Permeability is a key factor in the bioavailability of oral drugs. Therefore, in the early stage of drug discovery, accurate and efficient evaluation of drug permeability is essential. The parallel artificial membrane permeability assay (PAMPA) with Caco-2 cells model was used by the industry as early evaluation methods. At present, the Ussing chamber rat model is also widely used. This review summarizes the human data for the in vivo single-pass perfusion technique (Loc-I-Gut) – the gold standard, and then focuses on the basic principles, experimental operation, and efficiency of the three in vitro methods, with correlation to the effective permeability coefficient (P_eff) and fractional absorbed (Fa) in man. We provide recommendations for the use of proper permeability methods at different stages in drug discovery and development.

BACKGROUND: von Willebrand factor (vWF) is only released from endothelial cells and platelets and is an in vivo and in vitro marker of endothelial injury in septic patients with acute lung injury (ALI). Interleukin-8 (IL-8), as a proinflammatory mediator causing recruitment of inflammatory cells, induces an increase in oxidant stress mediators and makes it as a key parameter for localized inflammation. However, it has not been well established whether the level of serum IL-8 is associated with the severity of lung injury and whether it is a prognosis marker for severe lung contusion. This study was to investigate the expression of plasma vWF and IL-8 and their association with the severity and outcomes of severe pulmonary contusion. METHODS: A total of 63 patients were divided into a severe pulmonary contusion with acute respiratory distress syndrome (ARDS) group and a non-ARDS group, or a survivor group and a non-survivor group, or an injury severity score (ISS) <20 group and an ISS ≥20 group. Another 20 healthy volunteers served as controls. The levels of plasma vWF and serum IL-8 were measured by enzyme-linked immunosorbent assay (ELISA) at 1, 3, 5 and 7 days after injury. The expression patterns of the plasma vWF and serum IL-8 were compared between different groups. RESULTS: The concentrations of plasma vWF and serum IL-8 were significantly increased in all severe pulmonary contusion patients at all time points in comparison with the control group. The concentrations of plasma vWF in patients with ARDS increased during the whole study period, but vWF in patients with non-ARDS increased gradually until day 5 and then decreased at day 7. The concentration of serum IL-8 showed a similar expression pattern in both groups, but the expression increased more significantly in the ARDS group than in the non-ARDS group. Interestingly, both plasma vWF and serum IL-8 levels steadily increased in the non-survivor group. Furthermore, the level of plasma vWF was higher in the ISS≥20 group than in the ISS<20 group. The level of serum IL-8 in the ISS≥20 group was consistently high, while that in the ISS<20 group peaked at day 3 and decreased at day 5. In addition, the level of plasma vWF was positively correlated with platelet count, but negatively correlated with oxygen index. The level of serum IL-8 was positively correlated with white blood cell count and ISS score, and inversely correlated with oxygen index. CONCLUSION: The elevated levels of plasma vWF and serum IL-8 in severe pulmonary contusion patients reflect the severity of pulmonary injury and patients outcomes, suggesting that the plasma vWF and serum IL-8 are sensitive markers for clinical evaluation of the severity of pulmonary injury and predication of patient prognosis.

To investigate the effect of Jiawei Foshou San and its various combined administration on hepatic P450 enzyme activity and hepatocyte morphology in rats. Rats were orally administered with drugs for four weeks and then sacrificed to prepare liver microsomes. The liver microsomes were incubated with the cocktail method; The metabolites were determined with the rapid liquid chromatography with tandem mass spectrometry (LC-MS/MS) to investigate the hepatocyte P450 enzyme activity. In addition, the hepatic pathological changes were observed by using the hematoxylin and eosin (HE) staining. Compared with the control group, the enzyme activity of CYP1A2 and CYP3A4 in the Jiawei Foshou san group showed a significant rise (P < 0.05); the enzyme activity of CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in the ferulic acid + ligustrazine group and the ligustrazine + tetrahydropalmatine group showed a significant rise (P < 0.05) ; the enzyme activity of CYP1A2, CYP2D6 and CYP2E1 in the ligustrazine group showed a significant rise (P < 0.05); the enzyme activity of CYP3A4 in the ferulic acid group showed a significant reduction (P < 0.05). After the administration with various drugs, the hepatocyte morphologies in the ferulic acid group and the ligustrazine group were normal. The pathological changes were observed in the tetrahydropalmatine group, such as unclear boundary of hepatic lobules, disordered hepatic cell arrangement, blurred edge, anisokaryosis and infiltration of inflammatory cells. The ferulic acid + tetrahydropalmatine group, the ligustrazine + tetrahydropalmatine group and the Jiawei Foshou San group also showed inflammatory infiltration, but with less pathological changes, particularly the Jiawei Foshou San group. The study result shows that Jiawei Foshou San can induce the enzyme activity of CYP1A2 and CYP3A4, and ligustrazine may be the effective substance for inducing CYP1A2. Its combination with ferulic acid and ligustrazine can significantly reduce the liver toxicity of tetrahydropalmatine.
Animals ; Cytochrome P-450 Enzyme System ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Hepatocytes ; drug effects ; enzymology ; Microsomes, Liver ; drug effects ; enzymology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the absorption mechanism of genistein self-microemulsifying system in rat intestines.

METHODThe concentrations of phenol red and genistein by in situ perfusion in rats were determined by UV and HPLC, respectively. The effects of drug concentrations, pH, various intestinal segments and P-glycoprotein (P-gp) inhibitor verapamil on the absorption had been studied.

RESULTThe absorption rate constant (Ka) of genistein had no significant difference at concentrations of 0.05-0.5 mg x mL(-1) and pH of 5.4-7.8 in perfusion. It was Ka of jejunum > ileum > duodenum > colon. The absorption of genistein in jejunum had significant difference (P < 0.05) compared with other parts of intestines. Ka was increased obviously when verapamil was coper-fused with genistein (P < 0. 05).

CONCLUSIONThe absorption of genistein self-microemulsifying system is a first order process with passive diffusion mechanism related to P-gp efflux. It can be absorbed at all segments of rat intestine, and the jejunum is the best absorption segment, pH had no special effect on the absorption of genistein self-microemulsifying system in rat intestine.

ATP Binding Cassette Transporter, Sub-Family B ; antagonists & inhibitors ; Animals ; Chromatography, High Pressure Liquid ; Emulsions ; Genistein ; analysis ; metabolism ; pharmacokinetics ; Hydrogen-Ion Concentration ; Intestinal Absorption ; drug effects ; Intestines ; drug effects ; metabolism ; Male ; Organ Specificity ; Rats ; Rats, Wistar ; Temperature ; Verapamil ; pharmacology

Objective: To prepare monoclonal antibodies (McAb) with cardiac troponin I (cTnI) which was purified from fresh human cardiac muscle within 6 h. Methods: (1) Extraction and purification of human cTnI: cTnI was purified by high salt extraction, saltless precipitation, 65℃ treatment, ammonium sulfate fractionation and DEAE-cellulose chromatography, etc. (2) Preparation of anti human cTnI McAb: The purified cTnI was injected into the spleen of BALB/c mice. The cTnI-primed spleen cells were fused with Sp2/0 myoloma cell. The McAbs anti human cTnI were obtained by screening with indirect ELISA and 3 times clone. (3)The identification of anti cTnI McAb. Results: Five hybridoma cell lines, named 3A7，3A11，3D2，3F10 and 1H9 were developed, which could secret McAb stably. The 5 McAbs all were demonstrated to be IgG2a by double gel diffusion test. The number of hybridoma chromosomes was between 92 to 110 and the chromosomes were mainly telocentric. Five kinds of ascites had no cross-reaction to LDH，CK，CK-MB ，AST and cardiac troponin T(cTnT), and their titers were between 3.2×10-6 to 1.6×10-7. Conclusion: 3D2，3F10 and 3A7,3A11,1H9 react to different epitopes of cTnI.

Objective: To prepare monoclonal antibodies (McAb) with cardiac troponin I (cTnI) which was purified from fresh human cardiac muscle within 6 h. Methods: (1) Extraction and purification of human cTnI: cTnI was purified by high salt extraction, saltless precipitation, 65℃ treatment, ammonium sulfate fractionation and DEAE-cellulose chromatography, etc. (2) Preparation of anti human cTnI McAb: The purified cTnI was injected into the spleen of BALB/c mice. The cTnI-primed spleen cells were fused with Sp2/0 myoloma cell. The McAbs anti human cTnI were obtained by screening with indirect ELISA and 3 times clone. (3)The identification of anti cTnI McAb. Results: Five hybridoma cell lines, named 3A7，3A11，3D2，3F10 and 1H9 were developed, which could secret McAb stably. The 5 McAbs all were demonstrated to be IgG2a by double gel diffusion test. The number of hybridoma chromosomes was between 92 to 110 and the chromosomes were mainly telocentric. Five kinds of ascites had no cross-reaction to LDH，CK，CK-MB ，AST and cardiac troponin T(cTnT), and their titers were between 3.2×10-6 to 1.6×10-7. Conclusion: 3D2，3F10 and 3A7,3A11,1H9 react to different epitopes of cTnI.

Adult ; Blood Platelets ; chemistry ; CD40 Antigens ; blood ; CD40 Ligand ; blood ; Cholesterol ; blood ; Female ; Humans ; Hypercholesterolemia ; blood ; drug therapy ; Male ; Middle Aged ; Simvastatin ; therapeutic use

OBJECTIVETo investigate the hepatotoxic effects of N, N-dimethylformamide (DMF) in the workers of a synthetic leathers factory, and the effects on liver function of covariates such as alcohol consumption and other factors.

METHODSThe workers were classified into three groups (low, high and the control) by the concentration of DMF in workplace which was determined in the past two years. A questionnaire was drawn up for relevant demographic characteristics and other factors influencing liver function. The bloods were collected for laboratory test which included parameters especially relevant to the liver (ALT AST and gamma GT).

RESULTSLow and high-exposure groups were significantly associated with elevated ALT and gamma GT, and high-exposure group was significantly associated with elevated Liver index. Modeling by stepwise regression analysis demonstrated that high concentration of DMF and BMI were associated with and elevated ALT, gamma GT and Liver index, besides DMF and BMI, the elevation of ALT was also associated with high TRIG. AST was only associated with alcohol consumption. The AST/ALT ration < 1 was present in 86.7% of the exposure workers of liver function abnormal.

CONCLUSIONDMF can cause liver function alternations even if air concentration of DMF was below PC-TWA. Besides the levels of DMF exposure, obesity (BMI) and alcohol consumption are covariates alternating liver function. Liver index can be a parameter for assessment liver function, and the AST/ALT ration < 1 may serve as markers of risk in health screening programs.

Adult ; Alanine Transaminase ; blood ; Dimethylformamide ; toxicity ; Female ; Humans ; Liver ; metabolism ; physiopathology ; Liver Function Tests ; Male ; Occupational Exposure

OBJECTIVETo investigate abnormal liver function associated with polymorphism of GSTT1, GSTM1 and CYP2E1 in workers exposed to N, N-dimethylformamide.

METHODSSixty-nine workers with abnormal liver function in a synthetic leather factory were recruited as case. One hundred and twenty five control subjects with similar work tasks were selected from the same factory. Genotypes for GSTT1 and GSTM1 were determined by multiplex PCR, and for CYP2E1 PstI by PCR-RFLP assay.

RESULTSThe frequency of positive GSTM1 was 59.42% in cases and 38.40% in control, with an odds ratio (OR) of 2.34,95% CI: 1.29-4.29 (P=0.005). For GSTT1 and CYP2E1 PstI, the frequencies of genotypes showed no significant difference between case and control.

CONCLUSIONGSTM1 positive genotype may be genetic risk factors for development of abnormal liver function in workers exposed to N, N-dimethylformamide.

Adult ; Chemical and Drug Induced Liver Injury ; etiology ; genetics ; Cytochrome P-450 CYP2E1 ; genetics ; Dimethylformamide ; adverse effects ; Female ; Genotype ; Glutathione Transferase ; genetics ; Humans ; Male ; Occupational Exposure ; adverse effects ; Polymorphism, Genetic

OBJECTIVETo evaluate the efficacy of treatment with allogeneic peripheral blood stem cell transplantation for benzene-induced severe aplastic anemia.

METHODSHLA-compatible sibling (pregnancy) allogeneic peripheral blood stem cell transplantation (Allo-PBSCT) was successfully performed for a patient with severe aplastic anemia caused by benzene poisoning. 9.41 x 10(8) nucleated cells/kg, 12.49 x 10(6) CD(34) positive cells/kg and CFU-GM 8.2 x 10(5)/kg were infused. The patient was treated with cyclophosphamide (120 mg/kg), total body radiation (8 Gy) and anti-lymphocyte globulin (60 mg/kg) before transplantation. Donor buffy coat cells (9.02 x 10(8) nucleated cells/kg, 10.62 x 10(6) CD(34) positive cells/kg, 6.3 x 10(5) CFU-GM/kg) were infused again on day 18 after transplantation to prevent from graft failure. Graft versus host disease prophylaxis consisted of both methotrexate and cyclosporin A.

RESULTSThe lowest ANC was 0, the lowest platelet was 3 x 10(9)/L after transplantation. The patient achieved an ANC of greater than 0.5 x 10(9)/L from 21st day, and the platelet of greater than 50 x 10(9)/L from 28th day after transplantation. Grade I cGVHD was found the fourth month after grafting. Examination of recipient's bone marrow cells showed a normal 46, XX (presumably marrow donor) karyotype. Blood group changed from B to O.

CONCLUSIONThis is the first case reported in China showing a successful treatment of benzene-induced severe aplastic anemia with allo-PBSCT. Allo-PBSCT may be an effective remedy for this kind of patients.

Adult ; Anemia, Aplastic ; chemically induced ; therapy ; Benzene ; poisoning ; Humans ; Male ; Occupational Diseases ; chemically induced ; therapy ; Peripheral Blood Stem Cell Transplantation ; Transplantation, Homologous ; Treatment Outcome