kukoamine-b and Sepsis

To evaluate the safety, tolerability, pharmacokinetics, and efficacy of kukoamine B (KB), an alkaloid compound with high affinity for both lipopolysaccharide (LPS) and oligodeoxynucle-otides containing CpG motifs (CpG DNA), in patients with sepsis-induced organ failure.. This was a multicenter, randomized, double-blind, placebo-controlled phase IIa trial. Patients with sepsis-induced organ failure were randomized to receive either KB (0.06, 0.12, or 0.24 mg/kg) or placebo, every 8 h for 7 days. Primary endpoint was safety, and secondary endpoints included pharmacokinetic (PK) parameters, changes in inflammatory mediators' level, and prognostic parameters.. Of 44 patients enrolled, adverse events occurred in 28 patients [n = 20, 66.7% (KB pooled); n = 8, 57.1% (placebo)], while treatment emergent adverse events were reported in 14 patients [n = 10, 33.3% (KB pooled); n = 4, 28.6% (placebo)]. Seven patients died at 28-day follow-up [n = 4, 13.3% (KB pooled); n = 3, 21.4% (placebo)], none was related to study drug. PK parameters suggested dose-dependent drug exposure and no drug accumulation. KB did not affect clinical outcomes such as ΔSOFA score, vasopressor-free days or ventilator-free days.. In patients with sepsis-induced organ failure, KB was safe and well tolerated. Further investigation is warranted.. http://ClinicalTrials.gov, NCT03237728.

Topics: Caffeic Acids; Double-Blind Method; Humans; Sepsis; Spermine; Treatment Outcome; Vasoconstrictor Agents

To assess the appropriateness of the body weight or fixed dosing regimen, a population pharmacokinetic (PopPK) model of kukoamine B has been built in sepsis patients.. Plasma concentrations of kukoamine B and the covariates information were taken from 30 sepsis patients assigned into 0.06 mg/kg, 0.12 mg/kg and 0.24 mg/kg groups in a Phase IIa clinical trial. The PopPK model was built using a nonlinear mixed-effect (NLME) modelling approach. Based on the final model, PK profiles were respectively simulated 500 times applying the body weight and renal function information of 12 sepsis patients from the 0.24 mg/kg group on the body weight or the fixed dosing regimen. For each dosing regimen, PK profiles of 6000 virtual patients were obtained. Statistical analyses for C. The PopPK model was successfully developed using the NLME approach. A bi-compartmental model was selected as the basic model. Renal function was identified as a statistically significant covariate of systemic clearance with the objective function value (OFV) decreasing 8.6, resulting in a 5.2% decrease in inter-individual variability (IIV) of systemic clearance. Body weight was not identified as a statistically significant covariate. Simulation results demonstrated two methods had a bias of 8.1% for C. Based on the simulation results, a fixed dosing regimen was recommended in the subsequent clinical trials.

Topics: Body Weight; Caffeic Acids; Computer Simulation; Dose-Response Relationship, Drug; Humans; Models, Biological; Sepsis; Spermine

To study the role of Kukoamine B (KB) in inhibiting the inflammatory response of small intestine in septic mice and its molecular mechanisms.. Twenty-four male ICR mice were randomly divided into control group, model group, and KB intervention group (each, n = 8). Sepsis model was reproduced by intra-peritoneal injection of 20 mg/kg lipopolysaccharide (LPS), while equivalent normal saline was given in control group, and 20 μg/kg KB was injected through caudal vein 4 hours after LPS challenge in KB intervention group. The blood/tissue samples (jejunum and ileum) were harvested 8 hours after LPS injection. The levels of plasma LPS, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured. The pathological changes in small intestine tissues were observed under light microscope, while the levels of inflammatory cytokines TNF-α and IL-1β in the tissue homogenates (jejunum and ileum) were assessed by enzyme linked immunosorbent assay (ELISA). The activity of myeloperoxidase (MPO) was measured by colorimetry. The expression of intercellular adhesion molecule-1 (ICAM-1) was determined by immunohistochemistry. The expressions of inducible nitric oxide synthase (iNOS) mRNA was assayed by reverse transcription-polymerase chain reaction (RT-PCR). The activation of nuclear factor-Κβ (NF-Κβ) was determined by Western Blot.. The mice in model group were found to have an increase in microvascular permeability, interstitial edema, and infiltration of white blood cells, and the levels of LPS, TNF-α and IL-1β in their plasma, with an increase in concentrations of TNF-α and IL-1β, activity of MPO, positive expression of ICAM-1, expression of iNOS mRNA and NF-ΚB protein in small intestine (jejunum and ileum). Compared with model group, in mice with KB intervention, microvascular permeability, interstitial edema, and infiltration of white blood cells were reduced significantly, while the levels of LPS, TNF-α and IL-1β in plasma, the concentration of TNF-α and IL-1β, the activity of MPO, the positive expression of ICAM-1, the expression of iNOS mRNA and NF-ΚB protein in small intestine (jejunum and ileum) were significantly decreased [plasma LPS (kEU/L): 654.09±28.13 vs. 1 155.65±47.15, TNF-α (ng/L): 12.75±0.47 vs. 30.61±0.71, IL-1β (ng/L): 53.06±5.32 vs. 64.47±2.61; jejunum TNF-α (ng/L): 43.27±1.20 vs. 64.82±2.09, IL-1β (ng/L): 326.38±14.47 vs. 535.22±13.48, MPO (U/g): 0.14±0.01 vs. 0.32±0.02, iNOS mRNA (2(-ΔΔCt)): 2.39±0.13 vs. 10.80±0.22, NF-ΚB protein (gray value): 0.687±0.062 vs. 1.404±0.046; ileum TNF-α (ng/L): 62.75±3.92 vs. 104.24±2.82, IL-1β(ng/L): 408.06±1.70 vs. 521.97±1.16, MPO (U/g): 0.36±0.08 vs. 0.66±0.05, iNOS mRNA (2(-ΔΔCt)): 1.65±0.11 vs. 3.59±0.29, NF-ΚB protein (gray value): 0.830±0.114 vs. 1.609±0.051, all P <0.05].. KB can combine with LPS and inhibit LPS/Toll-like receptor 4 (TLR4) signaling pathway, thereby significantly inhibit the inflammatory response and protect the function of the small intestine in LPS-induced septic mice.

Topics: Animals; Caffeic Acids; Cytokines; Disease Models, Animal; Intercellular Adhesion Molecule-1; Interleukin-1beta; Intestine, Small; Lipopolysaccharides; Male; Mice; Mice, Inbred ICR; NF-kappa B; Nitric Oxide Synthase Type II; RNA, Messenger; Sepsis; Signal Transduction; Spermine; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

To investigate the inhibitory effect of kukoamine B (KB) on lung inflammatory responses in mice with sepsis and its possible molecular mechanism.. Twenty-eight male mice were randomly divided into control group (n=8), lipopolysaccharide (LPS) group (n=10), and LPS + KB group (n=10). Sepsis model was reproduced by intra-peritoneal injection of 20 mg/kg LPS, while equivalent normal saline was given in control group, and 20 μg/kg KB was injected through caudal vein 4 hours after LPS challenge in LPS + KB group. After 8 hours of LPS challenge, the concentration of LPS in plasma and the activity of myeloperoxidase (MPO) in the lung tissue were determined. The contents of tumor necrosis factor-α (TNF-α) and interleukin-1β(IL-1β) in plasma, alveolar lavage fluid and lung tissue homogenates were assessed by enzyme linked immunosorbent assay (ELISA). The activation of nuclear factor-ΚB (NF-ΚB) and the expression of inducible nitric oxide synthase (iNOS) in lung tissue were determined by Western Blot. The pathological changes in lung tissues were observed with hematoxylin-eosin (HE) staining. The expression of intercellular adhesion molecule-1 (ICAM-1) in lung tissue was determined by immunohistochemistry.. Compared with control group, the concentration of LPS in plasma (1 155.650±147.149 kEU/L vs. 31.390±18.859 kEU/L), MPO activity (1.177±0.093 U/g vs. 0.775±0.166 U/g), NF-ΚB activity (gray value: 1.557±0.105 vs. 0.824±0.032) and the expression of iNOS (gray value: 0.650±0.129 vs. 0.392±0.097) were significantly increased in LPS group (all P<0.05). After KB intervention, the concentration of LPS (624.461±149.012 kEU/L), MPO activity (0.919±0.023 U/g), NF-ΚB activity (1.127±0.074) and the expression of iNOS (0.425±0.066) were significantly lowered (all P<0.05). Compared with control group, the contents of TNF-α (47.325±13.864 ng/L vs. 6.534±0.544 ng/L, 13.382±2.231 ng/L vs. 3.748±0.692 ng/L, 31.127±7.399 ng/L vs. 14.948±4.673 ng/L) and IL-1β (74.329±11.890 ng/L vs. 29.921±6.487 ng/L, 9.422±2.674 ng/L vs. 1.105±0.364 ng/L, 528.509±32.073 ng/L vs. 109.945±13.561 ng/L) in plasma, alveolar lavage fluid and lung tissue homogenates were obviously enhanced in LPS group (all P<0.05). With KB intervention, the contents of TNF-α (20.331±7.789 ng/L, 7.145±1.202 ng/L, 15.966±2.946 ng/L) and IL-1β (57.707±8.098 ng/L, 2.212±0.878 ng/L, 426.154±11.270 ng/L) were markedly reduced (plasma TNF-α: F=16.052, P=0.002; IL-1β: F=20.649, P=0.000; lung tissue homogenates TNF-α: F=31.134, P=0.001; IL-1β: F=22.792, P=0.002; alveolar lavage fluid TNF-α: F=10.013, P=0.009; IL-1β: F=319.857, P=0.000). In addition, leukocyte infiltration to the lung tissue was attenuated, and the expression of ICAM-1 was reduced by KB in histological examination.. KB, as a neutralizer of LPS, can inhibit the release of inflammatory mediators, reduce the pulmonary inflammatory response and protect the function of lung in septic mice.

Topics: Animals; Caffeic Acids; Disease Models, Animal; Interleukin-1beta; Lung; Male; Mice; Mice, Inbred ICR; NF-kappa B; Nitric Oxide Synthase Type II; Peroxidase; Sepsis; Spermine; Tumor Necrosis Factor-alpha

Treating sepsis remains challenging at present. Bacterial lipopolysaccharide (LPS) and bacterial DNA/CpG DNA are important pathogenic molecules and drug targets for sepsis. It is thus a promising strategy to treat sepsis by discovering agents that neutralize LPS and CpG DNA simultaneously. In this study, we present evidences of the biosensor based screening and isolation of active anti-sepsis fractions and monomers from traditional Chinese herbs using dual targets (LPS and CpG DNA) guided drug discovery strategy. Firstly, LPS or CpG DNA was immobilized on surfaces of cuvettes in the biosensor to establish a screening platform. Then, Cortex lycii with both highest affinities was selected out from one hundred and fourteen traditional Chinese herbs. In subsequent experiments, chromatography was utilized and coupled with the biosensor to purify fractions with a higher affinity for LPS and CpG DNA. In line with affinity assay, these fractions were shown to neutralize LPS and CpG DNA and inhibit their activity in vitro and in vivo. Lastly, the contributing monomer Kukoamine B (KB) was purified. KB neutralized LPS and CpG DNA in vitro. It inhibited TLR4, TLR9 and MyD88 mRNA expressions up-regulated by LPS and CpG DNA, and also attenuated the LPS and CpG DNA elicited nuclear translocation of NF-κB p65 protein in RAW264.7 cells. It also protected mice from lethal challenge of heat-killed E. coli, a mixture of LPS and CpG DNA. In conclusion, we presented a dual target guided discovery of a novel anti-sepsis agent KB from traditional Chinese herbs via combination of biosensor technology and chromatography methods.

Topics: Animals; Biosensing Techniques; Caffeic Acids; Cell Line; CpG Islands; DNA, Bacterial; Drug Discovery; Drugs, Chinese Herbal; Lipopolysaccharides; Macrophages; Mice; Sepsis; Spermine

Lipopolysaccharides (LPS) and oligodeoxynucleotides containing CpG motifs (CpG DNA) are important pathogenic molecules for the induction of sepsis, and thus are drug targets for sepsis treatment. The present drugs for treating sepsis act only against either LPS or CpG DNA. Hence, they are not particularly efficient at combating sepsis as the latter two molecules usually cooperate during sepsis. In this study, a natural alkaloid compound kukoamine B (KB) is presented as a potent dual inhibitor for both LPS and CpG DNA.. The affinities of KB for LPS and CpG DNA were assessed using biosensor technology. Direct interaction of KB with LPS and CpG DNA were evaluated using neutralization assays. Selective inhibitory activities of KB on pro-inflammatory signal transduction and cytokine expression induced by LPS and CpG DNA were analysed by cellular assays. Protective effects of KB in a sepsis model in mice were elucidated by determining survival and circulatory LPS and tumour necrosis factor-alpha (TNF-α) concentrations.. KB had high affinities for LPS and CpG DNA. It neutralized LPS and CpG DNA and prevented them from interacting with mouse macrophages. KB selectively inhibited LPS- and CpG DNA-induced signal transduction and expression of pro-inflammatory mediators without interfering with signal pathways or cell viability in macrophages. KB protected mice challenged with heat-killed Escherichia coli, and reduced the circulatory levels of LPS and TNF-α.. This is the first report of a novel dual inhibitor of LPS and CpG DNA. KB is worthy of further investigation as a potential candidate to treat sepsis.

Topics: Animals; Caffeic Acids; CpG Islands; Cytokines; Disease Models, Animal; DNA-Binding Proteins; Escherichia coli; Female; Gene Expression; Lipopolysaccharides; Macrophages, Peritoneal; Male; Mice; Sepsis; Spermine; Survival Analysis