Protective effect of isosteviol sodium against LPS-induced multiple organ injury by regulating of glycerophospholipid metabolism and reducing macrophage-driven inflammation.

Sepsis is a severe inflammatory disorder that can lead to multiple organ injury. Isosteviol sodium (STV-Na) is a terpenoid derived from stevioside that exerts anti-inflammatory, antioxidant and antiapoptotic activities. However, the influence of STV-Na on sepsis remains unknown. Here, we assessed the potential effects of STV-Na on sepsis and multiple organ injury induced by lipopolysaccharide (LPS). We found that STV-Na increased the survival rate of mice treat with LPS, significantly improved the functions of the heart, lung, liver, and kidney, reduced the production of inflammatory cytokines and decreased macrophage infiltration. Moreover, Multiorgan metabolomics analysis demonstrated that glutathione metabolism, purine metabolism, glycerophospholipid metabolism and pantothenate and CoA biosynthesis, were significantly altered by STV-Na. This study provides novel insights into the metabolite changes of multiple organ injury in septic mice, which may help characterize the underlying mechanism and provide an improved understanding of the therapeutic effects of STV-Na on sepsis.

Enantioselective resolution of Rac-terbutaline and evaluation of optically pure R-terbutaline hydrochloride as an efficient anti-asthmatic drug.

Terbutaline is a ß2 -adrenoceptor agonist for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Among the two isomers of terbutaline (TBT 2), R-isomer was found to be the potent enantiomer in generating therapeutic effect, while S-isomer has been reported to show side effects. In this study, R-terbutaline hydrochloride (R-TBH 6) was synthesized through chiral resolution from the racemic terbutaline sulfate (rac-TBS 1) with 99.9% enantiomeric excess (ee) in good overall yield (53.6%). Further, R-TBH 6 nebulized solution was prepared in half dosage of Bricanyl®, which is a marketed product of racemic terbutaline and evaluated in vitro aerosol performance and in vivo anti-asthmatic effect on guinea pigs via. pulmonary delivery. From the investigation, it is evident that R-TBH 6 nebulized solution of half dosage performed similar fine aerosol characteristics and anti-asthmatic effect with Bricanyl®.

Preparation of a Sustained-Release Nebulized Aerosol of R-terbutaline Hydrochloride Liposome and Evaluation of Its Anti-asthmatic Effects via Pulmonary Delivery in Guinea Pigs.

An aerosolized liposome formulation for the pulmonary delivery of an anti-asthmatic medication was developed. Asthma treatment usually requires frequent administration of medication for a sustained bronchodilator response. Liposomes are known for their sustained drug release capability and thus would be a suitable delivery system for prolonging the therapeutic effect of anti-asthmatic medication. Liposomes prepared by thin film hydration were loaded with a model drug, R-terbutaline hydrochloride(R-TBH), using an ammonium sulfate-induced transmembrane electrochemical gradient. This technique provided an encapsulation efficiency of up to 71.35% and yielded R-TBH liposomes with a particle size of approximately 145 ± 20 nm. According to stability studies, these R-TBH liposomes should be stored at 4°C before usage. Compared to R-TBH solution, which showed 90.84% release within 8 h, liposomal R-TBH had a cumulative release of 73.53% at 37°C over 192 h. A next generation impactor (NGI) was used to analyze the particle size distribution in the lungs of R-TBH liposome aerosol in vitro at 5°C. The therapeutic efficacy of the nebulized aerosol of the R-TBH liposomes was assessed via pulmonary delivery in guinea pigs. The results showed that, compared to the R-TBH solution group, the R-TBH liposome group had a prolonged anti-asthma effect.

Effects of R-salbutamol on the inflammatory response and acute lung injury in endotoxemic mice.

Salbutamol, which consists of an R-isomer and S-isomer, is an effective and widely used ß2 adrenoreceptor agonist that may possess anti-inflammatory properties in addition to its bronchodilator activity. Whether the salbutamol R-isomer has advantages over its racemic mixture and effectiveness in treating endotoxemia and endotoxin-induced lung injury has not been well studied. In this study, we investigated the preventive and therapeutic effects of R-salbutamol (R-sal), S-salbutamol (S-sal), and their racemic mixture (Rac-sal) on a mouse model of lipopolysaccharide (LPS)-induced endotoxemia. Dexamethasone (Dex) was used for comparison. The results showed that R-sal markedly improved the 7-day survival rate of endotoxic mice when administered before and after LPS treatment. Dex was toxic and accelerated the death of endotoxic mice when administered before LPS injection. Histological examination of the lungs revealed that the LPS challenge resulted in acute lung damage, including inflammatory cell infiltration, thickened alveolar septa, and congestion. R-sal pre-treatment effectively inhibited these changes, accompanied by markedly reduced lung myeloperoxidase levels, serum cytokine levels, and lactate release, significant restoration of lymphocyte count, and reduction of monocyte count. This may have occurred through inhibition of M1 macrophage inflammatory responses by enhancement of ß-arrestin2 expression and suppression of NF-κB activation. Rac-sal exhibited diminished effects compared to that of R-sal, while S-sal showed enhanced release of some inflammatory cytokines. In addition, R-sal pre-treatment showed a better improvement in prognostic pulmonary function on day 4 compared to that by Rac-sal. Collectively, our results indicate the potential benefits of R-sal in regulating inflammatory responses to endotoxemia and endotoxin-induced lung injury.

Aerosol Characteristics and Physico-Chemical Compatibility of Combivent® (Containing Salbutamol and Ipratropium Bromide) Mixed with Three Other Inhalants: Budesonide, Beclomethasone or N-Acetylcysteine.

Inhalation therapy with a nebulizer is widely used in chronic respiratory disease. Mixing inhalation solutions/suspensions for simultaneous inhalation is more convenient and might simplify the administration procedure. However, there are no data available to address the in vitro aerosol characteristics and physico-chemical compatibility of Combivent® (containing Salbutamol and Ipratropium bromide) with other inhalation solutions/suspensions. In order to investigate the in vitro aerosol characteristics and physico-chemical compatibility of Combivent® with Budesonide, Beclomethasone, and N-acetylcysteine, the appearance, pH, osmotic pressure, chemical stability, mass median aerodynamic diameter (MMAD), fine particles fraction (FPF), particle size corresponding to X50 (particle size, which accounts for 50% of the total cumulative percentage of volume of all particles), delivery rate, and total delivery of the mixed inhalation solution/suspension were tested. There was no change in the appearance such as a change in color or precipitation formation at room temperature. The pH, osmolality, and chemicals of the mixtures were stable for 24 h after mixing. There were no significant differences between Combivent®, Budesonide, Beclomethasone, N-acetylcysteine, and the mixtures in MMAD, FPF, X50, the delivery rate, and the total delivery. This indicates that the mixtures were physically and chemically compatible. The mixing did not influence the particle size, distribution, or delivery compatibility of the mixtures.

Effects of flow pattern, device and formulation on particle size distribution of nebulized aerosol.

The evaluation of particle size recommended in the pharmacopeias requires a constant flow rate, and the method for pediatric inhaled drugs is the same as for adult drugs. In this study, the aerosol concentration and particle size distribution (PSD) were measured under a realistic breathing pattern and constant flow. Two types of nebulizer (i.e., breath-enhanced nebulizer and vibrating-mesh nebulizer) and two formulations (i.e., budesonide suspension and albuterol solution) were chosen for comparison. The aerosol concentration under the realistic pattern was not constant, which was different from the result at constant flow. The changing trend of aerosol concentration varied with the operation process of each device. The aerosol concentration profile was similar between budesonide suspension and albuterol solution. As to the PSD, as inspiratory flow increased, the X50 (50% undersize) increased with all nebulizers but Omron microAir NE-U22 nebulizer. There was good agreement between X50 obtained under the realistic inhalation patterns and their equivalent average flow rates by Bland-Altman analysis, although the X50 obtained under the realistic inhalation pattern was greater than value at constant flow. The agreement of the two breath-enhanced jet nebulizers was better than that of the vibrating-mesh nebulizers. The X50 of budesonide was not equal to that of albuterol when using the same nebulizer. Interestingly, a significant difference was observed in the X50 and Span when comparing the results of PSD under adult and child breathing patterns. Furthermore, all vibrating-mesh nebulizers produced aerosol droplets having larger mean diameter and narrower size distribution than those of the air-jet nebulizers. We conclude that it will be more conducive to the evaluation of particle size to use a laser diffractometer under a realistic pattern and make up for the shortcomings of cascade impactors. The effects of flow pattern, nebulizer and formulation should be taken into account in the evaluation of the qualities of nebulizer products in pharmaceutical practice.

Differential effects of inhaled R- and S-terbutaline in ovalbumin-induced asthmatic mice.

Inhaled terbutaline is commercially available ß2-agonist which consists of equivalent amount of R- and S-enantiomer. In this study, we aimed to investigate the effects of single enantiomers of terbutaline and its racemate in an ovalbumin (OVA)-induced mouse model of asthma via. seven days inhalation and the potential mechanisms involved. In a standard experimental asthma model, BALB/c mice were sensitized and challenged with OVA. R-terbutaline (R-ter), S-terbutaline (S-ter) or racemic terbutaline (rac-ter) was given via. nose-only inhalation for one week. Airway responsiveness to methacholine was measured by the plethysmography in conscious mice. Eosinophils counts in blood and bronchoalveolar (BAL) fluid were determined. The OVA-sIgE in plasma and inflammatory cytokines and mediators in BAL fluid or lung tissue were analyzed by ELISA, qRT-PCR or western blotting. Airway inflammation and remodeling were evaluated with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and Masson staining. Drug distribution and deposition after inhalation were determined by LC-MS/MS. Our data showed that R-ter efficiently ameliorated asthma responses, including airway hyperresponsiveness, eosinophils influx and IL-5 in BALF, plasma OVA-sIgE and significantly reduced pulmonary inflammation, peribronchial smooth muscle layer thickness, goblet cell hyperplasia, and deposition of collagen fibers, as well as downregulation of p38 MAPK phosphorylation and NF-κB expression. Racemic mixture exhibited diminished effects while S-ter enhanced airway responsiveness to methacholine and exerted pro-asthmatic effects.

Isosteviol prevents the development of isoprenaline­induced myocardial hypertrophy.

Isosteviol sodium (STVNa), which is a derivate of the natural sweet­tasting glycoside stevioside, has recently been developed and it has been determined that this compound exhibits neuro­ and cardio­protective properties. In the current study, whether STVNa interferes with the development of cardiac hypertrophy, which is induced by isoprenaline (Iso), was investigated in an experimental rat model. Rats were treated with a vehicle (0.9% NaCl; control), isoprenaline (Iso; 5 mg/kg) or Iso (5 mg/kg) with STVNa (4 mg/kg; Iso + STVNa). Cardiomyocytes were isolated using enzymatic dissociation and were treated with 5 µM Iso for 24 h and co­treated with 5 µM STVNa. Brain natriuretic peptide (BNP) mRNA expression was determined using PCR analysis. Cell surface area, intracellular reactive oxygen species (ROS), mitochondrial transmembrane potential (ΔΨm), cytoplasmic Ca2+ and Ca2+ and contractile function were examined using a laser scanning confocal microscope. The current study demonstrated that STVNa inhibited Iso­induced cardiac hypertrophy by inhibiting cardiomyocyte size. STVNa significantly reduced cell surface area and decreased BNP mRNA expression in ventricular cardiomyocyte Iso­induced hypertrophy. STVNa was also revealed to restore ΔΨm and reduce ROS generation and intracellular Ca2+ concentration when compared with the Iso­treated group. Additionally, STVNa preserved Ca2+ transients in hypertrophic cardiomyocytes. In conclusion, the present study demonstrated that STVNa protects against Iso­induced myocardial hypertrophy by reducing oxidative stress, restoring ΔΨm and maintaining Ca2+ homeostasis.

Isosteviol sodium injection improves outcomes by modulating TLRs/NF-κB-dependent inflammatory responses following experimental traumatic brain injury in rats.

Previous studies have shown that isosteviol sodium (STVNa) protects against permanent cerebral ischemia injury by inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated inflammatory responses. Overwhelming evidence shows that toll-like receptors (TLRs) are the upstream regulators of NF-κB. On the basis of the similarity of the pathology caused by traumatic brain injury (TBI) and stroke, we speculated that STVNa may have a therapeutic effect against TBI through regulation of the TLRs/NF-κB signaling-mediated inflammatory response. Thus, we studied the potential therapeutic effects of STVNa and the underlying mechanisms. Male rats, subjected to controlled cortical impact (CCI) injury, were injected intraperitoneally with STVNa (5, 10, 20, 40, and 80 mg/kg, daily for 3 or 7 days) after trauma. Neurobehavioral scores, relative numbers of cortical lesions, and histology were examined. We also measured the mRNA and protein expression levels of TLRs/NF-κB signaling pathway-related genes including TLR2, TLR4, and NF-κB by quantitative real-time-PCR and western blotting, respectively, and concentrations of tumor necrosis factor-α and interleukin-1ß by an enzyme-linked immunosorbent assay. The results indicated that STVNa (20 mg/kg) showed significant neuroprotective effects 3 and 7 days after TBI, including the reduction of cortical lesions, improvement of the neurological severity score, significantly increased number of restored neurons, decreased number of astrocytes, and lower concentrations of tumor necrosis factor-α and interleukin-1ß. Results from quantitative real-time-PCR and western blotting also show that the mRNA and protein expression levels of TLR2, TLR4, and NF-κB were significantly lower in STVNa-treated rats compared with the vehicle-treated rats. The administration of STVNa attenuates the TLR/NF-κB signaling pathway-mediated inflammatory responses in the injured rat brain, and this may be the mechanism by which STVNa improves the outcome following TBI.