[Effects of colpotomic approaches on prognosis and recurrence sites of stage â a2-â ¡a2 cervical cancer after laparoscopic radical hysterectomy].

Objective: To investigate the factors affecting the prognosis of stage Ⅰa2-Ⅱa2 cervical cancer after laparoscopic radical hysterectomy (LRH), and to compare the prognosis and recurrence sites of patients with different colpotomy paths. Methods: The clinical data of 965 patients with stage Ⅰa2-Ⅱa2 cervical cancer who underwent LRH in the First Affiliated Hospital of Army Medical University from January 2015 to December 2018 were collected. The median age was 47.0 years of all patients with a median follow-up of 62 months (48-74 months). Cox regression was used to perform the univariate and multivariate analysis of the clinicopathological factors associated with the prognosis that included disease-free survival (DFS) and overall survival (OS). Patients were categorized into LRH through vaginal colpotomy (VC group, n=475) and LRH through intracorporeal colpotomy (IC group, n=490) according to the colpotomic approaches. The prognosis and recurrence sites of patients in each group were compared. Results: (1) During the follow-up period, 137 cases recurred (14.2%, 137/965) and 98 cases died (10.2%, 98/965). The 5-year DFS and OS were 85.8% and 89.9%, respectively. In univariate analysis, positive vaginal margin (PVM) was significantly affected the 5-year OS of patients with cervical cancer (P=0.023), while clinical stage, maximum diameter of tumor, degree of pathological differentiation, lymph node metastasis (LNM), depth of cervical stromal invasion, parametrium involvement, and uterine corpus invasion (UCI) were significantly associated with 5-year DFS and OS in patients with cervical cancer (all P<0.05). In multivariate analysis, clinical stage (HR=1.882, 95%CI: 1.305-2.716), LNM (HR=2.178, 95%CI: 1.483-3.200) and UCI (HR=3.650, 95%CI: 1.906-6.988) were independent risk factors of 5-year DFS (all P<0.001). Clinical stage (HR=2.500, 95%CI: 1.580-3.956), LNM (HR=2.053, 95%CI: 1.309-3.218), UCI (HR=3.984, 95%CI: 1.917-8.280), PVM (HR=3.235, 95%CI: 1.021-10.244) were independent risk factors of 5-year OS (all P<0.05). (2) Different colpotomy paths did not significantly affect the 5-year DFS and OS of patients with stage Ⅰa2-Ⅱa2 cervical cancer. The 5-year DFS in VC group and IC group were 85.9% and 85.6% (P=0.794), and the 5-year OS were 90.8% and 89.3% (P=0.966), respectively. Recurrence patterns consisted of intraperitoneal recurrence, pelvic recurrence, vaginal stump recurrence, and lymph node and distant metastasis. The intraperitoneal recurrence rate of VC group was significantly lower than that of IC group [0.6%(3/468) vs 2.3% (11/485), P=0.037], while the rates of pelvic recurrence, vaginal stump recurrence, lymph node and distant metastasis and overall recurrence were not significantly different between two groups (all P>0.05). Subgroup analysis of patients with different clinical stages, LNM and UCI showed that statistical differences of the intraperitoneal recurrence rates between two groups were only in patients without LNM (0.5% vs 2.3%, P=0.030) or without UCI (0.7% vs 2.3%, P=0.037). Conclusions: Clinical stage, LNM, PVM and UCI are independent risk factors for the prognosis of patients with stage Ⅰa2-Ⅱa2 cervical cancer. For patients without LNM or UCI, LRH through VC could reduce the intraperitoneal recurrence rate, while it is not enough to improve 5-year DFS and OS of patients. Low proportion of intraperitoneal recurrence, intra-operative tumor cells spillage to vagina stump and pelvic cavity might be the explanation.

Expression of breast cancer anti-estrogen resistance 1 in relation to vascular endothelial growth factor, p53, and prognosis in esophageal squamous cell cancer.

The purpose of this study was to clarify the role of breast cancer anti-estrogen resistance 1 (BCAR1) expression in relation to vascular endothelial growth factor (VEGF), p53, and proliferation in esophageal squamous cell cancer (ESCC). Expression of BCAR1, VEGF, p53, and the ki-67 proliferative index were examined by tissue microarray and immunohistochemistry in 106 specimens with ESCC and matched adjacent normal tissues. Among them, 40 cases were simultaneously examined by Western blot. Both Western blot and immunohistochemistry showed that BCAR1 expression was substantially higher in ESCC than in adjacent normal tissues (P < 0.001). BCAR1 expression was significantly connected with degree of tumor differentiation, with poorly differentiated tumors showing higher BCAR1 expression (P < 0.001). BCAR1 expression was significantly and positively correlated with VEGF and p53 expression levels (r= 0.541, P < 0.001; r= 0.374; P < 0.001) but not proliferative index (r= 0.44; P= 0.066). Additionally, a significant relationship was also observed between VEGF and p53 (r= 0.321; P= 0.001). Kaplan-Meier survival analysis revealed that patients with high BCAR1 expression had significantly shorter survival times than those with low BCAR1 expression levels (median survival 40 months vs. 27 months, P= 0.09). Multivariate analysis also revealed that levels of BCAR1 expression (hazard ratio 2.250, P= 0.015) was a significant and independent prognostic indicator. High expression of BCAR1 is associated with elevated VEGF and p53 expression levels, as well as poor prognosis in ESCC. Therefore, BCAR1 may be a potential candidate for predicting prognosis and a new therapy target for ESCC.

Reconstruction of hypopharyngeal and cervical esophageal defect after resection of hypopharyngeal carcinoma: a new technique based on the use of bilateral platysma myocutaneous flaps.

Commonly used procedures for reconstructing hypopharyngeal and cervical esophageal defects resulting from total laryngopharyngectomy (TL) are the gastric conduit or colon transposition as well as microvascularized free flaps. Herein we designed an alternative procedure utilizing bilateral platysma myocutaneous flaps (PMCFs) for the reconstruction of hypopharyngeal and cervical esophageal defects. This report summarizes the technical description of this procedure. TL and cervical esophagectomy were performed and bilateral PMCFs were harvested for reconstruction of hypopharyngeal and cervical esophageal defects in 25 patients aged between 46 and 73 years (mean 58.7 ± 16.2 years). All these patients had advanced-stage (IV) cancer with involvement of the cervical esophagus. Operative time ranged from 176 to 382 minutes (average 243 ± 91 minutes) and the mean intraoperative blood loss was 294 ± 119mL. There were six cases of anastomotic leak (24.0%) and two of them (8.0%) developed anastomotic stricture. Neither flap necrosis nor postoperative death was observed. The majority of our patients (68.0%) were restored to a normal unrestricted oral diet after surgery. The 3-year and 5-year actuarial survival rates were approximately 54.7% and 26.1%, respectively. We conclude that reconstruction of the cervical esophagus with bilateral PMCFs is a valuable method for treating advanced hypopharyngeal carcinoma.

Management of delayed intrathoracic esophageal perforation with modified intraluminal esophageal stent.

In this article, we reviewed our experience of treatment of the delayed intrathoracic nonmalignant esophageal perforation employing modified intraluminal esophageal stent. Between February 1990 and August 2006, eight patients were included in this study. Five patients experienced sepsis. The interval time between perforation and stent placement ranged from 36 h to 27 days (average, 8.6 days). Esophageal stenting and throracotomy for foreign body removal were performed in four patients. The remaining four patients underwent stent placement and thoracostomy. Nutrition was initiated through gastrostomy after 7 to 10 days after the stenting. The stent was removed after the patients resumed oral intake of food and the esophagogram showed that perforation was closed. There was no death in this group. Signs of sepsis remitted 1 week after stent placement. Complications included stress ulcer, stimulative cough, and pneumonia each. Stent removal ranged 32 to 120 days (average 66.7) after its placement. The stent was kept in place for 4 months to prevent formation of esophageal stricture in one patient with caustic esophageal burns. The follow-up was completed in all the patients. The mean follow-up period was 59 months (range 12-180). One patient with caustic esophageal burn underwent cicatricial esophagectomy and gastric transposition 3 years later due to the esophageal stricture. Barium swallow demonstrated that there was a diverticulum-like outpouching in one patient and slight esophageal stricture at T2 and T3 level in another. One patient developed reflux esophagitis 5 years after stent removal. All the patients finally had a normal intake of food. Modified esophageal stenting is an effective method to manage the delayed intrathoracic esophageal perforation. Prevention of stent migration and its convenient adjustment might be the major advantages of this method.

Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: a case study with melanocortin-4 receptor agonists.

1-[(2R)-2-([[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl]amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a lambda(max) at approximately 450 nm. The species responsible for this absorbance required the presence of beta-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug-drug interaction projections based on the k(inact) and K(I) parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug-drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution alpha to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.

ZNRD1 might mediate UV irradiation related DNA damage and repair in human esophageal cancer cells by regulation of ERCC1.

The downregulation of zinc ribbon domain-containing 1 (ZNRD1) protein was recently found to partially reverse the resistance of human leukemia cells toward chemical therapeutic drugs. Therefore, the ZNRD1 protein might be involved in the process of DNA damage and repair. To explore the possible protective effects of ZNRD1 on DNA damage induced by ultraviolet (UV)-C irradiation in human esophageal squamous cancer cell line EC109, we designed and transfected a expression vector into EC109 cells, and established an overexpression cell line. The single-cell gel electrophoresis (comet assay) was used to investigate the DNA damage and repair in UV-C-irradiated control and transfected cells. It was found that the ZNRD1-expressing cells exhibited a significant enhanced DNA repair capacity. Moreover, the overexpression of ZNRD1 could upregulate the expression of excision repair cross-complementing 1 (ERCC1) gene. Collectively, these findings suggested that ZNRD1 might play an important role in the process of DNA damage and repair by regulating the expression of ERCC1.

Prevention and management of complications after colon interposition for corrosive esophageal burns.

We present our experience in the management of complications after a colon interposition for corrosive esophageal burns. From April 1976 to December 2006, 85 patients with caustic esophageal burns were included in this study. The superior belly median incision with an anterior border incision of the left sternocleidomastoid was used. Anastomosis between the colon and the cervical esophagus was performed in 68 and between the colon and pharyngeal portion in 14 patients. An esophageal scar part resection and gastric-esophageal anastomosis was performed in one patient who had been given an unsuccessful colon and jejunum interposition at another institute. An anastomotic modeling operation was performed in one patient with anastomotic stricture who had been managed with colon interposition at another institute. Exploratory thoracotomy and gastrostomy was performed in one patient who had an unsuccessful colon interposition at another institute. Seven of 14 patients (8.5% of 17.1%) died with serious complications such as aspirated pneumonia, interposition colon necrosis, abdominal wound dehiscence and degradation of swallowing and concordance function. However, others with such serious complications survived and were discharged for rehabilitation after corresponding treatment. The 25 patients (30.1%) with other mild complications were discharged for rehabilitation and corresponding management. Two patients from other institutes were discharged for rehabilitation and one was lost to follow-up. The most dangerous complication of this procedure is colon necrosis, and the stomach is the best organ for re-operation. Otherwise, aspiration in infants due to hypoplasia and degradation of swallowing co-ordination needs attention. Peri-operative management is very important, including the control of mediastinal and pulmonary infection and systemic nutritional support to avoid abdominal wound dehiscence. The platysma flap is an excellent method for the treatment of anastomotic stricture.

Management of achalasia with transabdominal esophagocardiomyotomy and partial posterior fundoplication.

In this article we present our experience in the management of achalasia. From May 1988 through August 2005, 71 patients with achalasia underwent transabdominal esophagocardiomyotomy and partial posterior fundoplication. Barium swallow, manometry, and 24-h pH studies were performed in all patients preoperatively. Manometry and 24-h pH monitoring were only carried out in 58 patients at the third post-operative week and in 43 patients during follow-up, even though 52 patients were included in the follow-up. There were no operative deaths or complications. All the 71 patients were able to eat semifluid or solid food without dysphagia and heartburn at discharge. Esophageal barium studies showed that the maximum esophageal diameter decreased 2.2 cm and the minimum gastroesophageal junction diameter increased 8.4 mm after operation. Manometry examination in 58 patients revealed that the lower esophageal sphincter resting pressure decreased 15.0 mmHg in the wake of the procedure. Twenty-four hour pH monitoring demonstrated that reflux events were within the normal post-operative range. Fifty-five of the 58 patients had normal DeMeester scores. Among the patients with a mean 90-month follow-up, 49 patients had normal intake of food without reflux, the remaining three had mild dysphagia without requiring treatment. All the patients resumed their preoperative work and social activities. The manometry and 24-h pH studies in the 43 patients showed there were no significant changes between the third post-operative week and during follow-up. Transabdominal esophagocardiomyotomy and posterior partial fundoplication are able to relieve the functional outflow obstruction of the lower esophageal sphincter, obviate the rehealing of the myotomy edge and prevent gastroesophageal reflux in patients who have undergone myotomy alone.

Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: evidence for selectivity towards CYP3A4.

1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ). 2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues. 3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (+/- TSEM) Km and Vmax = 4.6 +/- 0.3 microM and 20.0 +/- 3.8 pmol/min/mg protein, respectively. 4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of lO microM (i.e. around twice Km). Good correlations (r2 = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms. 5. While 10O microM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. The metabolism of 10 microM BFBFC in human liver microsomes was markedly inhibited by 5-50 microM troleandomycin and 0.2-5 microM ketoconazole, but stimulated by 0.2-10 microM alpha-naphthoflavone. The metabolism of 10 microM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4. 7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed Km and Vmax = 70 microM and 3.39 nmol/min/mg protein, respectively. 8. While 80 microM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYPIA2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.

In vitro stimulation of warfarin metabolism by quinidine: increases in the formation of 4'- and 10-hydroxywarfarin.

It has been demonstrated that the activity of cytochrome P450 (CYP)3A4 in certain cases is stimulated by quinidine (positive heterotropic cooperativity). We report herein that the 4'- and 10-hydroxylation of S- and R-warfarin are enhanced in human liver microsomal incubations containing quinidine. These reactions were catalyzed by CYP3A4, based on data derived from immunoinhibitory studies, with 4'-hydroxylation being preferentially associated with S-warfarin and 10-hydroxylation with R-warfarin. The 4'-hydroxylation of S-warfarin and 10-hydroxylation of R-warfarin increased with increasing quinidine concentrations and maximized at ~3- and 5-fold the values of controls, respectively. Stimulatory effects of quinidine also were observed with recombinant CYP3A4, suggesting that increases in warfarin metabolism were due to quinidine-mediated enhancement of CYP3A4 activity. This positive cooperativity of CYP3A4 was characterized by a 2.5-fold increase in V(max) for the 4'-hydroxylation of S-warfarin and a 5-fold increase in V(max) for the 10-hydroxylation of R-warfarin, with little change in K(m) values. Conversely, V(max) for the 3-hydroxylation of quinidine was not influenced by the presence of warfarin. These results are consistent with previous findings suggesting the existence of more than one binding site in CYP3A4 through which interactions may occur between substrate and effector at the active site of the enzyme. Such interactions were subsequently illustrated by a kinetic model containing two binding domains, and a good regression fit was obtained for the experimental data. Finally, stimulation of warfarin metabolism by quinidine was investigated in suspensions of human hepatocytes, and increases in the formation of 4'- and 10-hydroxywarfarin again were observed in the presence of quinidine, indicating that this type of drug-drug interaction occurs in intact cells.

Human cytochrome P-450 3A4: in vitro drug-drug interaction patterns are substrate-dependent.

Testosterone, terfenadine, midazolam, and nifedipine, four commonly used substrates for human cytochrome P-450 3A4 (CYP3A4), were studied in pairs in human liver microsomes and in microsomes from cells containing recombinant human CYP3A4 and P-450 reductase, to investigate in vitro substrate-substrate interaction with CYP3A4. The interaction patterns between compounds with CYP3A4 were found to be substrate-dependent. Mutual inhibition, partial inhibition, and activation were observed in the testosterone-terfenadine, testosterone-midazolam, or terfenadine-midazolam interactions. However, the most unusual result was the interaction between testosterone and nifedipine. Although nifedipine inhibited testosterone 6beta-hydroxylation in a concentration-dependent manner, testosterone did not inhibit nifedipine oxidation. Furthermore, the effect of testosterone and 7,8-benzoflavone on midazolam 1'-hydroxylation and 4-hydroxylation demonstrated different regiospecificities. These results may be explained by a model in which multiple substrates or ligands can bind concurrently to the active site of a single CYP3A4 molecule. However, the contribution of separate allosteric sites and conformational heterogeneity to the atypical kinetics of CYP3A4 can not be ruled out in this model.

[Comparative study of pulmonary magnetic resonance angiography (MRA)].

Imaging was performed using a 1.0 Tesla superconducting MR system with a body coil. Two-dimensional(2D) turbo FLASH sequence and/or three-dimensional(3D) FISP sequence were carried out in 46 volunteers with 113 pulmonary MRA. The total MRAs were divided into 4 groups and analysed. The results showed: After Gd-DTPA was injected, the signal-to-noise ratio(SNR) of pulmonary 2D- and 3D-TOF MRA was increased in 46 normal pulmonary MR angiography cases(P < 0.01); the number of left and right pulmonary artery divisions identified was much greater than that before Gd-DTPA injection both in 2D- and 3D-MOF(except lingual segment of left lobe). It is suggested that Gd-DTPA may markedly promote the efficiency of pulmonary MRA.

Modulation of rat cytochrome P-450 by an investigational HIV protease inhibitor.

Previous studies in vitro have revealed that L-754,394, an HIV protease inhibitor, is a potent suicide inhibitor of cytochrome P-450 enzymes. The present report examines the effect of chronic treatment of L-754,394 on hepatic cytochrome P-450s in adult male rats. L-754,394 was administered orally once a day for 7 days and resulted in significant changes in marker activities. An unusual parabolic (ascending, then descending) profile was observed for testosterone 2beta-/6beta-(CYP 3A1/2-catalyzed) hydroxylase activities during the 7-day treatment with 20 mg/kg L-754,394. These activities, which were elevated 2-fold on day 2, returned to basal levels by day 8. In contrast, testosterone 2alpha-/16alpha-(CYP2C11-catalyzed) hydroxylase activities showed an opposite parabolic (descending, then ascending) profile during the same period, reducing to 40% of control activities on day 4, followed by a rebounding trend. Immunoquantitation of CYP 3A1/2 and 2C11 showed that the expressed protein levels were in parallel with the associated activities. Furthermore, mRNA levels of CYP 3A2 and CYP2C11 showed the same trends as the protein expression of the respective isoforms. These observations show that L-754,394 perturbs the relative abundance of P-450 isoforms in rat liver by affecting the regulation at a pretranslational step. This may further involve a disturbance of hormonal homeostasis. Although serum levels of testosterone did not show a marked change during treatment, thyroxine and triiodothyronine markedly decreased on days 2 and 4, and subsequently increased to basal levels.

Disposition of ivermectin and cyclosporin A in CF-1 mice deficient in mdr1a P-glycoprotein.

The pharmacokinetics and hepatic metabolism of [3H] ivermectin (IVM) and [3H]cyclosporin A (CSA) were investigated in a subpopulation of the CF-1 mouse stock naturally deficient in mdr1a p-glycoprotein (PGP). A survey of key drug-metabolizing activities in liver fractions from PGP-deficient (-/-) or wild-type (+/+) animals indicated the two subpopulations are not different in hepatic metabolic activity and capacity. Intravenous pharmacokinetics of CSA were identical between the two groups, and results from microsomal incubations indicated similar biotransformation of IVM and CSA in liver. Intestinal excretion of [3H]IVM and [3H]CSA was enhanced in PGP (+/+) animals. Absence of PGP resulted in higher blood concentrations of IVM after oral dosing, suggesting enhanced absorption of IVM in (-/-) mice. Concentrations of [3H]IVM and [3H]CSA were always greater in the brains of (-/-) mice compared with (+/+) mice after either i.v. or oral administration. In contrast, liver concentrations of either compound were not different between (+/+) and (-/-) animals after an i.v. dose. These results show the PGP (-/-) and (+/+) subpopulations of CF-1 mice are useful for studying the role of mdr1a PGP in systemic exposure and tissue disposition of PGP substrates in the absence of metabolism differences.

Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac.

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.

Inhibitory anti-CYP3A4 peptide antibody: mapping of inhibitory epitope and specificity toward other CYP3A isoforms.

An antipeptide antibody has been produced that recognizes CYP3A4 and exhibits greater than 90-95% inhibition on CYP3A4-mediated reactions [Wang RW and Lu AYH (1997) Drug Metab Dispos 25:762-767]. The inhibitory epitope of the 21-amino acid peptide, corresponding to residues 253 to 273 of CYP3A4, has been identified to reside in a 7-amino acid sequence (LEDTQKH: residues 261-267 of CYP3A4). This conclusion was based on the reversal of antibody inhibition of testosterone 6beta-hydroxylation when peptides with overlapping sequence in this region were preincubated with the antibody. In immunoblotting analysis, this antibody did not recognize CYP3A5 or CYP3A7 in microsomes prepared from baculovirus-infected cells containing these two expressed isoforms. In addition, the antipeptide antibody did not inhibit testosterone 6beta-hydroxylation or midazolam 1'- and 4-hydroxylation in microsomes containing expressed CYP3A5 and CYP3A7. Because the corresponding sequence in CYP3A5 (LNDKQKH) and CYP3A7 (LKETQKH) differs from CYP3A4 by only two amino acids, six peptides with either one or two amino acid changes were used to determine which amino acid is essential for antibody-antigen interaction. Our data indicate that Glu, Asp, and Thr in the 7-amino acid sequence of CYP3A4 are critical determinants of selectivity among CYP3A isoforms.

Identification of cytochrome P4503A4 as the major enzyme responsible for the metabolism of ivermectin by human liver microsomes.

1. Ivermectin was extensively metabolized by human liver microsomes to at least 10 metabolites. The structure of many of them (mostly hydroxylated and demethylated) was determined by 1H-NMR and LC/MS. 2. To determine which human cytochrome P450 isoform(s) is responsible for the metabolism of ivermectin, chemical inhibitors including sulphaphenazole, quinidine, furafylline, troleandomycin (TAO) and diethyldithiocarbamate (DDC) were used to evaluate their effect on ivermectin metabolism. TAO, a specific inhibitor of cytochrome P4503A4, was the most potent inhibitor, inhibiting the total metabolism as well as formation of each metabolite. Metabolism was also inhibited by an anti-human cytochrome 3A4 antibody by 90%. 3. When ivermectin was incubated with microsomes from cells expressing CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 or 3A4 at 4 mg/ml protein concentrations, metabolic activity was only detected with the microsomes containing CYP3A4. The metabolic profile from cDNA-expressed CYP3A4 microsomes was qualitatively similar to that from human liver microsomes. 4. Thus, cytochrome P4503A4 is the predominant isoform responsible for the metabolism of ivermectin by human liver microsomes.

Economic reforms and the acute inpatient care of patients with schizophrenia: the Chinese experience.

OBJECTIVE: This study compared insured and uninsured schizophrenic inpatients in China and examined changes in the acute inpatient care of schizophrenic patients during China's economic reform era. METHOD: Detailed chart reviews of 50 randomly selected inpatients discharged from a hospital in central China each year from 1984 through 1993 identified 321 patients with schizophrenia. Demographic, insurance, treatment, and cost data of these patients were collected from the charts. RESULTS: With logistic regression models to control for confounding variables, the analyses showed that the 129 insured patients were significantly more likely than the 192 uninsured patients to be urban residents, to be older, to have had 7 or more years of schooling, and to have had more psychiatric hospitalizations; moreover, their index admissions were longer and were more likely to include use of traditional Chinese medications. The estimated 19% of schizophrenic individuals in the community with health insurance receive inpatient treatment 2.8 times more frequently than the 81% without insurance. Compared to admissions in 1984-1988, admissions in 1989-1993 were significantly shorter and involved longer periods of polypharmacy with multiple antipsychotic medications but included lower mean chlorpromazine-equivalent doses of medication. The relative cost of inpatient care for an acute episode of schizophrenia increased 3.5-fold over the 10-year period, from 11% of mean annual household income in 1984 to 37% in 1993. CONCLUSIONS: Changes in the incentive system for care providers and rapid increases in the cost of care during the economic reform era have resulted in increasingly restricted availability of services for the many schizophrenic patients without health insurance.

Inhibitory anti-peptide antibody against human CYP3A4.

An inhibitory anti-peptide antibody was raised against a 21-amino acid peptide (VKRMKESRLEDTQKHRVDFLQ) corresponding to residues 253-273 of human cytochrome P450 3A4. High titer antibodies were produced by rabbits immunized with this peptide coupled to keyhole limpet hemocyanin, as judged by ELISA. Anti-peptide antibody recognized a single protein band in microsomes prepared from cells expressing recombinant human CYP3A4 in immunoblotting analysis. No immunodetectable proteins were found in microsomes containing other cytochrome P450 isoforms. In addition, the antibody did not recognize CYP3A5, a closely related isoform in the CYP3A family. In human liver microsomes, only one protein band which comigrated with human CYP3A4 was recognized by this antibody and the relative blotting intensity of this protein band correlated significantly with human CYP3A4-catalyzed testosterone 6 beta-hydroxylase activities (r = 0.96). More importantly, this antibody exhibited greater than 90-95% inhibition of testosterone 6 beta-hydroxylation, while other cytochrome P450-mediated reactions in human liver microsomes were not inhibited. Because of its specificity and inhibitory potency, this anti-peptide antibody should be a valuable tool in evaluating the role of CYP3A in mediating in vitro metabolism of therapeutic agents.