Bufei Huoxue capsule attenuates COPD-related inflammation and regulates intestinal microflora, metabolites.

Background: Bufei Huoxue capsule (BFHX) is widely used for the clinical treatment of chronic obstructive pulmonary disease (COPD) in China. Objectives: The aim of this study is to explore the effects on COPD and the underlying mechanism of BFHX. The process and methods: In this study, we established a COPD mouse model through cigarette smoke (CS) exposure in combination with lipopolysaccharide (LPS) intratracheal instillation. Subsequently, BFHX was orally administrated to COPD mice, and their pulmonary function, lung pathology, and lung inflammation, including bronchoalveolar lavage fluid (BALF) cell count and classification and cytokines, were analyzed. In addition, the anti-oxidative stress ability of BFHX was detected by Western blotting, and the bacterial diversity, abundance, and fecal microbiome were examined using 16S rRNA sequencing technology. Outcome: BFHX was shown to improve pulmonary function, suppress lung inflammation, decrease emphysema, and increase anti-oxidative stress, whereas 16S rRNA sequencing indicated that BFHX can dynamically regulate the diversity, composition, and distribution of the intestinal flora microbiome and regulate the lysine degradation and phenylalanine metabolism of COPD mice. These results highlight another treatment option for COPD and provide insights into the mechanism of BFHX.

Quanzhen Yiqi decoction attenuates inflammation in mice with smoking-induced COPD by activating the Nrf2/HO-1 pathway and inhibiting the NLRP3 inflammasome.

OBJECTIVE: Quanzhen Yiqi decoction (QZYQ) is a traditional Chinese medicine for treating chronic obstructive pulmonary disease. METHODS: Mice were exposed to cigarette smoke (CS) 6 days/week (40 cigarettes/day) for 24 weeks and then intragastrically administered QZYQ (4.72, 9.45, or 18.89 g/kg) or dexamethasone (DEX, 0.6 mg/kg) for 6 weeks. We examined the lung function and collected bronchoalveolar lavage fluid for inflammatory cell and cytokine quantification. The pathological lung changes, ROS and oxidative biomarkers were measured. We used immunohistochemistry and western blotting to evaluate the levels of Nrf2/HO-1, NLRP3/ASC/Caspase1/IL-1ß/IL-18. RESULTS: The CS group showed significant increases in the forced vital capacity, lung resistance, and chord compliance and a lower FEV50/FVC compared with the control, and QZYQ improved these changes. In addition, QZYQ effectively reduced emphysema, immune cell infiltration, and airway remodeling. QZYQ stimulated HO-1 expression and reduced oxidative stress through the Nrf2 pathway. QZYQ inhibited the production of NLRP3/ASC/Caspase-1 to inhibit IL-1ß and IL-18. CONCLUSION: Our study suggested that QZYQ can improve the function and histology of the lungs and reduce inflammatory cell recruitment. QZYQ inhibits ROS production and NLRP3 inflammasome activation by upregulating Nrf2 to reduce lung injury. The anti-inflammatory effects of QZYQ are similar to those of DEX.

Development of an amoxicillin-radix scutellaria extract formulation and evaluation of its pharmacokinetics in pigs.

BACKGROUND: A new antibacterial compound powder of amoxicillin (AMO)/Radix Scutellaria extract (RSE) was developed, and its pharmacokinetics were determined in pigs following oral administration. RESULTS: The MIC ranges of AMO against Escherichia coli, Staphylococcus aureus and Streptococcus were 1-8 µg/mL, 0.5-4 µg/mL and 0.5-64 µg/mL, respectively. The MIC ranges of RSE against E. coli, S. aureus, and Streptococcus were greater than 2.5 mg/mL, 0.156-2.5 mg/mL, and greater than 2.5 mg/mL, respectively. For S. aureus, the combined drug susceptibility test showed that AMO and RSE had an additive or synergistic effect. The results of compatibility test, the excipient screening test and the drug quality control test showed that the formulation had stable quality and uniform properties under the test conditions. Two studies were conducted to investigate the pharmacokinetics of the compound product in pigs. First, the pharmacokinetics of the AMO-RSE powder were compared with those of their respective single products. The results showed no significant change in the main pharmacokinetic parameters when either component was removed from the compound formulation; thus, AMO and RSE have no pharmacokinetic interaction in pigs. Second, pigs were orally administered three different doses of AMO-RSE powder. The Cmax and AUC increased proportionally with increasing p.o. dose; thus, the λz, t1/2λ, MRT, and Tmax were unchanged for the doses of 10, 20, and 30 mg/kg AMO and the doses of 5, 10, and 15 mg/kg BCL, showing that AMO/baicalin in AMO-RSE powder showed linear pharmacokinetic characteristics in pigs. CONCLUSIONS: The combined drug sensitivity test of AMO and RSE against S. aureus showed that the combination was additive or synergistic. Pharmacokinetic studies indicated that AMO and BCL do not interfere with each other in pigs when used in a compound formulation. The pharmacokinetic parameters remained unchanged regardless of the dose for p.o. administration, indicating linear pharmacokinetic properties over the tested dose range. The quality of the AMO-RSE powder was good and stable, providing a foundation for its clinical application in veterinary medicine. Further bioavailability, PK/PD and clinical trials are still needed to determine the final dosage regimen.

Bufei huoxue capsule alleviates bleomycin-induced pulmonary fibrosis in mice via TGF-ß1/Smad2/3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Bufei huoxue (BFHX) is a Traditional Chinese Medicine formulation that consists of Astragalus Exscapus L, Paeonia Lactiflora Pall, and Psoralea Aphylla L. It can ameliorate collagen deposition and inhibit EMT. However, it remains unknown whether and how BFHX alleviates IPF. AIM OF THE STUDY: Our work aimed to explore the therapeutic efficacy of BFHX on IPF and dissect the mechanisms involved. MATERIALS AND METHODS: A mouse model of IPF was induced by bleomycin. BFHX was administered on the first day of modeling and maintained for 21 days. Pulmonary fibrosis and inflammation were evaluated by micro-CT, lung histopathology, pulmonary function assessment, and cytokines in BALF. In addition, we examined the signaling molecules involved in EMT and ECM by immunofluorescence, western Blot, EdU, and MMP (Δψm) assays. RESULTS: BFHX alleviated lung parenchyma fibrosis as evidenced by Hematoxylin-eosin (H&E), Masson's trichrome staining, and micro-CT, and it improved lung function. In addition, BFHX treatment not only decreased the levels of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), but also upregulated E-cadherin (E-Cad) and downregulated α-smooth muscle actin (α-SMA), collagen Ӏ (Col Ӏ), vimentin, and fibronectin (FN). Mechanistically, BFHX repressed TGF-ß1-driven Smad2/3 phosphorylation, which, in turn, suppressed EMT and transition of fibroblasts to myofibroblasts in vivo and in vitro. CONCLUSION: BFHX effectively reduces the occurrence of EMT and inhibits the production of ECM by inhibiting the TGF-ß1/Smad2/3 signaling pathway, which provides a potential novel therapeutic strategy for IPF.

Rosmarinic Acid Prevents Cisplatin-Induced Liver and Kidney Injury by Inhibiting Inflammatory Responses and Enhancing Total Antioxidant Capacity, Thereby Activating the Nrf2 Signaling Pathway.

Drug-induced liver and kidney damage is an emergent clinical issue that should be addressed. Rosmarinic acid (RA) has obvious anti-inflammatory and antioxidant effects, so we evaluated the anti-inflammatory and antioxidant effects of RA pretreatment on serum and liver and kidney tissues of cisplatin (CP)-treated mice and explored the possible mechanisms. The results showed that RA pretreatment effectively downregulated the serum, liver, and kidney levels of ALT, AST, BUN, and CRE and the inflammatory factors IL-1ß, IL-6, and TNF-α, and simultaneously enhanced the total antioxidant capacity of the liver and kidney. RA pretreatment significantly reduced the levels of MPO, MDA, and NO in liver and kidney tissue, inhibited the mRNA expression of IL-1ß, IL-6, and TNF-α in liver and kidney tissue, activated the Nrf2 signaling pathway, and upregulated the mRNA expression of downstream target genes. Our findings show that RA could effectively prevent and alleviate acute liver and kidney injury caused by CP.

Preparation, evaluation, and pharmacokinetics in beagle dogs of a taste-masked flunixin meglumine orally disintegrating tablet prepared using hot-melt extrusion technology and D-optimal mixture design.

Flunixin meglumine (FM) is a nonsteroidal anti-inflammatory drug limited by irritation of the respiratory tract and mucosa in veterinary tissue. This study aimed to develop a taste-masked FM solid dispersion (SD) by hot-melt extrusion (HME) and formulate an orally disintegrating tablet (ODT) with selected excipients by direct compression. Eudragit® E PO was chosen as the matrix, and HME parameters were optimized: extrusion temperature, 135℃; screw speed, 100 rpm; and drug loading, 20%. Characterization techniques proved that FM was rendered amorphous in the HME extrudate. In vitro dissolution studies showed that FM SD released significantly slower than the corresponding physical mixture in artificial saliva. Excipients were selected based on compression formability, disintegration, and solubility. A D-optimal mixture design was used to optimize the composition: 25% FM SD, 18.75% microcrystalline cellulose, 52.5% mannitol, 3.75% low-substituted hydroxypropyl cellulose, and 1% magnesium stearate. Taste-masked FM ODT had a tensile strength of 0.7 ± 0.01 MPa and a disintegration time of 17.6 ± 0.1 s. E-tongue and E-nose analysis showed that FM ODT had a better taste-masked effect than commercial granules. Finally, a pharmacokinetic study proved that the main pharmacokinetic parameters of FM ODT were not significantly different from those of commercial granules, which indicated that these formulations had similar pharmacokinetic behaviours in beagles.

Preparation and In Vitro/In Vivo Evaluation of Orally Disintegrating/Modified-Release Praziquantel Tablets.

This study was designed to develop orally disintegrating/sustained-release praziquantel (PZQ) tablets using the hot-melt extrusion (HME) technique and direct compression, and subsequently evaluate their release in in vitro and in vivo pharmacokinetics. For the extrusion process, hypromellose acetate succinate (HPMCAS)-LG was the carrier of pure PZQ, with a standard screw configuration used at an extrusion temperature of 140 °C and a screw rotation speed of 100 rpm. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), powder X-ray diffraction (PXRD) and Fourier-transform infrared spectroscopy (FTIR) were performed to characterize the extrudate. Orally disintegrating/sustained-release praziquantel tablets (PZQ ODSRTs) were prepared by direct compression after appropriate excipients were blended with the extrudate. The release amount was 5.10% in pH 1.0 hydrochloric acid at 2 h and over 90% in phosphoric acid buffer at 45 min, indicating the enteric-coating character of PZQ ODSRTs. Compared with the pharmacokinetics of marketed PZQ tablets (Aipuruike®) in dogs, the times to peak (Tmax), elimination half-life (t1/2λ) and mean residence time (MRT) were extended in PZQ ODSRTs, and the relative bioavailability of PZQ ODSRTs was up to 184.48% of that of Aipuruike®. This study suggested that PZQ ODSRTs may have potential for the clinical treatment of parasitosis.

Preparation, characterization, and pharmacokinetics of tilmicosin taste-masked formulation via hot-melt extrusion technology.

Tilmicosin (TMS) is a macrocyclic antibiotic specially used in veterinary clinics, but its extreme bitterness limits its use. This study aimed to obtain a taste-masked formulation of TMS by hot melt extrusion (HME) technology and to investigate the formulation's characterization, stability, and effects in vitro/in vivo. Eudragit® E PO was selected as the carrier, and TMS dissolution in artificial saliva was used as a reference. The HME parameters were optimized via an orthogonal design. The optimized results were as follows: 135 ℃ extrusion temperature, 100 rpm screw speed and 30 % drug load. The masking efficiency of the formulation was evaluated by both simulated oral drug release in vitro and electronic tongue tests. The release of the taste-masked formulation in artificial saliva medium was significantly reduced within 60 s (less than 2%), while the release in 0.1 M HCl buffer was fast (more than 80 %) within 30 min. As suggested by the results of the electronic tongue, the taste-masked formulation had a better taste-masked effect than the commercial premix and the commercial enteric granules. Finally, a pharmacokinetic study was performed. Analysis of variance demonstrated that the pharmacokinetic behavior of the TMS taste-masked formulation was similar to that of the commercial premix, while the absorption effect was better than that of the commercially available enteric granules. This research indicates that the taste-masked formulation has the potential for future commercialization.

The Antioxidant Rosmarinic Acid Ameliorates Oxidative Lung Damage in Experimental Allergic Asthma via Modulation of NADPH Oxidases and Antioxidant Enzymes.

Oxidative stress can induce lung damage and aggravate airway inflammation in asthma. Previously, we reported that rosmarinic acid (RA) exerted strong anti-inflammatory effects in a mouse allergic asthma model. Therefore, we hypothesized that RA might also have antioxidative effects in a superimposed asthma mouse model with oxidative lung damage challenged with ovalbumin (Ova) and hydrogen peroxide (H2O2). We evaluated the antioxidative and anti-asthmatic activity of RA and explored its possible mechanisms of action. Mice sensitized to Ova and challenged with Ova and H2O2 were treated with RA 1 h after challenge. RA treatment greatly diminished the number of inflammatory cells; decreased IL-4, IL-5, and IL-13 production; increased IFN-γ secretion; significantly downregulated ROS production; and markedly upregulated the activities of SOD, GPx, and CAT. Furthermore, RA treatment resulted in a significant increase in the expression of Cu/Zn SOD and a notable reduction in NOX-2 and NOX-4 expression in lung tissues. These findings suggest that RA may effectively alleviate oxidative lung damage and airway inflammation in asthma.

Curcumin/sunitinib co-loaded BSA-stabilized SPIOs for synergistic combination therapy for breast cancer.

Coating supermagnetic iron oxide nanoparticles (SPIOs) with albumin would not only improve their in vivo stability but also improve their drug loading capacity, but current methods are either inefficient or time consuming. Herein, a single step synthesis of bovine serum albumin (BSA)-stabilized SPIOs with high dispersity and stability via a modified co-precipitation method is reported. The benefits of albumin for coating of SPIOs, i.e. its long circulation life, low immunogenicity and drug binding ability to specific binding domains, were all retained in our mildly modified BSA. The BSA-SPIOs thus prepared displayed an excellent T2 contrast enhancing effect and drug loading capacity. Two cytotoxic drugs curcumin and sunitinib, where the former is a drug-resistance depressor and the latter is a tyrosine kinase inhibitor, were further co-loaded into the BSA-SPIOs (denoted SPIO-SC) to achieve combined synergistic therapy. SPIO-SC formulations displayed the most significant tumor inhibition yet least drug-induced toxicity both in vitro and in vivo when compared with free drug formulations. Through in vivo pharmacokinetic analysis, it was demonstrated that SPIO-SC most efficiently delivered the encapsulated drugs to the tumor site, and at the same time maintained the originally designed, optimal ratios of curcumin to sunitinib concentrations at the tumor target and yielded the most optimal synergistic effect and, subsequently, the more effective therapeutic outcomes. The prepared BSA-SPIOs are an extremely promising candidate for both MR imaging and drug delivery as a healthcare material.

Enzyme-triggered, cell penetrating peptide-mediated delivery of anti-tumor agents.

Conventional chemotherapy has little or no specificity for cancer cells, normally resulting in low drug accumulation at the tumor region (inefficacy) and drug-induced severe side effects (toxicity). Nowadays, new strategies have been developed to improve both the targeting ability and cellular drug uptake using active targeting ligands and drug internalization agents, which could recognize and interact with specific receptors overexpressed on tumor cells and then trigger a drug internalization process by transporting the cargos into cells. Among those strategies, enzyme-triggered cell penetrating peptide (CPP)-mediated systems seem to be a feasible approach. The expression level of specific enzymes like proteases, esterases or glycosidases is often higher in tumor cells than in normal tissues, and such concentration gradients can be exploited as a tool for targeted cancer therapy. CPPs are known to be effective in promoting membrane transportation of the drug cargos, rendering a deeper tumor permeation that could further enhance the therapeutic efficacy of the delivered drug. An enzyme-triggered, CPP-mediated system would combine these advantages to yield a system with the enhanced tumor targeting ability and internalization efficiency and so far many systems have been successfully exploited and applied to cancer therapy. In this review, typical enzymes applied in cancer theranostic systems were firstly reviewed, followed by analyzing pros and cons of cell penetrating peptides. Most importantly, different types of applications of enzyme-triggered CPP-mediated systems in tumor imaging were illustrated. Finally, the drug loaded applications, i.e. enzyme-triggered CPP-mediated systems in drug delivery were reviewed.

[Nonconserved hinge in Baeyer-Villiger monooxygenase affects catalytic activity and stereoselectivity].

Baeyer-Villiger monooxygenases (BVMOs) are important biocatalysts to synthesize a series of valuable esters and lactones. Based on protein sequence alignment and crystal structure analysis, a nonconserved hinge which linked NADPH domain and FAD domain was speculated to play an important role in substrate recognition and catalytic oxidation process. Cyclohexanone monooxygenase (CHMO) was selected as a model. Mutants obtained by homologous replacement of the whole hinge almost completely lost its original catalytic activity, demonstrating that the overall hinge structure was of great importance. Some significant sites were identified to greatly affect the catalytic activity and stereoselectivity by alanine scanning mutagenesis, accompanied by enzyme activity assessments and chiral kinetic resolutions. Altering K153 decreased the activity of the enzyme but enhanced the stereoselectivity. Changing L143 site reduced stereoselectivity but had little effect on enzyme activity. Mutation at L144 site dramatically weakened both activity and stereoselectivity. Subsequently, these corresponding sites in phenylacetone monooxygenase were also illustrated to follow a similar rule, revealing a universal importance of these sites in the BVMO family. These results expanded our understanding of the structure-activity relationship of these enzymes and provided more proofs for future directed evolution of BVMOs.

15 years of ATTEMPTS: a macromolecular drug delivery system based on the CPP-mediated intracellular drug delivery and antibody targeting.

Traditionally, any drug intended for combating the tumor would distribute profoundly to other organs and tissues as lack of targeting specificity, thus resulting in limited therapeutic effects toward the tumor but severe drug-induced toxic side effects. To prevail over this obstacle of drug-induced systemic toxicity, a novel approach termed "ATTEMPTS" (antibody targeted triggered electrically modified prodrug type strategy) was designed, which directly introduces both of the targeting and prodrug features onto the protein drugs. The ATTEMPTS system is composed of the antibody targeting component consisting of antibodies linked with heparin, and the cell penetrating peptide (CPP) modified drug component. The two components mentioned above self-assembled into a tight complex via the charge to charge interaction between the anionic heparin and cationic CPP. Once accumulated at the targeting site, the CPP modified drug is released from the blockage by a second triggering agent, while remaining inactive in the circulation during tumor targeting thus aborting its effect on normal tissues. We utilized the heparin-induced inhibition on the cell-penetrating activity of CPP to create the prodrug feature, and subsequently the protamine-induced reversal of heparin inhibition to resume cell transduction of the protein drug via the CPP function. Our approach is the first known system to overcome this selectivity issue, enabling CPP-mediated cellular drug delivery to be practically applicable clinically. In this review, we thoroughly discussed the historical and novel progress of the "ATTEMPTS" system.

Low molecular weight protamine (LMWP): a nontoxic protamine substitute and an effective cell-penetrating peptide.

Low molecular weight protamine (LMWP) is a peptide fragment produced in our laboratory from enzymatic digestion of native protamine. More than 30 papers studying the properties and applications of LMWP have been published by our group in various journals since its initial discovery in 1999. Results have shown that LMWP could completely neutralize the anticoagulant functions of both heparin and low molecular weight heparin (LMWH), with reduced antigenicity and cross-reactivity toward the mice-derived anti-protamine antibodies. Aside from its potential as a heparin/LMWH antagonist, LMWP also shows the ability to retard insulin adsorption by the formation of an insoluble complex, making it a less toxic long-lasting insulin product than the conventional neutral protamine Hagedorn (NPH) insulin for diabetic control. Importantly, LMWP (Sequence: VSRRRRRRGGRRRR), with 10 arginine residues in its structure, could function as a cell-penetrating peptide (CPP), also termed protein transduction domain (PTD), to achieve effective intracellular protein or gene delivery in clinical practice. In this paper, we present a thorough review of our work related to LMWP, with the aim of providing readers an insight into its potential to be a clinical protamine substitute as well as a non-toxic cell penetrating peptide applicable to achieve intracellular protein and gene delivery.

Structural insights into the substrate specificity of Streptococcus pneumoniae ß(1,3)-galactosidase BgaC.

The surface-exposed ß-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity toward the ß(1,3)-linked galactose and N-acetylglucosamine (Galß(1,3)NAG) moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at 1.8 Å and its complex with galactose at 1.95 Å. At pH 5.5-8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains: a catalytic domain of a classic (ß/α)(8) TIM barrel, followed by two all-ß domains (ABDs) of unknown function. The side walls of the TIM ß-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp-243 and Tyr-455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Galß(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp-240 and Tyr-455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp-243 is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35 family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain.

Characterization of a new cell line from caudal fin of koi, Cyprinus carpio koi, and first isolation of cyprinid herpesvirus 3 in China.

A new continuous cell line (KCF-1) from caudal fin of koi, Cyprinus carpio koi, was developed and sub-cultured more than 100 passages since the present study was initiated in March 2006. KCF-1 predominantly consisted of short fibroblast-like cells and grew well in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Chromosome analysis revealed that 56% of the KCF-1 cells maintained normal diploid chromosome number (2n=100) at Passage 82. Using the KCF-1 cell line, a strain of cyprinid herpesvirus 3 (designated as CyHV-3-QY08) was isolated from the diseased koi. CyHV-3-QY08 continuously propagated in the KCF-1 cells, as confirmed by immunofluorescence assay (IFA) and transmission electron microscopy (TEM). KCF-1 cells infected with CyHV-3-QY08 produced typical cytopathic effects characterized by severe vacuolation, deformation of nuclei, and marginalization of the nuclear chromatin, which are consistent with those of previous reports. CyHV-3-QY08 was purified and subsequently analyzed by SDS-PAGE and TEM. The results showed that the purified virions contained two types of morphologies and were composed of more than 30 obvious viral polypeptides. An infectivity experiment revealed that CyHV-3-QY08 could cause 100% mortality in the infected koi. Based on the genome sequence of CyHV-3-I/U, the CyHV-3(I/U)-ORF136 homologue in CyHV-3-QY08 was cloned and sequenced. Multiple sequence alignments of CyHV-3-I/U-ORF136 homologues showed that CyHV-3-QY08 belonged to the typical Asian genotype. The CyHV-3(I/U)-ORF136 homologue seems to be a novel molecule marker, which can be used to distinguish Asia isolates from Europe-America strains.