Preclinical characterization of danatinib as a novel FLT3 inhibitor with excellent efficacy against resistant acute myeloid leukemia.

The therapeutic benefits of available FLT3 inhibitors for AML are limited by drug resistance, which is related to mutations, as well toxicity caused by off-target effects. In this study, we introduce a new small molecule FLT3 inhibitor called danatinib, which was designed to overcome the limitations of currently approved agents. Danatinib demonstrated greater potency and selectivity, resulting in cytotoxic activity specific to FLT3-ITD and/or FLT3-TKD mutated models. It also showed a superior kinome inhibition profile compared to several currently approved FLT3 inhibitors. In diverse FLT3-TKD models, danatinib exhibited substantially improved activity at clinically relevant doses, outperforming approved FLT3 inhibitors. In vivo safety evaluations performed on the granulopoiesis of transgenic myeloperoxidase (MPO) zebrafish and mice models proved danatinib to have an acceptable safety profile. Danatinib holds promise as a new and improved FLT3 inhibitor for the treatment of AML, offering long-lasting remissions and improved overall survival rates.

Natural products as potential lead compounds to develop new antiviral drugs over the past decade.

Virus infection has been one of the main causes of human death since the ancient times. Even though more and more antiviral drugs have been approved in clinic, long-term use can easily lead to the emergence of drug resistance and side effects. Fortunately, there are many kinds of metabolites which were produced by plants, marine organisms and microorganisms in nature with rich structural skeletons, and they are natural treasure house for people to find antiviral active substances. Aiming at many types of viruses that had caused serious harm to human health in recent years, this review summarizes the natural products with antiviral activity that had been reported for the first time in the past ten years, we also sort out the source, chemical structure and safety indicators in order to provide potential lead compounds for the research and development of new antiviral drugs.

Bufotenine and its derivatives: synthesis, analgesic effects identification and computational target prediction.

Natural product bufotenine (5) which could be isolated from Venenum Bufonis, has been widely used as a tool in central nervous system (CNS) studies. We present here its quaternary ammonium salt (6) which was synthesized with high yields using 5-benzyloxyindole as raw materials, and we firstly discover its analgesic effects in vivo. The analgesic evaluation showed that compounds 5 and 6 had stronger effects on the behavior of formalin induced pain in mice. Moreover, the combination of compound 6 and morphine has a synergistic effect. We intended to explain the molecular mechanism of this effect. Therefore, 36 analgesic-related targets (including 15 G protein-coupled receptors, 6 enzymes, 13 ion channels, and 2 others) were systemically evaluated using reverse docking. The results indicate that bufotenine and its derivatives are closely related to acetyl cholinesterase (AChE) or α4ß2 nicotinic acetylcholine receptor (nAChR). This study provides practitioners a new insight of analgesic effects.

Design, synthesis, and biological activity evaluation of BACE1 inhibitors with antioxidant activity.

The proteolytic enzyme ß-secretase (BACE1) plays a central role in the synthesis of the pathogenic ß-amyloid peptides (Aß) in Alzheimer's disease (AD), antioxidants could attenuate the AD syndrome and prevent the disease progression. In this study, BACE1 inhibitors (D1-D18) with free radical-scavenging activities were synthesized by molecular hybridization of 2-aminopyridine with natural antioxidants. The biological activity evaluation showed that D1 had obvious inhibitory activity against BACE1, and strong antioxidant activity in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS+• ) assay, which could be used as a lead compound for further study.

Design, synthesis and evaluation of 2-amino-imidazol-4-one derivatives as potent ß-site amyloid precursor protein cleaving enzyme 1 (BACE-1) inhibitors.

Inhibition of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) to prevent brain ß-amyloid (Aß) peptide's formation is a potential effective approach to treat Alzheimer's disease. In this report we described a structure-based optimization of a series of BACE1 inhibitors derived from an iminopyrimidinone scaffold W-41 (IC50 = 7.1 µM) by Wyeth, which had good selectivity and brain permeability but low activity. The results showed that occupying the S3 cavity of BACE1 enzyme could be an effective strategy to increase the biological activity, and five compounds exhibited stronger inhibitory activity and higher liposolubility than W-41, with L-5 was the most potent inhibitor against BACE1 (IC50 = 0.12 µM, logP = 2.49).

Synthesis and Biological Evaluation of Scutellarein Alkyl Derivatives as Preventing Neurodegenerative Agents with Improved Lipid Soluble Properties.

BACKGROUND: Exogenous antioxidants are considered as a promising therapeutic approach to treat neurodegenerative diseases since they could prevent and/or minimize the neuronal damage by oxidation. OBJECTIVE: Three series of lipophilic compounds structurally based on scutellarein (2), which is one metabolite of scutellarin (1) in vivo, have been designed and synthesized. METHODS: Their antioxidant activity was evaluated by detecting the 2-thiobarbituric acid reactive substance (TBARS) produced in the ferrous salt/ascorbate-induced autoxidation of lipids, which were present in microsomal membranes of rat hepatocytes. The lipophilicity of these compounds indicated as partition coefficient between n-octanol and buffer was investigated by ultraviolet (UV) spectrophotometer. RESULTS: This study indicated that compound 5e which had a benzyl group substituted at the C4'- OH position showed a potent antioxidant activity and good lipophilicity. CONCLUSION: 5e could be an effective candidate for preventing or reducing the oxidative status associated with the neurodegenerative processes.

Protective effect of 6-O-methyl-scutellarein on repeated cerebral ischemia/reperfusion in rats.

Scutellarin (1) possesses protective effects against neuronal injury, while 6-O-methyl-scutellarein (3), as the main metabolite of scutellarin in vivo, has not been reported about its protective effects previously. The present study mainly investigated whether the neural injury caused by ischemia/reperfusion would be influenced by different doses of 6-O-methyl-scutellarein (3). The results of behavioral, neurological, and histological examinations indicated that 6-O-methyl-scutellarein (3) could improve neuronal injury, and exhibit significant difference among the various doses. More importantly, 6-O-methyl-scutellarein (3) had better protective effects than scutellarin in rat cerebral ischemia.

Synthesis and Bioactivity Characterization of Scutellarein Sulfonated Derivative.

Scutellarin (1) has been widely used to treat acute cerebral infarction in clinic, but poor aqueous solubility decreases its bioavailability. Interestingly, scutellarin (1) could be metabolized into scutellarein (2) in vivo. In this study, a sulfonic group was introduced at position C-8 of scutellarein (2) to enhance the aqueous solubility of the obtained derivative (3). DPPH (1,1-diphenyl-2-picrylhydrazyl)-radical scavenging ability and antithrombic activity were also conducted to determine its bioactivity. The result showed that scutellarein derivate (3) could be a better agent for ischemic cerebrovascular disease treatment.

Design, Synthesis, and Biological Evaluation of Scutellarein Derivatives Based on Scutellarin Metabolic Mechanism In Vivo.

Three series of scutellarein derivatives have been designed and synthesized based on metabolic mechanism of scutellarin (1) in vivo. Their thrombin inhibition activities were tested through the analyzation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB). The antioxidant activities of these target products were assessed by 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay and the ability to protect PC12 cells against H2 O2 -induced cytotoxicity, and their solubilities were evaluated by ultraviolet (UV) spectrophotometer. The results showed that the two isopropyl groups substituted derivative (18c) demonstrated stronger anticoagulant activity, better water solubility, and good antioxidant activity compared with scutellarein (2), which warrants further development of 18c as a promising agent for ischemic cerebrovascular disease treatment.

Synthesis of scutellarein derivatives to increase biological activity and water solubility.

In order to improve the biological activity and water solubility of scutellarin (1), some derivatives of its main metabolite (scutellarein) were designed and synthesized. All the compounds were tested for their thrombin inhibition activity through the analyzation of thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (FIB). Their antioxidant activities were assessed by measuring their scavenging capacities toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and the ability to protect PC12 cells against H2O2-induced cytotoxicity, their water solubility were also assessed by ultraviolet (UV) spectrophotometer. The results showed that compound 8b demonstrated stronger anticoagulant and antioxidant activity, better water solubility compared with scutellarein (2), which warrants it as a promising agent for the treatment of ischemic cerebrovascular disease.

A new and practical synthetic method for the synthesis of 6-O-methyl-scutellarein: one metabolite of scutellarin in vivo.

Scutellarin (1) has been used for the treatment of angina pectoris, cerebral infarction and coronary heart disease with a large market share in China. Pharmacokinetic studies on scutellarin showed that scutellarin (1) is readily converted into its metabolites in vivo. In this paper, a new and practical synthetic method for the synthesis of 6-O-methyl-scutellarein (3) (one metabolite of scutellarin in vivo) is reported. The benzyl bromide was firstly used to selectively replace the acetyl group at C-7 in 7, and was then used to protect the hydroxy groups at C-4' in 10, 6-O-methyl-scutellarein (3) is obtained in high yield through these methods.

[Comparison of activated carbon and ultrafiltration technique in the production process of huoxue tongluo injection].

OBJECTIVE: To research the applicability of activated carbon and ultrafiltration technique in the production process of Huoxue Tongluo Injection. METHODS: The kinetic-turbidimetric method was used to determine the content of bacterial endotoxins in Huoxue Tongluo solution. Particle size change in Huoxue Tongluo solution was determined by nanometer particle size instrument before and after the use of different concentration activated carbon and different molecular weight ultrafiltration membrane. RESULTS: The removal efficacy of bacterial endotoxins was 65.2%, 77.5%, 80.4% by using three concentrations of active carbon at 0.05%, 0.10%, 0.30% in Huoxue Tongluo Injection, respectively. It was above 95% by using cutoff molecular weight both 5 kDa and 10 kDa ultrafiltration membrane. Measure results by nanometer particle size instrument showed that particle size of filter liquor by 10 kDa cutoff molecular weight ultrafiltration membrane was much smaller than that of by use of different concentration activated carbon. CONCLUSION: Ultrafiltration method is more suitable to the removal of bacterial endotoxins. The solution is more clear after using ultrafiltration method, and large particles of solution is removed. The ultrafiltration method provides the basis for injection production.

[Effect on elimination of pyrogen and effectual components of Panax notoginseng saponins by ultrafiltration membranes of different cut-off molecular weight and different materials].

OBJECTIVE: To study the elimination effect of bacterial endotoxins and the transmittance of Panax notoginseng saponins by ultrafiltration membranes of different cut-off molecular weight and different materials. METHODS: The kinetic-turbidimetric method was used to determine the content of bacterial endotoxins in Panax notoginseng saponins solution before and after using the ultrafiltration. The change of the contents of active components was examined by HPLC,using notoginsennoside R1, ginsennoside Rg1, ginsennoside Rb1 and ginsennoside Rd as the mark components. RESULTS: The removal rate of bacterial endotoxin fell along with the increasing of membrane aperture. The removal rate was 20. 69% by ultrafiltration membranes of 100 KDa with polysulfone material,less than those of other ultrafiltration membranes with polysulfone material. But the removal rate of bacterial endotoxin by E membranes of blend materials was higher than those of other ultrafiltration membranes with polysulfone material. The contents of active components filtered by E membranes of blend materials was more than that of ultrafiltration membranes of 100 KDa with polysulfone material. CONCLUSION: The applicability of ultrafiltration membranes of large cut-off molecular weight and blend materials of effectual component in Panax notoginseng saponins and elimination of pyrogen is good.

[Evaluation on the analytical sensitivity of 31 HBsAg enzyme immunoassay kits].

OBJECTIVE: To study the analytical sensitivity on 31 HBsAg enzyme immunoassy (EIA) test kits. METHODS: Thirty one HBsAg EIA kits produced by domestic or overseas manufactories and applied for approval during May 2007 to May 2008, were evaluated using the national reference panels. The hyperbolic curve of the log A value and log concentration for the national sensitivity standards was established. The cut-off value of each kit was substituted into the curvilinear equation to determine the analytical sensitivity which was compared between different HBsAg EIA kits. RESULTS: Twenty seven (351 lots) domestic and 4 (27 lots) overseas kits were compared. Among 378 lots of the 31 HBsAg EIA kits, only 2 lots of the domestic kits had a lower sensitivity when tested with the national HBsAg reference panels, with an average approvalr ate of 99.43% (349/351). The mean analytical sensitivity of the domestic kits for adr, adw, ay serotypes were 0.307, 0.419, 0.513 ng/ml, respectively. There was a significant difference between serotypes (F = 97.30, P < 0.01). The mean analytical sensitivity of the overseas kits for adr, adw, ay serotypes were 0.054, 0.066, 0.050 ng/ml respectively, with no significant difference between serotypes (F = 0.65, P > 0.05). The analytical sensitivity of the overseas kits for all the three serotypes was higher than that of the domestic kits (P < 0.01). There was no significant difference found between the analytical sensitivities of the kits produced by the same manufactory using 30- or 60-minute incubation of detection (P > 0.05). In contrast, there was significant difference noticed between the analytical sensitivities of the kits produced by the same manufactory when tested for 10 or 15-minute coloration of the results (P < 0.01). CONCLUSION: Analytical sensitivity of the HBsAg EIA domestic kits should be further improved, especially for detecting adw and ay serotypes.

[Comparison of the kinesis of immune responses in mice vaccinated by different kinds of recombinant hepatitis B vaccines].

OBJECTIVE: To evaluate the kinesis of cellular and humoral immune responses to different kinds of recombinant hepatitis B(rHB) vaccines in the immunized mice. METHODS: At serial time points, the levels of IFN-gamma and IL-2 secreted by spleens mononuclear cells (MNC) of the vaccinated mice were detected by enzyme-linked immunospot methods (ELISPOT) after stimulation in vitro with HBsAg MHC class I peptide S28-39 or HBsAg. The lymphocytotoxicity of the immunized mice were also detected (CTL) by a specific lysis assay and the levels of anti-HBs were measured by the Abbott IMX kit. RESULTS: The peak values of IFN-gamma and IL-2 in vaccinated mice were detected by ELISPOT, 10 - 14 days after immunization. The CTL and the level of IFN-gamma induced by rHB vaccine derived from yeast cells (Hansenula polymorpha) (rHP vaccine) were significantly higher than the other two vaccines (P < 0.05). The maximum lysis of CTL appeared in the vaccinated mice on day 10 after immunization, with the percentage of 39.8%. The levels of IL-2 induced by rHP vaccine were significantly higher than the other two vaccines (P < 0.05). However, the IL-2 levels in the rSC (saccharomyces cerevisiae) vaccine group were higher as compared with the rCHO vaccine group at day 7 and day 14 (7 d t = 4.595, P = 0.001 < 0.05; 14 d t = 5.721, P = 0.000 < 0.05) after immunization. The cellular immune response to the rHP vaccine was the strongest while it was the lowest to the rCHO vaccine at day 7 after immunization. The sero-positive rates and the titers of anti-HBs in the vaccinated mice increased with time after vaccination. The titers of anti-HBs in the rCHO vaccine group at day 7 were similar to the rSC vaccine group, but significantly higher than that of the rHP vaccine group (P = 0.044 < 0.05). The anti-HBs titers of the rCHO vaccine group at day 14 were significantly higher as compared to the rSC (P = 0.012 < 0.05) and rHP (P = 0.009 < 0.05) vaccine groups. CONCLUSION: The immune responses induced by the three kinds of rHB vaccines were different in their patterns and levels. According to the intensity of early cellular immune response, the two yeast HB vaccines were superior to the rCHO vaccine, especially to the rHP vaccine. In contrast, the rCHO vaccine induced early seroconversion and high levels of anti-HBs.

[Preparation of the national reference panel for hepatitis B surface antigen].

OBJECTIVE: To establish the national quantity standard of hepatitis B surface antigen according to the world health organization' s standard material and prepare the national liner reference panel for hepatitis B surface antigen. METHODS: Sera from hepatitis B patients and health blood donors in different areas were collected and detected by domastic HBsAg kits, anti-HBs kits, HBeAg kits, anti-HBe kits, anti-HBc kits and anti-HCV, and then confirmed by the kits produced by Abbott, which was approved by WHO. One serum with high concentration of HBsAg was calibrated with the standard sample of WHO. And then it was diluted by 1.5 fold as the liner HBsAg reference panel. RESULTS: The HBsAg concentration of one serum was 1226 IU/ml calibrated by 21 independent standardization measurements with 7 kinds of kits. The coefficient of variation of each calibration were less then 15%. A panel contained 8 serial dilutions was established as the national liner HBsAg reference panel. The permitted range of every dilution was stipulated and the stability of the panel was detected by accelerated test. CONCLUSIONS: The national quantity standard of hepatitis B surface antigen was established and the national quantitative reference panel for HBsAg which contains eight liner serum was developed.

[Cellular immune responses of recombinant hepatitis B (rHB) vaccine and HBsAG derived from Hansenula polymorpha cells].

OBJECTIVE: To study the kinetics of immune response in mice and human immunized with rHB vaccine or rHBsAg derived from yeast cells (Hansenula polymorpha). METHODS: With different doses,the level of IFN-gamma secreted by spleen mononuclear cells (MNC) including CD8+ T cells by MACs of mice were detected by enzyme-linked immunospot (ELISPOT) methods after stimulation in vitro with HBsAg MHC class I peptide S28-39, respectively. At serial time points, the immunized mice were detected for IFN-gamma by ELISPOT as above and for the lymphocytotoxicity test (CTL) by specific lysis assay. The levels of IFN-gamma, IL-2, IL-5 and anti-HBs in mice induced by rHB vaccine were detected after single or three doses. Four adults were vaccinated with rHB vaccine according to 0, 1 and 2 month schedule. The peripheral blood mononuclear cells (PBMCs) were collected at the 3, 8, 21, 34 and 65 days after the first dose. The CD8+ T cells with high purity obtained by sorting from PBMCs were stimulated with rHBsAg or HBsAg peptides. The SFC of IFN-gamma, IL-2 and IL-4 of CD4+ and CD8+ T cells were determined by ELISPOT. RESULTS: The cytokine of IFN-gamma became detectable on day 7 and its peak value appeared on day 14 by ELISPOT. The CTL was detected on day 7 and the maximum lysis of CTL appeared on day 28. The cellular immune response of IFN-gamma of MNCs were significantly correlated with the doses vaccinated from 1 microg to 8 microg (r(positive rates) = 0.951, P(positive rates) = 0.049 < 0.05; r(SFC) = 0.996, P(SFC) = 0.000 < 0.05). IFN-gamma SFC of CD+ T cells were significantly associated with the doses from 1 microg to 4 microg (r = 0.999, P = 0.025 < 0.05). The HBsAg specific cellular immune an d humoral responses of mice immunized with three doses were significantly higher than that with a single dose (P < 0.05). The characteristics of IFN-gamma, IL-2 and IL-4 of CD4+ and CD8+ T cells were variable between individuals immunized with the same rHB vaccine. The level of IL-2 and IL-4 of responders were significantly related to the titer of anti-HBs. CONCLUSION: Data from this study showed the kinesis of cellular immunity in mice and adults vaccinated with rHBsAg or rHB vaccine respectively, and the characteristics of cellular immune response in adults induced by the vaccine. Our data provided the basis of standardizing the analysis of cellular immune response to rHB vaccine.

[Primary evaluation of anti-HEV diagnostic reagent by experimental infection animal model with hepatitis E virus].

OBJECTIVE: To evaluate anti-HEV diagnostic kits by experimental infecting rhesus monkeys with HEV. METHODS: Eight rhesus monkeys were infected with genotype 1 and 4 HEV separately. The alanine aminotransferase (ALT) level of all monkeys were detected before and after the process of infection. HEV RNA in stool specimens was tested by reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Anti-HEV IgG in serum was detected by GL-IgG and WT-IgG. RESULTS: HEV RNA presented in the stool of all the 8 monkeys after infection. The ALT level of 1 monkey infected with genotype 1 HEV and 2 monkeys infected with genotype 4 HEV appeared abnormally after infection. Tested by GL-IgG, 2 of the 4 monkeys infected with genotype 1 HEV and 1 of 4 monkeys infected with genotype 4 HEV seroconverted to anti-HEV IgG. However, when tested by WT-IgG, all the infected monkeys seroconverted to anti-HEV IgG. The anti-HEV IgG tested by WT-IgG was positive during the whole observation period,and the anti-HEV IgG measured by GL-IgG only remained 12 weeks after infection. Detected by GL-IgG and WT-IgG, seropositive conversion of the anti-HEV IgG happened almost at the same time. CONCLUSION: Both GL-IgG and WT-IgG could detect the anti-HEV IgG of experimentally infected rhesus monkeys but the WT-IgG had a higher sensitivity for detection of anti-HEV IgG than

[Sensitivity and specificity of 4 domestic ELISA kits for detection of hepatitis B virus markers].

OBJECTIVE: To compare and analyze the sensitivity, specificity of 4 domestic ELISA kits for detection of hepatitis B virus (HBV) markers (HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc). METHODS: Five hundred and ninety four serum samples collected from patients with chronic hepatitis B and abnormal blood donors were detected for HBV markers and by 4 domestic ELISA kits. Samples with conflicting results by different diagnostic kits were retested. Samples with the HBsAg values close to the cut-off point were detected by Abbott HBsAg confirmation kit (Architect HBsAg confirm). Sensitivity of the kits was determined, using the national sensitivity reference panels for HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc. RESULTS: The rates of sensitivity on 4 domestic kits for detection of HBsAg were 4 to 10 times lower, and on the 4 domestic kits for detection of anti-HBs, HBeAg, anti-HBe and anti-HBc were 4 to 16 times lower, as compared to Abbott Architect kits. In addition, the domestic HBV ELISA kits had some false positive results. The total coincidence rates of HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc were 96.46%-98.15%, 94.28%-98.15%, 98.15%-99.49%, 90.07%-96.30%, 92.09%-96.80%, respectively. CONCLUSION: Both sensitivity and specificity of the domestically produced HBV ELISA kits should be improved.

[Evaluation of multiplex nucleic acid testing assays for screening of hepatitis B virus DNA in blood donation process].

OBJECTIVE: To evaluate the multiplex nucleic acid testing (NAT) assays for HBV, HCV and HIV in detecting HBV DNA in plasma samples. METHODS: 534 plasma samples collected form several areas were detected with Abbott Architect i2000 HBsAg, ani-HBs, HBeAg, anti-HBe, anti-HBc and anti-HBc IgM diagnostic kits. HBV DNA levels of those samples were detected with Roche COBAS AmpliPrep/COBAS TaqMan HBV Test. Two kinds of multiplex NAT assays for HBV, HCV and HIV were used to test HBV DNA of those 534 samples. Results of serology-markers and quantitative HBV DNA levels with results of NAT were compared. RESULTS: HBV DNA was positive in all 81 HBsAg, HBeAg and anti-HBc positive samples, detected by both of NAT assays. HBV DNA was positive in 11 and 19 of 200 HBsAg negative samples when detected with the two kinds of NAT assays separately. Compared with the quantitative results detected by Roche COBAS AmpliPrep/COBAS TaqMan HBV Test, the HBV DNA positive rates were 96.9% and 94.3% in 193 samples of HBV DNA levels over 500 IU/ml while 40.2% and 45.3% in 117 samples of HBV DNA levels below 500 IU/ml while 99.3% and 96.0% in 151 samples of DNA negative HBV. CONCLUSION: There are some occult low level HBV DNA carriers with HBsAg negative results in China. NAT assays for HBV, HCV and HIV may be useful to improve the transfusion safety.