educational campaigns.

Chemical induction of hepatic apoptosis in rodents.

The urge of identifying new pharmacological interventions to prevent or attenuate liver injury is of critical importance and needs an expanded experimental toolbox. Hepatocyte injury and cellular death is a prominent feature behind the pathology of liver diseases. Several research activities focused on identifying chemicals and hepatotoxicants that induce cell death by apoptosis, in addition to presenting its corresponding signaling pathway. Although such efforts provided further understanding of the mechanisms of cell death, it has also raised confusion concerning identifying the involvement of several modes of cell death including apoptosis, necrosis and fibrosis. The current review highlights the ability of several chemicals and potential hepatotoxicants to induce liver damage in rodents by means of apoptosis while the probable involvement of other modes of cell death is also exposed. Thus, several chemical substances including hepatotoxins, mycotoxins, hyperglycemia inducers, metallic nanoparticles and immunosuppressant drugs are reviewed to explore the hepatic cytotoxic spectrum they could exert on hepatocytes of rodents. In addition, the current review address the mechanism by which hepatotoxicity is initiated in hepatocytes in different rodents aiding the researcher in choosing the right animal model for a better research outcome.

Critical pharmacokinetic and pharmacodynamic drug-herb interactions in rats between warfarin and pomegranate peel or guava leaves extracts.

BACKGROUND: In-depth information of potential drug-herb interactions between warfarin and herbal compounds with suspected anticoagulant blood thinning effects is needed to raise caution of concomitant administration. The current study aimed to investigate the impact of co-administration of pomegranate peel and guava leaves extracts, including their quality markers namely; ellagic acid and quercetin, respectively, on warfarin's in vivo dynamic activity and pharmacokinetic actions, in addition to potential in vitro cytochrome P450 enzymes (CYP) inhibition. METHODS: Influence of mentioned extracts and their key constituents on warfarin pharmacodynamic and kinetic actions and CYP activity were evaluated. The pharmacodynamic interactions were studied in Sprague Dawley rats through prothrombin time (PT) and International Normalized Ratio (INR) measurements, while pharmacokinetic interactions were detected in vivo using a validated HPLC method. Furthermore, potential involvement in CYP inhibition was also investigated in vitro on isolated primary rat hepatocytes. RESULTS: Preparations of pomegranate peel guava leaf extract, ellagic acid and quercetin in combination with warfarin were found to exert further significant increase on PT and INR values (p < 0.01) than when used alone (p < 0.05). Pomegranate peel extract showed insignificant effects on warfarin pharmacokinetics (p > 0.05), however, its constituent, namely, ellagic acid significantly increased warfarin Cmax (p < 0.05). Guava leaves extract and quercetin resulted in significant increase in warfarin Cmax when compared to control (p < 0.01). Furthermore, guava leaves extract showed a significant effect on changing the AUC, CL and Vz. Significant reduction in CYP2C8, 2C9, and 3A4 was seen upon concomitant use of warfarin with ellagic acid, guava leaves and quercetin, unlike pomegranate that insignificantly affected CYP activities. CONCLUSION: All combinations enhanced the anticoagulant activity of warfarin as the results of in vivo and in vitro studies were consistent. The current investigation confirmed serious drug herb interactions between warfarin and pomegranate peel or guava leaf extracts. Such results might conclude a high risk of bleeding from the co-administration of the investigated herbal drugs with warfarin therapy. In addition, the results raise attention to the blood-thinning effects of pomegranate peel and guava leaves when used alone.

Hepatoprotective Actions of Ascorbic Acid, Alpha Lipoic Acid and Silymarin or Their Combination Against Acetaminophen-Induced Hepatotoxicity in Rats.

Background and objectives: Ascorbic acid, alpha lipoic acid (ALA) and silymarin are well-known antioxidants that have hepatoprotective effects. This study aims to investigate the effects of these three compounds combined with attenuating drug-induced oxidative stress and cellular damage, taking acetaminophen (APAP)-induced toxicity in rats as a model both in vivo and in vitro. Materials and Methods: Freshly cultured primary rat hepatocytes were treated with ascorbic acid, ALA, silymarin and their combination, both with and without the addition of APAP to evaluate their in vitro impact on cell proliferation and mitochondrial activity. In vivo study was performed on rats supplemented with the test compounds or their combination for one week followed by two toxic doses of APAP. Results: Selected liver function tests and oxidative stress markers including superoxide dismutase (SOD), malondialdehyde (MDA) and oxidized glutathione (GSSG) were detected. The in vivo results showed that all three pretreatment compounds and their combination prevented elevation of SOD and GSSG serum levels indicating a diminished burden of oxidative stress. Moreover, ascorbic acid, ALA and silymarin in combination reduced serum levels of liver enzymes; however, silymarin markedly maintained levels of all parameters to normal ranges. Silymarin either alone or combined with ascorbic acid and ALA protected cultured rat hepatocytes and increased cellular metabolic activity. The subjected agents were capable of significantly inhibiting the presence of oxidative stress induced by APAP toxicity and the best result for protection was seen with the use of silymarin. Conclusions: The measured liver function tests may suggest an augmented hepatoprotection of the combination preparation than when compared individually.

Low Molecular Weight Chitosan-Insulin Complexes Solubilized in a Mixture of Self-Assembled Labrosol and Plurol Oleaque and Their Glucose Reduction Activity in Rats.

Oral insulin delivery that better mimics physiological pathways is a necessity as it ensures patient comfort and compliance. A system which is based on a vehicle of nano order where positively charged chitosan interacts with negatively charged insulin and forms a polyelectrolyte complex (PEC) solubilizate, which is then solubilized into an oily phase of oleic acid, labrasol, and plurol oleaque-protects insulin against enzymatic gastrointestinal reduction. The use of an anionic fatty acid in the oily phase, such as oleic acid, is thought to allow an interaction with cationic chitosan, hence reducing particle size. Formulations were assessed based on their hypoglycaemic capacities in diabetic rats as compared to conventional subcutaneous dosage forms. 50 IU/kg oral insulin strength could only induce blood glucose reduction equivalent to that of 5 IU/kg (1 International unit = 0.0347 mg of human insulin). Parameters that influence the pharmacological availability were evaluated. A preliminary investigation of the mechanism of absorption suggests the involvement of the lymphatic route.

Effect of waterpipe tobacco smoking on airway inflammation in murine model of asthma.

OBJECTIVE: There has been an increase in the popularity of waterpipe tobacco smoking (WTS) worldwide, especially in the younger population, including asthma patients. In this study, we investigated the effects of waterpipe smoking on airway inflammation, cytokine levels and oxidative stress markers in an antigen-driven murine model of asthma. MATERIALS AND METHODS: Balb/c mice were divided into four groups; (1) control (received fresh air, ovalbumin sensitization and saline challenge), (2) WTS (received WTS, ovalbumin sensitization and saline challenge), (3) Ova S/C (received fresh air, ovalbumin sensitization and ovalbumin challenge) and (4) simultaneous WTS and Ova S/C (received WTS, ovalbumin sensitization and ovalbumin challenge). Airway inflammatory cells were evaluated in the broncho-alveolar lavage fluid. Cytokines [interleukin (IL)-13, 10 and 18] and oxidative stress markers [superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx)] were evaluated in the lung homogenates. RESULTS: Chronic exposure to WTS significantly increased the number of airway inflammatory cells in mice, specifically: eosinophils, neutrophils, macrophages and lymphocytes. The level of IL-13 in the lungs was increased and the level of IL-10 was reduced (p < 0.05) by WTS. Chronic WTS potentiated the increase in inflammatory cells induced by Ova S/C (p < 0.05). The level of IL-13 in the lungs was increased by simultaneous WTS and Ova S/C (p < 0.05) while, levels of IL-10, IL-18, SOD, catalase and GPx in the lungs were not affected. CONCLUSIONS: Chronic WTS exposure induced airway inflammation in control mice and enhanced airway inflammation in murine model of asthma.

Eriobotrya japonica Water Extract Characterization: An Inducer of Interferon-Gamma Production Mainly by the JAK-STAT Pathway.

Eriobotrya japonica (Thunb.) Lindl. (Loquat) (EJ) has been used as a medicinal plant to treat chronic bronchitis, coughs, phlegm, high fever and gastro-enteric disorders. Since the traditional use of EJ is related to modulating inflammation processes, our earlier studies on EJ leaves were performed on the water extract to investigate specific cytokines' modulation. These earlier studies, however, have shown that EJ leaf water extract (WE) and the water phase (WP) induce cytokines' production in in vitro and in vivo models. Therefore, the aim of this study was to specify the group(s) of compounds in EJ leaves that have this immunomodulatory activity and their mechanism of action. WE was obtained from boiling the leaves followed by butanol extraction, yielding a butanol-water phase (WP). WP was then subjected to methanol:acetone fractionation, yielding upper (MAU) and lower (MAL) phases. For further fractionation, MAU was subjected to column chromatography followed by elution with ethanol:water (EW), methanol:ethanol (ME) and, lastly, acetone:water (AW), respectively, to reveal three sub-fractions; MAU-EW, MAU-ME and MAU-AW. MAU-AW significantly increased IFN-γ production from unstimulated and stimulated mouse spleen cells, as well as CD3+ T cells and natural killer cells. Furthermore, the fold increase of IFN-γ production by MAU-AW was concentration dependent, higher than the parent extract or any of the other sub-fractions, and such an IFN-γ increase was reversed by two JAK-STAT inhibitors. In addition, MALDI-TOF-MS analysis of the extracts and sub-fractions showed compounds with molecular weights of >500 Daltons. The MAU-AW sub-fraction contained more polar compounds, such as flavonol and caffeic glycosides. In conclusion, these polar compounds in the EJ extract are responsible for inducing IFN-γ production. Further chemical elucidation is warranted to lead to a specific IFN-γ inducer and an immunomodulator in polarizing immune cells and balancing immune responses in certain diseases.

Influence of molecular weight and degree of deacetylation of low molecular weight chitosan on the bioactivity of oral insulin preparations.

The objective of the present study was to prepare and characterize low molecular weight chitosan (LMWC) with different molecular weight and degrees of deacetylation (DDA) and to optimize their use in oral insulin nano delivery systems. Water in oil nanosized systems containing LMWC-insulin polyelectrolyte complexes were constructed and their ability to reduce blood glucose was assessed in vivo on diabetic rats. Upon acid depolymerization and testing by viscosity method, three molecular weights of LMWC namely, 1.3, 13 and 18 kDa were obtained. As for the DDA, three LMWCs of 55%, 80% and 100% DDA were prepared and characterized by spectroscopic methods for each molecular weight. The obtained LMWCs showed different morphological and in silico patterns. Following complexation of LMWCs with insulin, different aggregation sizes were obtained. Moreover, the in vivo tested formulations showed different activities of blood glucose reduction. The highest glucose reduction was achieved with 1.3 kDa LMWC of 55% DDA. The current study emphasizes the importance of optimizing the molecular weight along with the DDA of the incorporated LMWC in oral insulin delivery preparations in order to ensure the highest performance of such delivery systems.

Protective Effects of a Dihydrodiazepine Against Endotoxin Shock Through Suppression of TLR4/NF-κB/IRF3 Signaling Pathways.

Sepsis and septic shock are life-threatening systemic inflammatory conditions and among the most frequent causes of morbidity and mortality globally. Preclinical evidence has identified a number of diazepine-based compounds with therapeutic potential in inflammatory diseases. However, the potential anti-inflammatory properties of diazepines in the overwhelming immune response during sepsis have been rarely examined. Thus, the current study aimed to identify a new diazepine compound with therapeutic potential in sepsis. Assessing the inflammatory response of macrophages to Lipopolysaccharides (LPS) in vitro identified 2-[7-(trifluoromethyl)-2,3-dihydro-1H-1,4-diazepin-5-yl]phenol (2-TDDP) as a potential anti-inflammatory agent. It reduced secretion of Interleukin-1ß (IL-1ß), IL-6, IL-12p70, IL-18, Tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ), IFN-ß, and increased the secretion of IL-10. In a mouse model of LPS-induced endotoxin shock, 2-TDDP reduced mortality and attenuated inflammation-induced tissue injury in the spleen, liver, kidney, and lung. This was accompanied by reduced serum levels of IL-1ß, IL-6, IL-12p70, TNF-α, IFN-γ, IFN-ß, and increased levels of IL-10. Importantly, 2-TDDP suppressed the Toll-like receptor 4 (TLR4)/Nuclear factor-κB (NF-κB) and TLR4/Interferon regulatory factor 3 (IRF3) signaling pathways through a reduction in the expression of TLR4, Myeloid differentiation primary response 88 (MyD88), P65, and TNF receptor-associated factor 3 (Traf3). Moreover, 2-TDDP suppressed the expression of CD86, Programmed death-ligand 1 (PD-L1) and C5a receptor (C5aR), but not Major histocompatibility complex II (MHCII). Analysis of splenic lymphocyte populations revealed a decrease in the number of CD4+, CD8+, and B cells. Collectively, these findings introduced the dihydrodiazepine 2-TDDP as a new anti-inflammatory agent with potent therapeutic potential in endotoxin shock, paving an avenue for future clinical application.

Structural insights into novel therapeutic deep eutectic systems with capric acid using 1D, 2D NMR and DSC techniques with superior gut permeability.

Therapeutic deep eutectic solvents (THEDSs) are the best exemplification of green alternative formulations of active pharmaceutical ingredients (APIs) that offer superlative properties of APIs. Previously, THEDESs of risperidone, fentanyl and levofloxacin with capric acid (CA) were developed by our group. These APIs share cyclic tertiary amine nuclei. Herein, DESs of two drugs bearing cyclic tertiary amine nucleus, namely, droperidol and aripiprazole, in the presence of CA, were investigated as model drugs. Comprehensive analyses were conducted using liquid-state 1D and 2D NMR and differential scanning calorimetry (DSC) to elucidate the regiochemistry and thermodynamic mechanisms bringing about those THEDESs. Everted gut sac technique was used to study the flux of the developed THEDESs. 1D and 2D NMR techniques analyses revealed the importance of cyclic tertiary amine nuclei in forming interactions with CA. This was confirmed by the downfield shift of the protons proximal to the tertiary amine groups compared to the individual drugs. Diffusion NMR analysis (DOSY) showed a significant reduction in the diffusion coefficient of CA in the mixed system compared with CA in isolation. Thermal analysis of the two drugs revealed that the drugs have a low tendency to recrystallise upon melting but rather vitrify from a melt to form an amorphous solid. Interestingly, the superior absorption and flux of the THEDES formulation of droperidol was demonstrated using the ERIS. Collectively, this work provides a green method to attain liquid formulations of APIs with enhanced pharmacokinetic features.