AIMTo isolate and identify a glucuronide metabolite of SFZ-47 [3H-1,2-dihydro-2-(4-methyl-phenylamino)methyl-1-pyrrolizinone], which is difficult to synthesize because it undergoes hydrolysis and intramolecular acyl migration at physiological pH, in rabbit urine.

METHODSTwo rabbits were ig 200 mg doses of SFZ-47. Urine was collected for 24 h, adjusted to pH 4.0 with acetic acid and lyophilized. The residues were reconstituted in 25 mL methanol and centrifuged at 5,000 r.min-1 for 10 min. The supernatant was filtered (0.45 micron) and then isolated with semi-preparative reversed phase HPLC. The eluent collected from individual peaks was evaporated by rotary evaporation and freeze-drying. Compounds were then identified with electrospray ion trap mass spectrometry and 1HNMR spectroscopy.

RESULTSThe 1HNMR and ESI-MSn results indicate that the metabolite is the 1-O-acyl beta-D-glucuronide conjugate of 4-(3H-1,2-dihydro-1-pyrrolizinone-2-methylamino) benzoic acid.

CONCLUSIONThis method was shown to be rapid and simple and gave excellent resolution from endogenous constituents in urine, and it is suitable for preparation of the glucuronide metabolites of SFZ-47 and its analogues.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; metabolism ; urine ; Chromatography, High Pressure Liquid ; Male ; Molecular Structure ; Pyrroles ; chemistry ; metabolism ; urine ; Rabbits ; Spectrometry, Mass, Electrospray Ionization

AIMTo investigate the fragmentation behavior of triazolobenzodiazepines and to develop a specific, sensitive and rapid LC/MSn assay for simultaneous determination of estazolam, alprazolam and triazolam in human urine.

METHODSAfter oral administration of a single 4 mg dose of the drugs to each of three healthy volunteers, urine samples were purified by solid-phase extraction, and then injected into an ODS column (150 mm x 4.6 mm) with a mobile phase of methanol-water (8:2) for LC/MSn analysis. The structures of estazolam, alprazolam and triazolam in human urine were identified by direct comparison of the observed mass spectra and the chromatographic retention time with those of the reference substance. The mass spectrometer (Finnigan LCQ) was operated in positive mode and in two scan modes including SIM and full scan MS/MS mode. The obtained mass spectra was analyzed assisted with the software Mass Frontier 1.0 for their fragmentation pathways.

RESULTSThe full scan MS/MS spectra of each compound gave characteristic fragment ions of [M + H - N2]+ and [M + H - Cl]+. The detection limit was below 0.5 ng.mL-1 for estazolam, alprazolam and triazolam in human urine.

CONCLUSIONThe method is useful in forensic and clinical toxicology in which unequivocal identification of eatazolam, alprazolam and triazolam is desired.

Alprazolam ; urine ; Anti-Anxiety Agents ; urine ; Chromatography, Liquid ; Estazolam ; urine ; Humans ; Male ; Spectrometry, Mass, Electrospray Ionization ; Triazolam ; urine

AIMTo develop a sensitive and specific LC/MS/MS method for direct determination of dextrorphan in human plasma and to study the pharmacokinetics of dextrorphan.

METHODSAfter a single oral dose of 60 mg dextromethorphan hydrobromide to 18 healthy Chinese male volunteers, the plasma concentration of dextrorphan, an active metabolite of dextromethorphan, was determined. Dextrorphan and internal standard chlorpheniramine were extracted from plasma using liquid-liquid extraction, then separated on a Zorbax Extend C18 column. The mobile phase consisted of methanol-water-formic acid (70:30:1), at a flow-rate of 0.5 mL x min(-1). A Finnigan TSQ tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring (SRM) using the precursor to product ion combinations of m/z 258 to 157 and m/z 275 to 230 was performed to quantify dextrorphan. The pharmacokinetic parameters of dextrorphan were calculated by non-compartment model statistics.

RESULTSThe linear calibration curves were obtained in the concentration range of 0.2 - 80 microg x L(-1) Each plasma sample was chromatographed within 3.0 min. The intra- and inter-day relative standard deviation (RSD) across three validation runs over the entire concentration range was less than 8%. Accuracy determined at three concentrations (0.5, 6.0 and 70 microg x L(-1) for dextrorphan) ranged from 98.8% to 100.6%. Pharmacokinetic parameters of dextrorphan was obtained as follows: Tmax was (2.1 +/- 0.7) h, Cmax was (14 +/- 8) microg x L(-1), T1/2 was (3.8 +/- 1.8) h, AUC0-t was (60 +/- 37) microg x h x L(-1).

CONCLUSIONPlasma concentration of the active metablite dextrorphan was directly determined. The method is sensitive and convenient, and is proved to be suitable for clinical investigation of dextrorphan pharmacokinetics and bioequivalence evaluation of formulations containing dextromethorphan.

Administration, Oral ; Adult ; Area Under Curve ; Dextromethorphan ; administration & dosage ; pharmacokinetics ; Dextrorphan ; blood ; Gas Chromatography-Mass Spectrometry ; Humans ; Male ; Spectrometry, Mass, Electrospray Ionization ; Therapeutic Equivalency

OBJECTIVETo construct a novel kind of nonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability.

METHODSThe synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo.

RESULTSThe polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides.

CONCLUSIONThe synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.

Animals ; Binding Sites ; Carcinoma ; therapy ; Cell Line, Tumor ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Female ; Fibroblast Growth Factors ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; chemical synthesis ; chemistry ; pharmacology ; Humans ; In Vitro Techniques ; Ligands ; Liver Neoplasms ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Particle Size ; Peptides ; chemistry ; metabolism ; pharmacology ; Polyethyleneimine ; chemistry ; metabolism ; pharmacology ; Prostatic Neoplasms ; therapy ; Receptors, Fibroblast Growth Factor ; drug effects ; genetics ; metabolism ; Structure-Activity Relationship ; Surface Properties ; Transfection ; Transplantation, Heterologous ; Xenograft Model Antitumor Assays

OBJECTIVETo evaluate the clinical characteristics, reasonable mode of treatment and prognostic factors in patients with primary central nervous system lymphoma (PCNSL).

METHODSTwenty-eight patients with PCNSL treated from 1989 to 2002 were retrospectively reviewed. The clinical characteristics, results of treatment and prognostic factors were analyzed by SPSS10.0 statistic software.

RESULTSOf 28 patients, 18 men and 10 women with a median age of 52 years. The median survival time was 2 years (range 6 months-6 years). The 5-year survival rate was 21.4%. Nineteen patients had single-locus lesion and 9 multi-locus lesion, 78.6% of the patients were diagnosed as having B-cell origin, its main type being diffuse large cell lymphoma. According to international working formulation (WF), moderate-grade of histopathology was observed in 57.7% (15/28). Cox regression analysis revealed that single- or multi-locus lesion was only independent prognostic factor (P = 0.0417). Combined chemotherapy showed significant efficacy for those patients who had lesion of B-cell origin, high grade or multi-locus lesion, and the efficacy of irregular chemotherapy was better than that of regular chemotherapy.

CONCLUSIONPrimary central nervous system lymphoma has a special prognostic factor. Chemotherapy plays a very important role in comprehensive treatment, irregular chemotherapy should be adopted as a regular treatment.

Adolescent ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Central Nervous System Neoplasms ; drug therapy ; radiotherapy ; surgery ; Combined Modality Therapy ; Cyclophosphamide ; administration & dosage ; Doxorubicin ; administration & dosage ; Female ; Follow-Up Studies ; Humans ; Lymphoma, Large B-Cell, Diffuse ; drug therapy ; radiotherapy ; surgery ; Lymphoma, Non-Hodgkin ; drug therapy ; radiotherapy ; surgery ; Male ; Middle Aged ; Prednisone ; administration & dosage ; Prognosis ; Retrospective Studies ; Survival Rate ; Teniposide ; administration & dosage

BACKGROUNDIt is known that the hypothalamic-pituitary-adrenal (HPA) axis is highlighted by stimulation, such as sepsis, trauma, etc, when corticortropin increases and plasma cortisol levels enhance. Relative adrenal insufficiency is not uncommon in critically ill patients and may occur in severe sepsis patients with high plasma cortisol levels. It has been demonstrated that a short corticotropin test has a good prognostic value and is helpful in identifying patients with septic shock at high risk for death, but it has not been established for all severe sepsis patients, especially in China. The aim of this study is to explore the relationship between adrenal function and prognosis in patients with severe sepsis.

METHODSThis prospective study was conducted between July and December 2004 in 6 teaching hospitals. Two hundred and forty patients with severe sepsis were enrolled in this study. A short corticotropin stimulation test was performed in all patients by intravenous injection of 250 microg of corticotropin. Blood samples were taken immediately before the test (T0), 30 (T30) and 60 (T60) minutes afterward, and the plasma cortisol concentration was measured by radio-immunoassay. At the onset of severe sepsis, the following parameters were recorded: age, sex, Acute Physiology and Chronic Health Evaluation (APACHE) II, heart rate, mean arterial pressure (MAP), arterial partial pressure of oxygen (PaO(2))/fraction of inspired oxygen (FiO(2)), peripheral blood of hemoglobin, platelets and leukocyte concentration and the number of organ failure. Patients were designated into two groups (survival and non-survival groups) according to the 28-day mortality. Relative adrenal insufficiency was defined as the difference between T0 and the highest value of T30 or T60 (DeltaTmax) < or = 9 microg/dl.

RESULTS(1) Two hundred and forty patients with severe sepsis were included in this study, with 134 patients in the survival group and 106 in the non-survival group. The 28-day mortality was 44.2%. (2) Between the survival group and non-survival group age, APACHE II, peripheral blood of platelets, the number of organ failures, T0 and DeltaTmax showed significant differences. T0 was (23 +/- 10) microg/dl and (36 +/- 18) microg/dl in the survival group and nonsurvival group respectively. DeltaTmax was (18 +/- 9) and (10 +/- 8) microg/dl in the survival group and non-survival group respectively. The areas under the ROC curve for T0 and DeltaTmax were both 0.72, and the area under the ROC curve for APACHE II was 0.70. By multivariate analysis age, T0, the number of organ failures and relative adrenal insufficiency (DeltaTmax < or = 9 microg/dl) were independent predictors of death. (3) The incidence of relative adrenal insufficiency was 38.3% in total, 19.4% in the survival group and 62.3% in the non-survival group (P < 0.001). The 28-day mortality was 71.7% among the relative adrenal insufficiency patients but 27.0% among normal adrenal function patients.

CONCLUSIONSThe prevalence of relative adrenal insufficiency is high in severe sepsis. Relative adrenal insufficiency has a good prognostic value for severe sepsis.

APACHE ; Adrenal Glands ; physiopathology ; Adrenal Insufficiency ; epidemiology ; Adrenocorticotropic Hormone ; Adult ; Aged ; Humans ; Middle Aged ; Prognosis ; Prospective Studies ; Sepsis ; complications ; mortality ; physiopathology

OBJECTIVETo explore the relationship between the adrenal function and the prognosis of acute respiratory distress syndrome (ARDS).

METHODSOne hundred and fifty-eight patients with ARDS were enrolled in this study and were divided into two groups based on the prognosis: survival group and death group. Every patient was given one shot of corticotrophin 250 microg intravenously, plasma cortisol level was detected by radio-immunoassay before the shot (T0) and 30 minutes (T30) and 60 minutes (T60) after. And meanwhile the following parameters in the patients were recorded: age, APACH II, heart rate, mean arterial pressure, PaO(2)/FiO(2), arterial pH, hemoglobin, platelets and WBC, the number of failed organ and 28-day mortality. Relative adrenal insufficiency was defined as the difference between T0 and the highest value of T30 or T60 (DeltaTmax)

RESULTSThe total 28-day mortality was 54.4% (86/158) and the total incidence of relative adrenal insufficiency was 42.7% (68/158). The incidence of relative adrenal insufficiency in death group was significantly higher than that in survival group (62.8% vs 19.4%, P < 0.01). The 28-day mortality in patients complicated with relative adrenal insufficiency was significantly higher than that in those did not (76.5% vs 36.8%, P < 0.001). The area under the ROC curve for DeltaTmax was 0.655. With the multivariate analysis, the number of failed organ and relative adrenal insufficiency were independent risk factors of the death in patients with ARDS.

CONCLUSIONSAdrenal function is valuable in predicting the prognosis of the ARDS.

Adolescent ; Adrenal Glands ; physiopathology ; Adrenal Insufficiency ; etiology ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Prognosis ; Prospective Studies ; Respiratory Distress Syndrome, Adult ; complications ; mortality ; physiopathology

OBJECTIVETo compare the efficacy of chemotherapy alone, radiotherapy alone and combined-modality therapy in the treatment for early-stage Hodgkin's lymphoma (HL).

METHODSFrom 1999 to 2002, totally 150 patients with stage I or II HL were treated in our hospital. They were stratified into several groups based on initial treatment strategy: chemotherapy alone (CT group, n = 22), radiotherapy alone (RT group, n = 18), combined-modality therapy (CMT group, n = 109) and surgical resection (SR group, n = 1). Chemotherapy regimens were mainly ABVD (adriamycin, bleomycin, vinblastine and dacarbazine) and MOPP (mechlorethamine, vincristine, procarbazine and prednisone). Radiotherapy modes included involved field radiotherapy (IFRT), extended field radiotherapy (EFRT) and sub-total nodal irradiation (STNI).

RESULTSThe pathological types included nodular sclerosis (NS, n = 84), mixed-cellularity (MC, n = 39), lymphocyte-predominant (LP, n = 23), lymphocyte-depleted (LD, n = 3) and nodular lymphocyte predominant Hodgkin's disease (NLPHD, n = 1). Of those, 72 were evaluble in terms of prognostic factors. No poor prognostic factor was found in 36.1% or 29.2% of the patients according to EORTC or GHSG criteria, respectively. There were 33 patients with complete response (CR), 109 with partial response (PR), 5 with stable disease (SD) and 3 with progressive disease (PD) after initial therapy. The median follow-up period was 71.5 months. The overall 7-yr survival rate was 89.3%, and treatment failure rate at 6 years was 18.8%. The response rate of CMT group was superior to that of CT group, and the patients with nodular sclerosis or mixed-cellularity type had significantly lower risk of treatment failure (P = 0.009 and 0.019, respectively). The multivariate analysis revealed that the treatment strategies affected the prognosis significantly. The risk of failure of chemotherapy alone was 2.52 times higher than that of combined-modality therapy (P = 0.004). No predictive factor affecting OS was identified by either univariate or multivariate analysis. The patients in CMT group suffered more adverse effects than those in either CT or RT groups, which mainly consisted of leucopenia, alopecia and gastrointestinal symptoms.

CONCLUSIONCombined-modality therapy is more effective than chemotherapy alone or radiotherapy alone in the treatment for early stage Hodgkin's lymphoma. Though its acute adverse effects are more severe than that of chemotherapy or radiotherapy alone, it may reduce the risk of treatment failure.

Adolescent ; Adult ; Aged ; Alopecia ; chemically induced ; Antineoplastic Combined Chemotherapy Protocols ; adverse effects ; therapeutic use ; Bleomycin ; adverse effects ; therapeutic use ; Child ; Child, Preschool ; Combined Modality Therapy ; Dacarbazine ; adverse effects ; therapeutic use ; Doxorubicin ; adverse effects ; therapeutic use ; Female ; Follow-Up Studies ; Hodgkin Disease ; drug therapy ; pathology ; radiotherapy ; Humans ; Leukopenia ; chemically induced ; Male ; Mechlorethamine ; adverse effects ; therapeutic use ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Prednisone ; adverse effects ; therapeutic use ; Procarbazine ; adverse effects ; therapeutic use ; Proportional Hazards Models ; Radiotherapy ; adverse effects ; methods ; Remission Induction ; Retrospective Studies ; Survival Rate ; Vinblastine ; adverse effects ; therapeutic use ; Vincristine ; adverse effects ; therapeutic use ; Young Adult

The possibility of using a subunit or fragment of human chorionic gonadotropin (hCG) as an immunogen for birth control has been actively explored for many years. This protein homone is produced by the fertilized egg and is required for implantation of the blastocyst into the maternal uterus and the maitenance of pregnancy. In previous studies, several bio-synthesized hCG chimeric peptides (CP) that contain three linear B-cell epitopes (beta5, beta9 and beta8) of beta-hCG subunit together with various foreign 'promiscuous' T-cell epitopes were constructed and expressed as potential new hCG vaccine immunogens. In order to detect antibodies to each of the individual B-cell epitopes present in the animal antiserum raised against the hCG CPs, we decided to construct three recombinant proteins, each contains a single target B-cell epitope (betaE) of beta-hCG. Two sets of DNA fragments were chemically synthesized encoding the beta5, beta9 and beta8 epitopes (betaE) 45 approximately 52, 113 approximately 116 or 133 approximately 144 of beta-hCG subunit and were inserted into the downstream of streptavidin (Stv) gene in pTSA18 separately, with or without an extra TAA codon at the 3'-terminals of the genes. SDS-PAGE analysis revealed that only Stv-betaE (-beta5, -beta9 or -beta8) fusion genes set with the TAA codon can be expressed in E. coli BL21 (DE3) pLysS strain at high level after 1mM IPTG induction for 4 hours. Additionally, these fusion proteins can all be recognized by specific polyclonal antiserum (RS-4157) generated upon immunization with the loop peptide 38 approximately 57 of beta-hCG, monoclonal antibody (mAb) FB12 to beta9 epitope and mAb OT3A that specially recognizes reporter sequence 133 approximately 139 of beta8 epitope 137 approximately 144. Each of the proteins can be purified to 95% relative homogeneity using an improved method of preparative gel polyacrylamide gel electrophoresis. The yields were 5 mg per 1 L culture. The three target Stv-betaE fusion proteins will be useful in determining the immunogenicity of designed hCG CPs and hCG vaccines, including hCG DNA vaccines.
Chorionic Gonadotropin, beta Subunit, Human ; genetics ; immunology ; Epitopes, B-Lymphocyte ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification ; Streptavidin ; genetics ; Vaccines, Synthetic ; immunology

OBJECTIVETo perform in vitro magnetic resonance imaging on magnetic iron oxide (Fe(2)O(3)-PLL) labeled rabbit peripheral blood endothelial progenitor cells (EPCs).

METHODSFe(2)O(3) was incubated with PLL for 2 hours to form Fe(2)O(3)-PLL. Rabbit peripheral blood mononuclear cells (MNCs) were isolated and EPCs were selected by adherence method, expanded and incubated with Fe(2)O(3)-PLL. Intracellular iron was detected by Prussian blue stain and under electron microscope. MTT assay was used to evaluate cell survival and proliferation of Fe(2)O(3)-PLL labeled EPCs. Flow cytometry was used to analysis cell cycle and apoptosis. The cells underwent in vitro MR imaging with various sequences.

RESULTSIron-containing intracytoplasmatic vesicles could be observed clearly with Prussian blue staining and electron microscope observation. Survival, life cycle and apoptosis values obtained by MTT and flow cytometry analysis were similar among unlabelled EPCs and EPCs labeled with various concentrations Fe(2)O(3)-PLL. The signal intensity on MRI was significantly decreased in labeled cells compared with that in unlabeled cells. The percentage change in signal intensity (DeltaSI) was most significant on T(2)*WI and DeltaSI was significantly lower in cells labeled for 7 days than that labeled for 1 day.

CONCLUSIONSThe rabbit peripheral blood EPCs can be labeled with Fe(2)O(3)-PLL without significant change in viability and proliferation. The labeled EPCs can be imaged with standard 1.5 T MR equipment. The degree of MR signal decreasing may indirectly reflect the cells count, growth state and division.

Animals ; Biomarkers ; Blood Cells ; Cells, Cultured ; Endothelial Cells ; cytology ; Ferric Compounds ; Magnetic Resonance Imaging ; methods ; Male ; Rabbits ; Stem Cells ; cytology