[Effect of recombinant human thrombin for hemostasis in liver resection: a randomized controlled phase â ¢ clinical trial].

Objective: To evaluate the hemostatic efficacy, safety and immunogenicity of recombinant human thrombin in the treatment of liver wounds that still ooze after conventional surgical hemostasis. Methods: A multicenter, stratified randomized, double-blind, placebo-controlled phase Ⅲ trial with a planned enrollment of 510 subjects at 33 centers, with a 2∶1 randomization to the thrombin group versus the placebo group. An interim analysis will be conducted after approximately 70% of the subjects have completed the observation period. The primary efficacy endpoint was the rate of hemostasis within 6 minutes at the point of bleeding that could be evaluated. Safety analysis was performed one month after surgery, and the positive rates of anti-drug antibody (ADA) and neutralizing antibody were evaluated. Results: At the interim analysis, a total of 348 subjects had been randomized and received the study drug (215 were male and 133 were female). They were aged 19-69 (52.9±10.9)years. Among them, 232 were in the thrombin group and 116 were in the placebo group, with balanced and comparable demographics and baseline characteristics between the two groups. The hemostasis rate at 6 minutes was 71.6% (95%CI:65.75%-77.36%) in the thrombin group and 44.0% (95%CI: 34.93%-53.00%) in the placebo group, respectively (P<0.001). No grade≥3 drug-related adverse events and no drug-related deaths were reported from the study.No recombinant human thrombin-induced immunologically-enhanced ADA or immunologically-induced ADA was detected after topical use in subjects. Conclusion: Recombinant human thrombin has shown significant hemostatic efficacy and good safety in controlling bleeding during liver resection surgery, while also demonstrating low immunogenicity characteristics.

Autophagy-dependent generation of Axin2+ cancer stem-like cells promotes hepatocarcinogenesis in liver cirrhosis.

Autophagy is a pathophysiological phenomenon in liver cirrhosis that can further progress into hepatocarcinoma. Liver cancer stem cells (CSCs) are believed to initiate hepatocarcinogenesis. To investigate the precise mechanism related to the origin of CSCs in liver cirrhosis and hepatocarcinogenesis, we labeled Axin2+ hepatic cells with EGFP in Axin2Cre;Rosa26EGFP transgenic rats, and then stratified clinical and rat liver cirrhosis samples by autophagy flux. Clinical follow-up and lineage tracing in transgenic rat liver cirrhosis revealed that while Axin2/EGFP+ hepatic cells were present in normal livers and cirrhotic livers without aberrant autophagy, hepatic Axin2/EGFP+CD90+ cells were generated exclusively in cirrhotic livers with aberrant autophagy and promoted hepatocarcinogenesis. Aberrant autophagy in liver cirrhosis resulted in hepatocyte growth factor (HGF) expression, leading to activation of Met/JNK and Met/STAT3 signaling in sorted hepatic Axin2/EGFP+ cells and their transition into Axin2/EGFP+CD90+ cells that possess CSC properties. In a transgenic rat liver cirrhosis model, induction or inhibition of autophagy in cirrhotic livers by systemic administration of rapamycin or chloroquine or transfection with Atg3- and Atg7-shRNAs significantly induced or suppressed HGF expression, which in turn increased or reduced generation of EGFP+CD90+ hepatic cells by activating or inactivating Met/JNK and Met/STAT3 signaling, thereby promoting or preventing hepatocarcinogenesis. Systemic treatment with HGF-shRNA, SP600125 or stattic also reduced generation of EGFP(Axin2)+ hepatic cell-originated CD90+ CSCs in aberrant autophagic cirrhotic livers by inactivating HGF/Met/JNK or HGF/Met/STAT3 signaling, further preventing hepatocarcinogenesis. These data suggest that activation of Met/JNK and Met/STAT3 signaling in Axin2+ hepatic cells via autophagy-dependent HGF expression and the resultant generation of Axin2+CD90+ CSCs is a major mechanism of hepatocarcinogenesis in cirrhotic livers.

On the human CYP2C9*13 variant activity reduction: a molecular dynamics simulation and docking study.

Cytochrome P450 2C9 (CYP2C9) plays a key role in the metabolism of clinical drugs. CYP2C9 is a genetically polymorphic enzyme and some of its allelic variants have less activity compared to the wild-type form. Drugs with a narrow therapeutic index may cause serious toxicity to the individuals who carry such allele. CYP2C9*13, firstly identified by some of the present authors in a Chinese poor metabolizer of lornoxicam, is characterized by mutation encoding Leu90Pro substitution. Kinetic experiments show that CYP2C9*13 has less catalytic activity in elimination of diclofenac and lornoxicam in vitro. In order to explore the structure-activity relationship of CYP2C9*13, the three-dimensional structure models of the substrate-free CYP2C9*1 and its variant CYP2C9*13 are constructed on the basis of the X-ray crystal structure of human CYP2C9*1 (PDB code 1R9O) by molecular dynamics simulations. The structure change caused by Leu90Pro replacement is revealed and used to explain the dramatic decrease of the enzymatic activity in clearance of the two CYP2C9 substrates: diclofenac and lornoxicam. The trans configuration of the bond between Pro90 and Asp89 in CYP2C9*13 is firstly identified. The backbone of residues 106-108 in CYP2C9*13 turns over and their side chains block the entrance for substrates accessing so that the entrance of *13 shrinks greatly than that in the wild-type, which is believed to be the dominant mechanism of the catalytic activity reduction. Consequent docking study which is consistent with the results of the kinetic experiments by Guo et al. identifies the most important residues for enzyme-substrate complexes.

Changes in cardiovascular activity during an increase in biliary tract pressure in rabbits.

In order to study the mechanism of decreased blood pressure caused by an acute increase in biliary tract pressure, we observed house rabbit model of self-made caecus for changes in cardiovascular function when biliary tract pressure was increased. It was found that both the blood pressure and cardiac output evidently decreased (P < 0.05) parallelly, and the systolic pressure decreased more markedly than diastolic pressure. At the same time there was fluctuation in heart rate and in central venous pressure; but there was no significant difference between them (P > 0.05), suggesting that in the absence of infective agents, the increased biliary tract pressure can bring about a decrease in cardiac output, which is an important factor contributing to an early blood pressure decrease in acute cholangitis of severe type (ACST).

Effect of increasing biliary tract pressure on house rabbit blood dynamics in acute cholangitis of severe type.

In this study 12 Japanese long ear rabbits were used as models of acute cholangitis of severe type (ACST), and also an increasing pressure apparatus of self-made caecus to form high pressure of the biliary tract. The animals were observed for changes in blood dynamics in an attempt to explore the effect and relation of high pressure of biliary tract and infective element in pathogenesis of ACST. It was found that when the biliary pressure was increased within 120 min in the 20 kPa group, the blood endotoxin level showed no obvious increment (P > 0.05), but the decreased range of average MAP (mean artery pressure) was over 4 kPa, and the cardiac output also decreased evidently (P < 0.05), and that when the biliary pressure was decreased, MAP and cardiac output were restored to normal gradually. Of these animals 3 didn't restore their normal condition when the blood pressure decreased to zero and died finally. Meanwhile the electric discharge frequency of the right greater splanchnic nerves increased (P < 0.05), but when pressure was reduced, the frequency slowed down. From the above findings, the authors came to the conclusion that the rapid increase of the biliary tract pressure is the important factor leading to a decrease in blood pressure of ACST, and even bringing about irreversible shock, which is involved in the activity of splanchnic nerves.

Effect of experimental acute hypertension of the biliary tract on hemodynamics and activity of the major splanchnic nerve in rabbits.

An experimental study of the effect of acute biliary hypertension on hemodynamics and activity of the major splanchnic nerve was conducted in Japanese big ear white rabbits. A catheter with an inflatable rubber bag fixed to its anterior end was inserted into the extrahepatic biliary duct via the duodenum, and a biliary high pressure of 20 kFa (150 mmHg) was created and maintained for 2 h by inflating the bag with water. The right major splanchnic nerve was isolated and the impulse frequencies of the nerve were recorded during the study. Arterial blood pressure was also dynamically monitored. A significant fall of the arterial blood pressure (P less than 0.01) and an increase in impulse frequency of the nerve (P less than 0.01) were found in this "simple type" acute biliary high pressure without infection. Biliary decompression immediately eliminated these abnormalities (P less than 0.01). The drop in blood pressure was much less prominent if the right splanchnic nerve was blocked prior to biliary high pressure.