Progesterone oxidation by cytochrome P450 2D isoforms in the brain.

The existence of cytochrome P450 2D isoforms in the brain has been demonstrated, although their physiological functions remain to be elucidated. In this study we demonstrated that recombinant rat cytochrome P450 2D1 and 2D4 and human cytochrome P450 2D6 possess progesterone 6 beta- and 16 alpha- hydroxylation activities; 2 beta- and 21-hydroxylation activities; and 2 beta-, 6 beta-, 16 alpha- and 21-hydroxylation activities, respectively. Cytochrome P450 2D4 had the lowest K(m) value and the highest maximum velocity value toward these activities. Progesterone 2 beta- and 21-hydroxylation activities were also detected in rat brain microsomes, and these activities were completely inhibited by anticytochrome P450 2D antibodies. The presence of endogenous 2 beta- and 21-hydroxyprogesterones in rat brain tissues was also demonstrated. The mRNAs of cytochrome P450 2D4, CYP11A, and 3 beta-hydroxysteroid dehydrogenase were detected in the rat brain, suggesting that progesterone was generated from cholesterol by CYP11A and 3 beta-hydroxysteroid dehydrogenase and then underwent hydroxylation to hydroxyprogesterones by cytochrome P450 2D4 in rat brain. Collectively, our findings support the idea that cytochrome P450 2D may be involved in the regulation (metabolism and/or synthesis) of endogenous neuroactive steroids, such as progesterone and its derivatives, in brain tissues.

Quantitative prediction of in vivo drug interactions between nevirapine and antifungal agents from in vitro data in rats.

We investigated the effects of ketoconazole (KCZ) and fluconazole (FCZ) on rat liver microsomal nevirapine (NVP) metabolism in vitro and on NVP plasma profiles in vivo in order to determine whether the in vivo drug interactions could be predicted quantitatively from the in vitro data. The Ki values of KCZ and FCZ for NVP 12-hydroxylation were 1.59 microm and 11.5 microM, respectively, indicating that KCZ inhibited this activity more strongly than FCZ in vitro. In contrast, FCZ orally pre-administered at 20 mg/kg to rats increased the area under the plasma concentration-time curve (AUC) of NVP 7.4-fold, whereas KCZ increased it 2.1-fold, compared to the vehicle. We next investigated the inhibitory potency and unbound concentrations of KCZ and FCZ in microsomal mixtures with or without rat albumin. In the presence of albumin, the inhibition by KCZ was greatly decreased. Further, the unbound fraction of KCZ was decreased dramatically to around 3%, whereas more than 90% of FCZ remained in unbound form. When the increase in the AUC for NVP was calculated based on the concentrations of unbound inhibitors in the portal vein, good agreement with the observed in vivo values was obtained.

Identification of CYP3A4 as the predominant isoform responsible for the metabolism of ambroxol in human liver microsomes.

1. In humans, ambroxol is metabolized to dibromoanthranilic acid (DBAA) and 6,8-dibromo-3-(trans-4-hydroxycyclohexyl)-1,2,3,4-tetrahydroquinazoli ne (DHTQ). The formation of DHTQ proceeds non-enzymatically, whereas that of DBAA requires NADPH. Studies have been performed to identify the CYP isozyme(s) involved in the formation of DBAA using human liver microsomes and microsomes expressing recombinant human CYP isozymes (1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 4A11). 2. The apparent Vmax and Km for the formation of DBAA were 472+/-192 pmol/ min/mg protein and 248+/-40.6 microM respectively (mean +/- S.D., n = 3). 3. Of the recombinant CYP examined, only CYP3A4 metabolized ambroxol to DBAA. The apparent Vmax and Km were 1.42 pmol/min/pmol P450 and 287 microM respectively. 4. Among the CYP inhibitors examined (furafylline, sulphaphenazole, quinidine, diethyldithiocarbamic acid, ketoconazole), only ketoconazole inhibited the production of DBAA (> 80%) at 1 microM and anti-CYP3A antiserum almost completely inhibited the formation of DBAA. 5. These results suggest that CYP3A4 is predominantly involved in the metabolism of ambroxol to DBAA in humans.

Identification of human cytochrome P450 isoforms involved in the metabolism of brotizolam.

1. To identify the cytochrome P450 (CYP) isoenzyme(s) responsible for the major metabolic pathways of brotizolam in man, we examined the metabolism of brotizolam using human liver microsomes and microsomes expressing individual human CYP isoenzymes (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4). 2. Brotizolam was metabolized to alpha-OH- and 6-OH-brotizolam by human liver microsomes (n = 3). Vmax for alpha- and 6-hydroxylation of brotizolam were 1470 +/- 259 and 8983 +/- 7740 pmol/min/mg protein respectively, and the corresponding Km were 49 +/- 9.3 and 595 +/- 580 microM respectively. 3. Among CYP inhibitors examined (furafylline, sulphaphenazole, quinidine, ketoconazole and cimetidine), ketoconazole showed the most potent inhibitory effect on brotizolam metabolism by human liver microsomes. Ki of ketoconazole for alpha- and 6-hydroxylation were 0.05 and 0.07 microM respectively. 4. When incubated with microsomes expressing individual human CYP isoenzymes (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4), brotizolam was metabolized only by CYP3A4. 5. Brotizolam metabolism in human liver microsomes was almost completely inhibited by anti-CYP3A4 antiserum. 6. These results suggest that CYP3A4 is predominantly responsible for both alpha- and 6-hydroxylation of brotizolam in human liver microsomes.

Metabolism of epinastine, a histamine H1 receptor antagonist, in human liver microsomes in comparison with that of terfenadine.

Epinastine is a non-sedative second-generation antiallergic drug, like terfenadine. In the present study, the metabolism of epinastine in human liver microsomes was investigated and compared with that of terfenadine. Terfenadine was extensively metabolized to terfenadine acid with a Km value of 1.78 microM, a Vmax value of 173.8 pmol/min/mg and a metabolic clearance (Vmax/Km) of 103.9. Epinastine, in contrast, was poorly metabolized by microsomes from the same source with a high Km value of 232 microM. Metabolic clearance of epinastine was only 0.832, which was lower by three orders of magnitude than that of terfenadine. Studies with microsomes expressing recombinant cytochrome P450 (CYP) species revealed that the CYP isoforms responsible for epinastine metabolism are CYP3A4, 2D6 and (to a minor extent) 2B6. Epinastine and terfenadine had no effect on CYP1A2 (theophylline 1-demethylation), 2C8/9 (tolbutamide hydroxylation) or 2E1 (chlorzoxazone 6-hydroxylation) activity, but weakly inhibited CYP2D6 (debrisoquine 4-hydroxylation) activity. CYP3A4 (testosterone 6 beta-hydroxylation) activity was strongly inhibited by terfenadine with a Ki value of 25 microM, whereas epinastine had no effect at up to 100 microM. Thus, epinastine is very poorly metabolized compared to terfenadine in human liver microsomes and does not inhibit CYP3A4 activity in vitro, unlike terfenadine.

The involvement and sources of active oxygen in experimentally induced acute pancreatitis.

The involvement of active oxygen has been suggested in the development of cerulein-induced acute pancreatitis in rats. Previously, we directly detected pancreatic active oxygen (O2-) production in rats with cerulein-induced pancreatitis by using a supersensitive photon counter and a cypridina luciferin analogue (MCLA) that reacts specifically with O2- by emitting luminescence. In the present study, with the specific aim of determining the source of O2-, we prepared two groups of animals with cerulein-induced pancreatitis: those treated with allopurinol, a xanthine oxidase inhibitor; and those treated with nitrogen mustard, a leukopenia-inducing substance. In each of these two groups, pancreatic O2- production and the severity of pancreatic injuries were comparatively studied. In the leukopenic animal group, decreases in O2- dependent chemiluminescence and improvement in the pancreatic condition coincided. This suggests that neutrophils might be involved in experimentally induced pancreatitis as a source of active oxygen.

LFA-1 (CD11a/CD18) and ICAM-1 (CD54) antibodies attenuate superoxide anion release from polymorphonuclear leukocytes in rats with experimental acute pancreatitis.

The inhibitive effects of anti-CD11a/CD18 (LFA-1) and anti-CD54 (ICAM-1) antibodies on the generation of superoxide anion (O2-) by polymorphonuclear leukocytes (PMNs) was elucidated in rats induced with experimental acute pancreatitis. We investigated the generation of O2- by PMNs in two protocols: in the first, we measured the active oxygen-producing ability of PMNs isolated from blood in normal rats; in the second, we measured it from blood, peritoneal cavity, and bronchial alveolar lavage fluid in rats 3 h after the induction of pancreatitis. In normal rats, although LFA-1 antibody attenuated the generation of O2-, ICAM-1 antibody did not. However, in pancreatitis rats, both LFA-1 and ICAM-1 antibodies reduced the generation of O2- by PMNs isolated from blood and the peritoneal cavity. These results showed not only that both LFA-1 and ICAM-1 antibodies have a protective effect on the generation of O2-, but also that LFA-1 has a direct inhibitive effect on the generation of O2- by PMNs in this model. Furthermore, histological studies showed there to be less neutrophil accumulation in the lungs of LFA-1- and ICAM-1-treated animals compared to control animals.

Detection of O2- generation and neutrophil accumulation in rat lungs after acute necrotizing pancreatitis.

BACKGROUND: Polymorphonuclear neutrophils (PMNs) and PMN-derived superoxide anions (O2-) have been found to mediate acute respiratory distress syndrome (ARDS). But the evidence has been obtained only indirectly, (for example, by treatment with free radical scavengers or by measuring lipid peroxides). METHODS: We used chemiluminescence technique to measure O2- generation directly from the surface of rat lungs with necrotizing pancreatitis. Myeloperoxidase activity in the lung tissue was measured by a new chemiluminescence method to quantitate pulmonary neutrophil sequestration. RESULTS: Transient bursts of chemiluminescence that reflected O2- generation were observed at the lung surface 3 to 5 hours after necrotizing pancreatitis was induced. No O2- dependent chemiluminescence was detected at the lung surfaces of the neutropenic rat made by pretreatment with polyclonal antineutrophil antibody. Myeloperoxidase activity was significantly high (mean, 1690 cell numbers/mg wet tissue) in the necrotizing pancreatitis rat lung (p < 0.005). CONCLUSIONS: These data confirmed that activated neutrophils and neutrophil-derived superoxide anions were implicated in lung injury after acute severe pancreatitis.

Detection of superoxide free radicals in rats with acute pancreatitis.

To study the importance of oxygen-derived free radicals in acute pancreatitis, an experimental study of in vivo detection of superoxide free radicals (O2-) was performed using rats. Using a new chemiluminescence probe (2-methyl-6-[p-methoxyphenyl]-3,7-dihydro-imidazol[1,2-1]pyrazin- 3-one; MCLA; a Cypridina luciferin analogue) and a highly sensitive photon counting system, O2- from the pancreatic surface of rats with experimental acute pancreatitis induced by 180 micrograms cerulein/kg was detected. The time course of MCLA-dependent luminescence suggested that O2- production began 2-3 h after cerulein injection and then decreased gradually. Superoxide free radical production in the pancreas of rats with cerulein-induced acute pancreatitis was confirmed using MCLA-dependent chemiluminescence. This new method allows direct observation of the behavior of oxygen-derived free radicals.

Anti-neutrophil antibody attenuates the severity of acute lung injury in rats with experimental acute pancreatitis.

OBJECTIVE: To examine the role of activated neutrophils in microvascular injury after severe acute pancreatitis. We used the polyclonal anti-rat neutrophil antibody (PoAb) to deplete peripheral neutrophil counts and the anti-rat monoclonal antibody (MoAb) CD18 to block neutrophil adherence functions. DESIGN: Prospective, controlled trial. SETTING: Laboratory. PARTICIPANTS: Anesthetized male Wistar breeder rats, in which necrotizing pancreatitis was induced by injecting necrotizing agents into the pancreatic duct. INTERVENTIONS: Treatment groups received an infusion of PoAb, 8 mL/kg, before induction of pancreatitis or MoAb CD18, 2 mg/kg, after induction of pancreatitis. Control animals received 2 mL of rabbit serum or 1 mL of saline solution. MAIN OUTCOME MEASURES: Survival rate, white blood cell count, levels of serum amylase and lipase, myeloperoxidase activity in the lung, lipid peroxide levels in the pancreas, and results of histological studies. RESULTS: The survival rate of rats treated either with PoAb before or MoAb CD18 after induction of sepsis improved significantly (P < .01). Histologically and according to the levels of neutrophil myeloperoxidase in their lungs, rats treated with the antibodies 24 hours after inducing pancreatitis improved significantly (P < .05). Moreover, the serum lipase concentrations and lipid peroxide levels in the pancreas of these rats decreased significantly (P < .05). CONCLUSIONS: Both the depletion of peripheral neutrophils by PoAb and blocking of neutrophil adherence functions by MoAb CD18 may help to prevent acute lung injury caused by severe acute pancreatitis in this model.

Heparin effects on superoxide production by neutrophils.

Heparin is widely used in the treatment of various diseases, but the mechanisms of its biologic actions remain largely obscure. Recently, oxygen radicals, which are produced in a variety of conditions and cause tissue damage, have been implicated in the pathophysiology of various diseases. To investigate the relationship between heparin and oxygen radical production by neutrophils, we compared the effects of standard heparin (heparin sodium), which has been widely used, and a recently developed low molecular weight heparin (LMWH) which has potent anti-Xa activity, on neutrophil oxygen radical production in vitro. Standard heparin increased neutrophil oxygen radical production slightly at the low concentrations used clinically but reduced it at high concentrations, so that the effect of heparin on neutrophil oxygen radical production was biphasic. The effects of LMWH on neutrophil oxygen radical production were slight at both low and high concentrations. In disseminated intravascular coagulation (DIC) locally activated neutrophils produce oxygen radicals and have toxic effects in vivo. Thus we concluded that LMWH should be indicated for the treatment of DIC.

Prognosis of cancer of the duodenal papilla of Vater in relation to clinicopathological tumor extension.

Between 1981 and 1991, cancer of the duodenal papilla of Vater was surgically treated in 26 out of 28 cases (92%). Twenty-five patients underwent pancreatoduodenectomy and one had a total pancreatectomy. There were no surgical or hospital deaths in these 26 patients. Their five-year cumulative survival rate was 52%. In the 26 patients, we submitted the surgically removed tissue specimens to histopathological examination and performed immunohistochemical analysis using antibodies against various gastrointestinal cancer antigens. We analyzed the results in relation to the prognosis. The histological stage of the cancer was found to correlate with the prognosis. Patients having cancer invading the pancreatic parenchyma had a particularly poor prognosis. Some exhibited perineural invasion. Patients with cancer invasion into the pancreatic parenchyma frequently showed a grade II positive response to immunohistochemical staining, with a staining pattern similar to that of pancreatic duct cell carcinoma. In patients in whom cancer invasion did not extend beyond the sphincter of Oddi, the five-year survival rate was 100%. Therefore, cancer of the papilla of Vater which has not invaded beyond the the sphincter of Oddi, may be regarded as early cancer of the papilla of Vater. These results indicate that surgical procedures for cancer of the papilla of Vater should be selected on the basis of an accurate preoperative evaluation of cancer invasion by various imaging techniques (chiefly endoscopic ultrasonography), and that the treatment of advanced cancer of the papilla of Vater requires not only extensive surgical resection but also a more effective multi-disciplinary treatment plan.

Decreased production of active oxygen species by neutrophils in patients with liver cirrhosis and hepatocellular carcinoma.

Adult respiratory distress syndrome (ARDS) following infection is one of the postoperative complications of hepatectomy of cirrhosis. In this study we focused on the anti-microbial activity of neutrophils. We measured production of active oxygen species by neutrophils, and simultaneously examined their nutritional status, immunity and ICG (K-ICG) disappearance ratio. When compared to the controls, the patients with cirrhosis had significantly lower production of O2- upon stimulation by N-formyl-Met-Leu-Phe (fMLP) or opsonic zymosan (OZ). The presence of cancer did not affect results for the groups studied. Overall H2O2 production was lower in cirrhotic patients than in controls. There was a positive correlation between O2- production and K-ICG, which was used to estimate the severity of cirrhosis. Indicators of nutrition and immunity were also lower in cirrhotic patients, but neither of these indicators correlated with the production of active oxygen species. From these results, we concluded that the production of active oxygen species by neutrophils is lower in cirrhotic patients than in controls. Moreover, this decline correlates with the severity of cirrhosis.

13C-labeled trioctanoin breath test for exocrine pancreatic function test in patients after pancreatoduodenectomy.

To assess exocrine pancreatic function in patients before and after pancreatoduodenectomy (PD), we used the breath test, with nonradioactive 13C-labeled trioctanoin, in 14 patients before pancreatic resection because of localized pancreatic mass (preop-PD group), and in 13 patients who had undergone pancreatoduodenectomy more than 5 yr before (post-PD group). The results were compared with those of the secretin test, N-benzolyl-L-tyrosyl-p-amino benzoic acid (BT-PABA) test, and fecal chymotrypsin. Means +/- SD and frequencies of low values of the recovery of the breath test were 42.0 +/- 3.4%, 0/5 in the control; 24.2 +/- 10.5%, 14/14 in the preop-PD group; and 18.6 +/- 8.0, 13/13 in the post-PD group. The overall sensitivities in the preop- and post-PD groups were 100% for the recovery and 93% for the maximal mass ratio of the breath test, 89% for the secretin test, 67% for the BT-PABA test, and 64% for fecal chymotrypsin. The recovery of the breath test correlated significantly with the duodenal outputs of lipase, amylase, and chymotrypsin, and was not affected in patients with obstructive jaundice or with low D-xylose absorption. The breath test is as sensitive as the secretin test, more reliable than the conventional tubeless tests, and is available to follow up the exocrine pancreatic function before and after pancreatoduodenectomy.

The ability of polymorphonuclear leukocytes to produce active oxygen in a model of peritonitis in rats.

To elucidate the mechanism of enhancing survival in peritonitis rats treated with lentinan, a fully purified beta-1,3-glucan, we measured the active oxygen-producing ability of polymorphonuclear leukocytes (PMNs). Four groups of rats (group I, fecal peritonitis control; II, rats receiving 3 mg/kg lentinan intraperitoneally at the same time as peritonitis induction; III, rats receiving 1 mg/kg gentamicin intramuscularly; and IV, rats receiving combined lentinan-gentamicin treatment) were used. The survival period was significantly longer in group IV than in the other three groups. The ability of ascitic PMNs to produce active oxygen (superoxide, H2O2, myeloperoxidase) was significantly more than that of blood PMNs in each group at 20 h after peritonitis induction. The increase in active oxygen production in ascitic PMNs was higher in group IV compared with that in the other three groups. The concentration of lentinan in the blood was high at 24 h after administering lentinan intraperitoneally to both the normal and peritonitis rats. In the in vitro study, the superoxide production in normal rat blood PMNs was significantly higher in the presence of cytokines (IL-1 beta, IL-6, TNF-alpha) without dose-dependence but was not higher for the lentinan group than in the control. This study therefore suggests that lentinan activated the peritoneal macrophage secretory activity and produced cytokines which thus enhanced the ability of PMNs to produce active oxygen, which possesses a bactericidal ability in PMNs.

[Long-term results of terminal esophago-proximal gastrectomy for esophageal varices].

Between 1973 and 1991, 193 patients underwent terminal esophagoproximal gastrectomy (TEPG) for esophageal varices. One hundred and sixty patients (84%) were cirrhotics. Ten patients (5.2%) were died within hospital stay. In 116 elective patients who had been free from hepatocellular carcinoma during therapeutic courses, the 5- and 10-year survival rates were 77% and 62% in Child A, and 62% and 38% in Child B, respectively. There was a significant difference between the two groups (p < 0.05). Postoperative rebleeding from esophageal varices was infrequent within 5 years but increased after 5 years especially in Child B and Child C. For patients with these recurrent varices, endoscopic injection sclerotherapy was very effective and improved the prognosis. The survival rate of patients with extremely decreased platelet counts was not different from that of patients without. There were no other severe complications after TEPG. We conclude that TEPG would be indicated firstly for elective or prophylactic cases unless they have severe hepatic damage.

[The results and problems of surgical treatment of cancer of the duodenal papilla of Vater].

Twenty six of 28 cases (92.9%) of cancer of duodenal papilla of Vater were resected from 1981 to 1990. Twenty five pancreatoduodenectomy and one total pancreatectomy were performed. Operative death and hospital death were not observed. Three year and five year cumulative survival rates of resected cases were 58% and 52% respectively. There was relatively good correlation between pathological cancer extension and postoperative prognosis. Postoperative prognosis of the cases which had cancer extension to the pancreas was extremely poor. But the prognosis of the cases of cancer within Oddi muscle was extremely good. Immunohistochemical staining using anti CEA, anti CA19-9 and anti DUPAN 2 was studied and Grade III staining pattern was observed only in the cases which had cancer invasion to the pancreas. Preoperative diagnosis of cancer extension using endoscopic ultrasonography and appropriate choice of the operative procedure are important. Extended operation and effective adjuvant therapy are necessary for advanced cases of cancer of the duodenal papilla.

Results of hepatic resection and postoperative arterial chemotherapy for hepatocellular carcinoma.

To improve the outcome of patients who had undergone hepatic resection for hepatocellular carcinoma (HCC), we employed postoperative adjuvant hepatic arterial infusion chemotherapy (AHAI) in 23 patients. Patients showing various risk factors for the recurrence of HCC were given one shot of doxorubicin and mitomycin C suspended in an oily medium (lipiodol) and an infusion of 5-fluorouracil. The 3-year survival value calculated for patients who were treated with AHAI was 75%, which was significantly higher than that found for patients who did not receive AHAI (n = 156; P < 0.05). In addition, among the patients who underwent hepatic lobectomy, the survival of those who received AHAI was also significantly greater than that of those who did not (n = 46; P < 0.01). AHAI did not cause any severe complications. These results indicate that AHAI may be an effective therapy for patients with HCC.

[The significance of extent of resection in the treatment of hepatoma].

The relationship between the types of hepatectomy in each stage of hepatoma and the outcome was examined in 222 patients with hepatoma, according to the rules established by Liver Cancer Study Group of Japan. In Stage 1, the survival rate after absolute curative resection was better than that after relative curative resection. In Stage II, the survival after segmentectomy or lobectomy was significantly better than that after subsegmentectomy or less. Tumor recurrence rates in the remaining liver after segmentectomy or lobectomy were significantly lower than that after smaller resections. In Stages III and IV, there was no difference in survival among the various extents of hepatectomy. Incidence and cause of death after hepatectomy were not influenced by the extent of hepatectomy, as far as it was not beyond the preoperatively estimated safety limits. These results indicate the following: 1) In Stage I, absolute curative resection must be carried out. (2) In Stage II, segmentectomy or lobectomy should be applied when feasible. (3) The patients treated with subsegmentectomy or less for Stage II tumor, and the patients with Stage III or Stage IV tumor are at high risk of recurrence, and those patients need adjuvant therapy after hepatectomy.