Development and validation of a simple and sensitive HPLC method for the determination of related substances in regorafenib tablets.

Regorafenib as an oral multi-kinase inhibitor has displayed a promising future in the anticancer drug market. However, there are no articles reporting the method for the determination of related substances in regorafenib tablets. A quality standard was first included in the Ph. Eur. 10.4 until April 2021 but could not detect seven known impurities A, C, D, E, FP-A, FP-B, and FP-C simultaneously. In this paper, a simple and sensitive HPLC method was established for the determination of related substances in regorafenib tablets. The determination was performed on a Polar-RP column with dual wavelength detection set at 230 nm and 260 nm. This method was validated according to the ICH guidelines. Furthermore, the possible sources of impurities were analyzed and forced degradation tests were performed, which provided guidance for formulation development and storage conditions. The established method is simple, sensitive and accurate for the determination of related substances in regorafenib tablets. A specified and sensitive HPLC method for the determination of related substances in regorafenib tablets.

Protective effects of the combination of sodium ferulate and oxymatrine on cecal ligation and puncture-induced sepsis in mice.

The aim of this study was to investigate the effects of the combination of sodium ferulate (SF) and oxymatrine (OMT) on mice with cecal ligation and puncture (CLP)-induced sepsis. Swiss male mice were randomly divided into a control group, CLP group, three SF + OMT groups (3.1+6.9; 6.2+13.8 and 12.3+27.7 mg/kg), SF (6.2 mg/kg) group and OMT (13.8 mg/kg) group. Eight hours after the administration of the drugs, the survival rates and survival times of the animals were monitored. In addition, the lung wet/dry weight (W/D) ratio; alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels in the serum; the C-reactive protein (CRP), interleukin-6 (IL-6) and interferon-γ (IFN-γ) levels in the serum and lung and liver homogenates; and the malondialdehyde (MDA) and superoxidase dismutase (SOD) levels in the lung and liver homogenates were measured. The bacterial load in the serum was also studied. Following treatment with the combination of SF and OMT, the survival rate increased and the survival time was prolonged; CLP-induced increases in the lung W/D ratio and the levels of ALT, AST, LDH, CRP, IL-6, IFN-γ and MDA were significantly reduced; and the SOD activity levels were increased, compared with those of the untreated animals with CLP-induced sepsis. These results indicated that the combination of SF and OMT induced protective effects against CLP-induced lethal sepsis of mice. The possible mechanism of these effects may be associated with the alleviation of systemic inflammation and diminishment of oxidative injury.

Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats.

BACKGROUND: Diabetes is one of the most prevalent human metabolic diseases. Wound healing in diabetes is frequently impaired and treatment remains challenging. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway. S1P/SPK plays a pivotal role in the signalling pathways of diverse cellular processes including proliferation, differentiation, migration, apoptosis in diverse cell types. METHODS: To investigate the role of sphingosine kinase 1 (SPK1) in skin injury, plasmids containing the SPK1 gene (pcDNA3-FLAG-SPK1) were applied to cutaneous wounds on a streptozotocin-induced diabetic rat model over a 21-day period. The wound area and rate of wound healing were determined. The histopathological features of the healed wounds were also observed, and SPK1 expression in the skin was detected by immunohistochemistry. RESULTS: There was a significant decrease in wound area in diabetic rats treated with 125 and 60µg/wound pcDNA3-FLAG-SPK1 (P<0.001-0.01). The mean sizes of the wounds were 0.67±0.15cm(2), 0.83±0.18cm(2), and 1.09±0.23cm(2) in both treated and diabetic control group at the 7th day post-treatment respectively. In addition, wound healing in diabetic rats of test group was accelerated. At the 7th day, the mean rates of healing were 73.2±5.7% and 66±7.3% in test group of 125 and 60µg/wound respectively, and 55.4±9.9% in diabetic control group (P<0.001-0.01). Histology revealed that tissue sections from the treated diabetic rats contained more granulation tissue and capillaries than that of the control rats. There was high SPK1 expression in the skin of the treated diabetic rats. CONCLUSIONS: SPK1 gene therapy may represent a novel approach to cutaneous wound healing.

The protective effects of the combination of sodium ferulate and oxymatrine on ethanol-induced liver damage in mice.

The aim of this study was to investigate the effects of the combination of SF and OMT on ethanol-induced liver damage in mice. The animal liver wet/dry weight (W/D) ratio and liver tissue histopathology, alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), malondialdehyde (MDA), superoxidase dismutase (SOD), C-reactive protein (CRP), interleukin-6 (IL-6), and nuclear factor κB (NF-κB) levels were measured. The data indicated that the levels of ALT, AST, TG, CRP, IL-6, NF-κB and MDA significantly decreased and that SOD activity improved after treatment with the combination of SF and OMT; the same effects were not observed with the same dose of SF or OMT when used alone. These results indicated that the combination of SF and OMT had a protective effect on ethanol-induced liver damage in mice and that antioxidation and anti-inflammatory effects might be involved in this protective mechanism.

[Stable and efficient expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells].

Hepatitis B surface antigen (HBsAg) carrying preS sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS1 production at 17-20 mg/L with viable cell density at 7 x 10(6)-10 x 10(6) cells/mL.

Observation of antinociceptive effects of oxymatrine and its effect on delayed rectifier K⁺ currents (Ik) in PC12 cells.

In order to observe antinociceptive effect of Oxymatrine (OMT) and its effect on voltage-activated K(+) channel, the acetic acid-induced abdominal contraction model of mouse was used to test the antinociceptive effect in vivo, and in vitro, the delayed rectifier K(+) currents (Ik) in PC12 cells (rat pheochromocytoma cells) was recorded using the automated patch-clamp method. The results indicated that after application of OMT, the number of acetic acid-induced animal abdominal contraction was significantly decreased, Ik in PC12 cells was significantly decreased, and showed a concentration-dependent manner. After application of OMT, both the activation and inactivation curves of Ik of PC12 cells were shifted to negative potentials. This study revealed that OMT showed antinociceptive effect in mice. The inhibition of voltage-activated K(+) channel might be one of mechanisms in which the enhanced both activation and inactivation of K(+) channel were involved and might play important roles.

[Preparation and characterization of recombinant protein A affinity packing].

To obtain an excellent antibody purification medium, affinity chromatographic packing with recombinant staphylococcal protein A (rProtein A) was synthesized and verified. With E. coli cells harboring the recombinant plasmid, the rProtein A was expressed and purified, then was conjugated to epichlorohydrin-activated Sepharose 4 Fast Flow to prepare an affinity chromatographic packing. The performances of the packing were validated with rabbit antiurate oxidase. After the reaction, the concentration of rProtein A coupled to Sepharose 4 Fast Flow was 1.5 x 10(-4) mol/L. Scatchard analysis of the binding isotherm for IgG showed excellent binding capacity on the adsorbent, giving a dissociation constant (Kd) of 2.28 x 10(-7) mol/L and a theoretical maximum adsorption capacity of 20. 697 g/L. The identification showed the packing was stable in 0.1 mol/L NaOH solution at 1 h. By using the packing, the pure antibody exhibited on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained from rabbit serum after one-step elution, with 96. 1% of yield and 19 mg IgG for one milliliter of gel. The research laid the foundation of the localization of rProtein A affinity packing.

The effects of the combination of sodium ferulate and oxymatrine on lipopolysaccharide-induced acute lung injury in mice.

In our previous study, the remarkable analgesic and anti-inflammatory effects of the combination of sodium ferulate (SF) and oxymatrine (OMT) had been found. In this study, we investigated the effect of the combination of SF and OMT on acute lung injury using lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. The cell counting and the protein concentration in the bronchoalveolar lavage fluid (BALF) were measured. The animal lung edema degree was evaluated by wet/dry weight (W/D) ratio. The superoxidase dismutase (SOD) activity and myeloperoxidase (MPO) activity was assayed by SOD and MPO kits, respectively. The levels of inflammatory mediators including C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α) were assayed by enzyme-linked immunosorbent assay method. The data showed that treatment with the combination of SF and OMT markedly attenuated inflammatory cell numbers and protein concentration in the BALF and improved SOD activity and inhibited MPO activity compared to LPS group. Moreover, the combination significantly inhibited the production of CRP and TNF-α in lung homogenate. The histological changes of the lungs were also more significantly improved by the combination. At the same dose, the obvious protective effect was not found in SF or OMT-treated alone group except that the protein concentration slightly decreased in SF group. The results indicated that the combination SF and OMT had a protective effect on LPS-induced ALI in mice, and the effect was much better than that of SF or OMT used alone.

The synergistic anti-inflammatory effect of the combination of sodium ferulate and oxymatrine and its modulation on inflammation-associated mediators in RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The combination of Radix Angelicae sinensis (Oliv.) Diels and Radix Sophora flavescens Ait. was extensively used in traditional Chinese medicine to treat inflammatory diseases, such as acne, heart disease, and hepatitis. Sodium ferulate (SF) and oxymatrine (OMT) were effective component of Radix Angelicae sinensis (Oliv.) Diels and Radix Sophora flavescens Ait., respectively. AIM OF THE STUDY: In this study, we investigated the synergistic anti-inflammatory effect of the combination of SF and OMT, and its modulation on inflammation-associated mediators in RAW 264.7 cells. MATERIALS AND METHODS: In vivo, the anti-inflammatory effects of the combination of SF and OMT were evaluated with the xylene-induced mouse ear edema model and the carrageenan-induced rat paw edema model. In vitro, chemokines and cytokines mRNA expressions in lipopolysaccharide (LPS)-activated RAW 264.7 cells were determined by real-time PCR (RT-PCR) microarray analysis. The levels of interleukin-11 (IL-11), C-reactive protein (CRP) and interferon-γ (INF-γ) in the supernatant of LPS-stimulated RAW 264.7 cells were measured by enzyme-linked immune-sorbent assay (ELISA). RESULTS: The combination of SF and OMT could significantly inhibit the edema in the xylene-induced mouse ear edema and carrageenan-induced rat paw edema, but no effect was found when each drug was used alone according to above doses. The combination exhibited a better effect in down-regulating mRNA expressions of inflammation-associated mediators in LPS-stimulated RAW 264.7 cells than SF or OMT alone. The ELISA results showed that the combination synergistically inhibited LPS-induced IL-11, CRP and INF-γ production in a dose-dependent manner. CONCLUSION: The combination of SF and OMT showed synergistic anti-inflammatory effect, and the activity was probably related to its modulation on inflammation-associated mediators, especially IL-11, CRP and INF-γ.

The analgesic effect and mechanism of the combination of sodium ferulate and oxymatrine.

Sodium ferulate (SF) and Oxymatrine (OMT) were compounds extracted from Chinese herbs, and have been used in clinical treatment of heart and hepatic diseases, respectively, in China for many years. The objective of this study was to examine the analgesic effect and the mechanism of the combined treatment of SF and OMT. Using the animal pain models by applying Acetic Acid Writhing Test and Formalin Test, the combination of SF and OMT showed significant analgesic effect in dose-dependent manner. In vitro, the combined treatment inhibited the increase in intracellular calcium concentration evoked by capsaicin in the dorsal root ganglion neurons. Importantly, a synergistic inhibitory effect of SF and OMT on the capsaicin-induced currents was demonstrated by whole-cell patch-clamp. Our results suggest that SF and OMT cause significant analgesic effect which maybe related to the synergistic inhibition of transient receptor potential vanilloid-1.

Imaging with random 3D reference structures.

We describe a sensor system based on 3D 'reference structures' which implements a mapping from a 3D source volume on to a 2D sensor plane. The reference structure used here is a random three dimensional distribution of polystyrene beads. We show how this bead structure spatially segments the source volume and present some simple experimental results of 2D and 3D imaging.

[Promotion of Thrombopoiesis by rhIL-11 in Normal and Myelosuppressed Mice]

Hematological effects of rhIL-11 on normal and myelosuppressed male BALB/c mice were observed. Mice were subcutaneously injected with rhIL-11 for 7 consecutive days, at the dose of 200 or 400 micro g/kg per day, peripheral blood platelet counts were moderately elevated on 5 days after administration and returned to base level within 4 days after discontinuation of injection. In myelosuppressed mice, treatment with rhIL-11 significantly ameliorated the degree of thrombocytopenia, the recovery of thrombocytopenia was also significantly accelerated at the dose range of 100 - 400 micro g/kg per day, and blood platelet counts reached pre-irradiated level after 13 - 15 days of treatment. The magnitudes of platelet count elevation were similar among groups of 100, 200 and 400 micro g/kg per day, although recovery appeared earlier in group of 400 micro g/kg per day. Significant increases in CFU-Meg were observed both in normal and myelosuppressed mice. Our results suggest that rhIL-11 promotes the increase of peripheral blood platelets both in normal and myelosuppressed mice, and can be used as a potential therapeutic agent for thrombocytopenia induced by chemotherapy.

[Therapeutic Effects of rhIL-11 on Carboplatin-Induced Thrombocytopenia in Monkeys]

A model of myelosuppression with thrombocytopenia was produced in monkey by i.v. administration of carboplatin to the evaluate effects of rhIL-11 treatment in monkeys. Following myelosuppression, rhIL-11 was subcutaneously injected for 19 consecutive days at the dose of 50 or 100 micro g/kg. In myelosuppressed monkeys treated with rhIL-11, peripheral blood platelet started to drop at the day 8 after the administration of carboplatin, and reaching the nadir between the day 12 - 14, the decrease in blood platelet was less severe compared with untreated monkeys; peripheral blood platelet began to recovery on day 11 - 13 (D14 - D16) after rhIL-11 treatment, and reached or surpassed the baseline value before carboplatin administration after 13 - 15 days rhIL-11 treatment. Blood platelet counts remained high level after discontinuation of rhIL-11 administration and returned to baseline after 4 days. The results demonstrated that rhIL-11 has a significant thrombopoietic activity, it can reduce the severity of thrombocytopenia as well as shorten the duration of thrombocytopenia caused by myeloablastive agents, and is likely to become an effective agent against thrombocytopenia induced by chemotherapy.