[A four-year prospective study on the influence of serum elevated lipoprotein (a) concentration on ischemic heart disease and cerebral infarction in elderly patients with type 2 diabetes].

To clarify the influence of elevated serum lipoprotein (a) (Lp(a)) concentration on ischemic heart disease (IHD) and the perforating artery occlusion type of cerebral infarction (CI) in elderly patients with type 2 diabetes, we measured the serum levels of Lp(a) of type 2 diabetic patients (n = 158, 81 men and 77 women). The group was followed up prospectively for 4 years and the incidence of IHD or CI was monitored. The diagnosis of CI was confirmed by computed tomography and that of IHD, which includes myocardial infarction and angina pectoris, was diagnosed by electrocardiogram and blood chemistry examination, Lp(a) concentrations of 20 mg/dl or more were identified as elevated Lp(a) levels and Lp(a) concentrations of less than 20 mg/dl were identified as normal Lp(a) levels. A Kaplan-Meier survival analysis (log-rank test) assessed the time to event rate stratified by an Lp(a) cutoff point of 20 mg/dl. The predictive value for CI or IHD events was assessed by multiple logistic regression analysis. The probability of IHD events was significantly higher in the elevated Lp(a) group than in the normal Lp(a) group without a history of IHD but was similar in the two groups for those patients with a history of IHD. There was no significant difference between the elevated Lp(a) group and the normal Lp(a) group with regard to CI events in patients without a history of CI and with a history of CI. On multiple logistic regression analysis, Lp(a), hyperlipidemia and a history of IHD were significant predictors of IHD and hypertension, hyperlipidemia and a history of CI were significant predictors of CI. These results show that elevated serum Lp(a) concentrations is an independent risk factor for IHD, but not for the perforating artery occlusion type of CI in type 2 elderly diabetic patients.

Pharmacologic effect of recombinant human IFN-alpha, continuously released from a matrix prepared from a polyglycerol ester of fatty acids, on 2',5'-oligoadenylate synthetase activity in murine liver.

The objective of this study was to assess the pharmacologic effect of continuously released recombinant human interferon-alpha (rHuIFN-alpha) in the liver, the target organ of chronic hepatitis B and C, using 2',5'-oligoadenylate synthetase (2',5'-OAS) activity as an indicator of an antiviral state. A cylindrical matrix prepared from tetraglycerol dipalmitate (TGDP), a polyglycerol ester of fatty acids (PGEF), released rHuIFN-alpha in a pseudo-zero-order manner for about 1 week after implantation into mice, without any major loss of rHuIFN-alpha biologic activity during the release period. To evaluate the pharmacologic effect of the rHuIFN-alpha continuously released from this type of matrix, we established a murine test system. Bolus injections of rHuIFN-alpha solution at three doses increased 2',5'-OAS activities in murine liver extract and serum in a dose-dependent manner, indicating that this system is suitable for evaluating rHuIFN-alpha activity. After subcutaneous insertion of TGDP-matrix implants containing 5.5x10(7) IU rHuIFN-alpha per animal, 2',5'-OAS activities in both liver extracts and serum increased rapidly and remained high for over 1 week. Subcutaneous injections of an equivalent total dose (5.0x10(7) IU/animal per week) of rHuIFN-alpha solution in three or seven fractions prolonged 2',5'-OAS activities compared with a single bolus injection. Comparing 2',5'-OAS activity on day 7 and the portion of the area under the 2',5'-OAS activity-time curve above the normal level (deltaAUC) between the TGDP-matrix implant and multiple injections of the solution revealed that continuously released rHuIFN-alpha has an effect almost equivalent to that of three or seven injections of the solution per week.

[Sulfonylureas and daily blood glucose profiles in elderly diabetics].

The aim of this study is to clarify the relationship between the efficacy of sulfonylureas and duration of diabetes in elderly diabetics. Daily blood glucose profiles were measured in 87 Type 2 elderly diabetic patients on sulfonylureas (tolbutamide, gliclazide or glibenclamide). Plasma glucose concentrations were determined at 08.00 (before breakfast), 10.00, 12.00 (before lunch), 14.00, 18.00 (before dinner), 20.00, 24.00, 03.00, 06.00, 08.00 hours. The subjects were divided into 4 sub-groups, according to their duration of the diabetes (< 10, 10-14, 15-19, 20 or more years). Mean plasma glucose values at 08.00, 10.00, 20.00, 03.00 and 06.00 hours were not significantly different among the four groups. However, mean plasma glucose values at 12.00, 14.00, 18.00, 00.00 hours and mean total blood glucose area under the daily profile (total BG) were significantly different among the four groups and the values in patients with a history of diabetes of 15 years or more increased. Duration of diabetes positively correlated with blood glucose values at 12.00, 14.00, 18.00, 00.00, 03.00 hours and total BG, and the dose of sulfonylureas positively correlated with blood glucose values at 12.00, 14.00, 18.00, 00.00 hours and total BG in multiple regression analysis. These results suggest that duration of diabetes and dose of sulfonylureas are important determinants of blood glucose control with sulfonylureas in elderly diabetic patients.

[An elderly case of giant infected liver cyst that decreased dramatically without percutaneous aspiration].

A 70-year-old woman was referred to our hospital for treatment of cholelithiasis. A giant liver cyst (6 cm in diameter) had been diagnosed three years earlier. On admission, she had low grade fever and hepatomegaly. High values were observed for WBC (9900/microliter), CRP (8.9 mg/dl), GPT (45 IU/l), ALP (1399 IU/l), gamma-GTP (333 IU/l) and LAP (249 IU/l). The diagnosis of infected liver cyst (8 cm in diameter) was made based on contrast-enhanced CT scan. Endoscopic retrograde cholangiopancreaticography showed no communication between the cyst and the intrahepatic bile duct. She was successfully managed with antibiotics and discharged without percutaneous aspiration the cyst. On abdominal CT scan 4 months after the discharge, the liver cyst had decreased dramatically in size (1 cm in diameter). The patient remains healthy without symptoms.

A novel UDP-Glc-specific glucosyltransferase catalyzing the biosynthesis of 6-O-glucosides of bile acids in human liver microsomes.

Two active site-directed photoaffinity analogs, 5-[beta-32P]azido-UDP-glucuronic acid and 5-[beta-32P]azido-UDP-glucose, were used for the characterization of UDP-sugar-utilizing enzymes in human liver microsomes. Both compounds were recognized by human microsomal proteins: major photolabeled bands of 50-56 kDa were detected. Both photoincorporations were competitively decreased by increasing concentrations of either UDP-Glc or UDP-GlcUA, indicating a high affinity for both nucleotides. The patterns of photoaffinity labeling in the 50-56-kDa range by the two probes were significantly different, indicating the presence of different UDP-GlcUA- and UDP-Glc-specific enzymes of similar molecular mass. The presence of a UDP-Glc-dependent transferase was confirmed by the identification of an enzymatic activity catalyzing the formation of glucosides of the 6 alpha-hydroxylated bile acid hyodeoxycholic acid (3 alpha, 6 alpha-diOH (HDCA)) in the presence of UDP-Glc. The specific activity of 1.5-3.2 nmol/min/mg of protein was similar to that of 6 alpha-glucuronidation of HDCA. The apparent Km for UDP-Glc estimated with HDCA was 280 microM, and the formation of HDCA glucosides was strongly inhibited by UDP-GlcUA (apparent Ki = 7 microM). Evidence is presented that HDCA-specific UDP-glucuronosyltransferase (clone UGT2B4) expressed in V79 cells is not involved in glucosidation of HDCA and is not photolabeled with 5-[beta-32P]azido-UDP-Glc. Rigorous structure identification of the biosynthetic product proved that HDCA was glucosidated at the 6-position. Thus, this UDP-Glc-dependent activity catalyzing the biosynthesis of 6-O-glucosides of 6 alpha-hydroxylated bile acids represents a new pathway in the metabolism of these bile acids.

Application of 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid photoaffinity probes for the determination of the active site orientation of microsomal UDP-glucosyltransferases and UDP-glucuronosyltransferases.

A new approach to determining the active site orientation of microsomal glycosyltransferases is presented which utilizes the photoaffinity analogs [32P]5-Azido-UDP-glucose ([32P]5N3UDP-Glc) and [32P]5-Azido-UDP-glucuronic acid ([32P]5N3UDP-GlcA). It was previously shown that both photoprobes could be used to photolabel UDP-glucose:dolichol phosphate glucosyltransferase (Glc-P-Dol synthase), as well as the family of UDP-glucuronosyltransferases in rat liver microsomes. The effects of detergents, proteases, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the photolabeling of these enzymes were examined in intact rat liver microsomes. Photolabeling of Glc-P-Dol synthase by either photoprobe was the same in intact or disrupted vesicles, was susceptible to trypsin digestion, and was inhibited by the nonpenetrating inhibitor DIDS. Photolabeling of the UDP-glucuronosyltransferases by [32P]5N3UDP-GlcA was stimulated 1.3-fold in disrupted vesicles as compared to intact vesicles, whereas photolabeling of these enzymes by [32P]5N3UDP-Glc showed a 14-fold increase when vesicles were disrupted. Photolabeled UDP-glucuronosyltransferases were only susceptible to trypsin digestion in disrupted vesicles, and this was further verified by Western blot analyses. The results indicate a cytoplasmic orientation for access of UDP-sugars to Glc-P-Dol synthase and a lumenal orientation of most UDP-glucuronosyltransferases.

Enhanced antitumor activity in mice after administration of thermosensitive liposome encapsulating cisplatin with hyperthermia.

The antitumor effect of cisplatin-(CDDP)-encapsulated thermosensitive large unilamellar liposome (ThLip) administration with hyperthermia (HT) was examined in mice bearing Meth A fibrosarcoma. The tumor Pt levels after ThLip administration were increased in response to HT. The targeting index was approximately 3. The antitumor activity of ThLip + HT, as measured by tumor growth delay or tumor weight inhibition, was larger than that of ThLip without HT or a solution with or without HT. The CDDP dose in ThLip + HT to give equivalent tumor growth delay in solution (40 micrograms/mouse) + HT was about 10 micrograms/mouse, and therefore the targeted drug delivery enhancement ratio was about 4. The ratio correlates with the targeting index. The blood urea nitrogen level, as an indicator of CDDP nephrotoxicity, was increased 7 days after the administration of ThLip (40 micrograms CDDP/mouse) with HT. However, this blood urea nitrogen level rise was independent of the activity enhancement by the liposome. These findings suggest that the HT combined CDDP delivery system using ThLip can decrease the effective CDDP dose, thereby increasing its therapeutic index.

Increased tumor cisplatin levels in heated tumors in mice after administration of thermosensitive, large unilamellar vesicles encapsulating cisplatin.

Hyperthermia (HT)-dependent cisplatin (CDDP) release and tumor CDDP level increase after the administration of thermosensitive, large unilamellar vesicles (LUVs: LUV-1 and LUV-2) and a thermosensitive, small unilamellar vesicle (SUV: SUV-1) were examined in comparison with those following administration of a non-thermosensitive LUV (LUV-3) and a CDDP solution (Sol) in tumor bearing mice. The LUV-1 and LUV-2 released CDDP at a faster rate than SUV-1 when incubated in saline at temperatures between 41 and 44 degrees C. The blood CDDP levels after liposome administration were higher than those after Sol administration. The systemic clearance of LUV-2 was slightly larger than those of the other liposomes. The tumor CDDP levels after thermosensitive liposome administration were increased in response to HT in comparison to LUV-3 or Sol. The increased ratio for LUV-1 was the largest. The ratio of the area under the tumor CDDP level versus time curve (AUC) for LUV-1 + HT to the AUC for Sol + HT was approximately 5. The results indicate that (1) the tumor-CDDP level increase after thermosensitive liposome administration is due to CDDP release from the liposome in the blood at or adjacent to the heated tumor, (2) the increase is highly dependent on the heat sensitivity and systemic stability of the liposome, and (3) LUV, such as LUV-1, exhibit higher heat sensitivity and larger, targeted drug delivery efficiency than SUV.

Enzymatically activated microencapsulated liposomes can provide pulsatile drug release.

A system for the delayed or pulsed release of biologically active substances was achieved by encapsulating liposomes containing the substance of interest inside microcapsules. The microcapsules retain the liposomes but allow controlled diffusion of the active substance when it is released from the liposomes. Furthermore, by coating the liposomes with phospholipase A2 (an enzyme that removes an acyl group from the 2 position of phospholipids) before placing them within the microcapsule, a pulsatile release pattern was achieved both in vitro and in vivo. The time of onset of the pulse as well as the release rate can be controlled by the amount of phospholipase A2, the molecular weight of the poly(L-lysine) that is used to coat the microencapsulated liposomes, and the composition of the phospholipid bilayer membrane. Even at 37 degrees C the system would protect a model enzyme (horseradish peroxidase). When not placed inside the microencapsulated liposomes, the enzyme lost its activity in solution at 37 degrees C in a few days, whereas it retained 40% of the initial activity after 30 days of incubation at 37 degrees C inside the microencapsulated liposomes.

[Acute myeloblastic leukemia development in a patient with small cell lung cancer in complete remission].

A 67 year-old man was admitted to our hospital because of cough and sputum. He smoke one pack of cigarettes a day for more than twenty years and the chest X-ray film revealed a mass in the left hilum and left sided pleural effusion. The diagnosis of small carcinoma of the lung (limited disease, T4N1MO, stage 3B) was made by trans-bronchial lung biopsy and radiographic studies. Both chemotherapy (nimustine (ACNU), cyclophosphamide, vincristine, and methotrexate) and radiation therapy was started, however, the chemotherapy was discontinued in July 1987 because of severe anemia. The diagnosis of refractory anemia with excess of blasts in transformation (RAEB in T) was made by bone marrow aspiration and the patient was treated by transfusion (400-800 ml/week). In December 1987 transition to acute myeloblastic leukemia was confirmed by another bone marrow aspiration biopsy and the patient was given low dose cytosine arabinoside (Ara-C). The response was favorable in the beginning but in about two months pancytopenia became refractory and the patient died in June 1988. Clinically there was no sign of local or distal recurrences of lung cancer, and the complete remission of small cell lung cancer (SCLC) was confirmed by autopsy. Survival in SCLC remains poor, so that the choice of treatment is still the primary concern, however, development of other malignancies which include acute leukemia is another problem which should be taken into account when the treatment is extensive.

[An autopsy case of a patient with myasthenia gravis who showed various symptoms of collagen diseases and complicated with malignant thymoma].

A 37-year-old man suffered from photosensitivity and urinary casts with serological findings of positive anti-DNA antibody, LE cells and false positive VD reaction in September of 1979. He developed general fatigue, dyspnea and diplopia with ptosis of bilateral eyelids in November of 1979, which were improved by the anti-cholinesterase drugs. In January of 1980, he had an attack of unconsciousness and his chest X-ray film showed several tumorous shadows in the anterior mediastinum and middle and lower lung fields. Treating him with chemotherapy of VEMP, the pulmonary shadows disappeared. However, he developed severe muscle weakness with an elevated CPK (430 mU/ml) and a myogenic EMG pattern along with an increased anti-acetylcholine receptor antibody (243 n Mol/l), dysphagia and eyelid-ptosis. He died in September of 1985 and his autopsy disclosed a malignant thymoma of mixed type in the anterior mediastinum and an atrophy and fibrosis with infiltration of inflammatory cells in the striated muscles.

Antitumor activity on line 10 hepatoma in strain 2 guinea pigs and pharmaceutical properties of quinonyl-MDP preparations.

The pharmaceutical properties of quinonyl-MDP-66 were discussed with special reference to the stability of oil-in-water emulsion, distribution capacity into regional lymph nodes and tumor regressive activity. The oil-in-water emulsion of quinonyl-MDP-66, which was prepared by treatment of quinonyl-MDP-66 with squalane (25 x) and emulsified with aqueous solution of 5% HCO-60 and 5.6% d-mannitol, was kept in lyophylized state and used after reconstitution by the addition of water before use. The reconstituted suspension of quinonyl-MDP-66 in oil-in-water emulsion was stable for more than 24 hrs. The oil-in-water emulsion of quinonyl-MDP-66 as prepared above was effective for the distribution of quinonyl-MDP-66 into regional lymph node in rats and for the regression of line 10 hepatoma in strain 2 guinea pigs by intralesional injection.

Kinetics of receptor occupation and anticonvulsive effects of diazepam in rats.

Time courses of anticonvulsive effect nd benzodiazepine receptor occupation in the brain were determined after iv administration of diazepam (DZP) (1.2 mg/kg) to rats. Score for the anticonvulsive effect was assigned to each DZP-treated rat depending on the degree of protection against pentylenetetrazole-induced seizures. Measurement of in vivo receptor occupation was made by comparing the specific 3H-DZP binding in vitro to the crude synaptosomal fractions between vehicle-treated rats (control) and DZP-injected rats. The receptor occupation declined in parallel with the anticonvulsive effect with approximate half-life of 1 hr. A good linear correlation (r = 0.977) was observed between the receptor occupation and the anticonvulsive effect. The time course of anticonvulsive effect was further compared with that of DZP concentration in the brain which was previously reported [Igari et al., Drug Metab. Dispos. 10, 676 (1982)]. The anticonvulsive effect was nonlinearly related to the DZP concentration in the brain with the half-maximum concentration, Kd in vivo of 793 nM. The half-maximum concentration for the unbound DZP, Kd in vivo was also estimated to be 113 nM, which was still 50 times the values of Kd (2.3 nM) determined at 0 degree C from an in vitro binding study of 3H-DZP to the receptor in the crude synaptosomal fraction.

In vitro and in vivo assessment of hepatic and extrahepatic metabolism of diazepam in the rat.

Since diazepam is metabolized by many organs in the rat, the microsomal fractions of the liver, kidney, and lung from male Wistar rats were assayed for NADPH-dependent metabolism of diazepam and enzymatic parameters. The predicted extraction ratios were obtained from this in vitro experimental system. The organ clearances of the liver, kidney, and lung were then calculated for the determination of the relative contribution of each eliminating organ to the total body clearance (CLtot) of diazepam in the rat. The liver was the most effective eliminating organ, followed by the kidney and the lung, in that order. The hepatic extraction ratio of diazepam was determined in vivo after portal and femoral vein administrations of diazepam. The validity of the in vitro experimental system for the liver was demonstrated by a good agreement between the calculated hepatic extraction ratio of diazepam from in vitro enzymatic parameters (0.616) and that derived in vivo (0.648). However, the sum of organ clearances of the liver, kidney, and lung did not account for CLtot of diazepam in the rat, suggesting the possible contribution of the metabolism in the other organs or tissues, or an underestimation of the pulmonary and renal metabolism.

Prediction of diazepam disposition in the rat and man by a physiologically based pharmacokinetic model.

A physiologically based pharmacokinetic model for diazepam disposition was developed in the rat, incorporating anatomical, physiological, and biochemical parameters, i.e., tissue volume, blood flow rate, serum free fraction, distribution of diazepam into red blood cells, drug metabolism and tissue-to-blood distribution ratio. The serum free fraction of diazepam was determined by equilibrium dialysis at 37 degrees C and was constant over a wide concentration range. Partition of diazepam between plasma and erythrocytes was determined in vitro at 37 degrees C, and the resultant blood-to-plasma concentration ratio was constant over a wide concentration range. The enzymatic parameters (Km, Vmax) of the eliminating organs, i.e., liver, kidney, and lung, previously determined using microsomes, were used for the prediction. The tissue-to-blood distribution ratios inferred by inspection of the data when pseudoequilibrium is reached after i.v. bolus injection of 1.2 mg/kg diazepam were corrected according to the method of Chen and Gross. Predicted diazepam concentration time-course profiles in plasma and various organs or tissues, using an 11-compartmental model, were compared with those observed. Prediction was successful in all compartments including brain, the target organ of diazepam. Scale-up of the disposition kinetics of diazepam from rat to man was also successful.